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16 pages, 2847 KiB  
Article
Regulation of Isoleucine on Colonic Barrier Function in Rotavirus-Infected Weanling Piglets and Analysis of Gut Microbiota and Metabolomics
by Changsheng Jiang, Weiying Chen, Yanan Yang, Xiaojin Li, Mengmeng Jin, Ahmed H. Ghonaim, Shenghe Li and Man Ren
Microorganisms 2024, 12(12), 2396; https://doi.org/10.3390/microorganisms12122396 (registering DOI) - 22 Nov 2024
Abstract
Rotavirus (RV) is a significant contributor to diarrhea in both young children and animals, especially in piglets, resulting in considerable economic impacts on the global pig industry. Isoleucine (Ile), a branched-chain amino acid, is crucial for regulating nutrient metabolism and has been found [...] Read more.
Rotavirus (RV) is a significant contributor to diarrhea in both young children and animals, especially in piglets, resulting in considerable economic impacts on the global pig industry. Isoleucine (Ile), a branched-chain amino acid, is crucial for regulating nutrient metabolism and has been found to help mitigate diarrhea. This study aimed to assess the impact of isoleucine supplementation in feed on colonic barrier function, colonic microbiota, and metabolism in RV-infected weanling piglets. A total of thirty-two weaned piglets, aged 21 days, were randomly assigned to two dietary groups (each further divided into two subgroups, with eight replicates in each subgroup), receiving diets with either 0% or 1% isoleucine for a duration of 14 days. One group from each treatment was then challenged with RV, and the experimental period lasted for 19 days. The results showed that dietary Ile significantly increased the secretion of IL-4, IL-10, and sIgA in the colon of RV-infected weanling piglets (p < 0.05). In addition, Ile supplementation notably increased the expression of tight junction proteins, including Claudin-3, Occludin, and ZO-1 (p < 0.01), as well as the mucin protein MUC-1 in the colon of RV-infected weanling piglets (p < 0.05). Gut microbiota analysis revealed that dietary Ile increased the relative abundance of Prevotella and decreased the relative abundance of Rikenellaceae in the colons of RV-infected weanling piglets. Compared with the RV+CON, metabolic pathways in the RV+ILE group were significantly enriched in vitamin digestion and absorption, steroid biosynthesis, purine metabolism, pantothenate and CoA biosynthesis, cutin, suberine, and wax biosynthesis, as well as fatty acid biosynthesis, and unsaturated fatty acid biosynthesis. In conclusion, dietary Ile supplementation can improve immunity, colonic barrier function, colonic microbiota, and colonic metabolism of RV-infected weaned piglets. These findings provide valuable insights into the role of isoleucine in the prevention and control of RV. Full article
(This article belongs to the Special Issue Enteric Disease-Associated Pathogens)
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<p>Timeline of the events in the experimental study.</p>
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<p>The histological structure of the colon in RV-infected weaned piglets (HE, 200×). (<b>A</b>) The RV-CON group, (<b>B</b>) the RV-Ile group, (<b>C</b>) the RV+CON group, and (<b>D</b>) the RV+Ile group. Bar = 200 μm.</p>
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<p>Effect of isoleucine on the expression of colonic tight junction protein, defensin and mucin protein in weaned Piglets. The proteins expression of Claudin 3 (<b>A</b>), Occludin (<b>B</b>), MUC-1 (<b>C</b>) and ZO-1 (<b>D</b>), respectively, in colon of RV-infected piglets. Data are presented as the mean ± S.D, * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>Principal coordinate analysis (<b>A</b>) and non-metric multidimensional scaling analysis (<b>B</b>).</p>
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<p>Taxonomy. Phylum-level (<b>A</b>) and genus-level (<b>B</b>) taxonomic distribution of gut microbiota.</p>
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<p>Bioinformatics analysis of the metabolomics. (<b>A</b>) The volcano plot of differential metabolites between the RV-ILE group and the RV-CON group. (<b>B</b>) The volcano plot of differential metabolites between the RV+ILE group and the RV+CON group. (<b>C</b>) KEGG analysis of the differential metabolites between the RV-ILE group and the RV-CON group. (<b>D</b>) KEGG analysis of the differential metabolites between the RV+ILE group and the RV+CON group.</p>
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17 pages, 35306 KiB  
Article
Replacing Hydrolyzed Soybean Meal with Recombinant β-Glucosidase Enhances Resistance to Clostridium perfringens in Broilers Through Immune Modulation
by Jingxi Huang, Qihang Hou and Ying Yang
Int. J. Mol. Sci. 2024, 25(21), 11700; https://doi.org/10.3390/ijms252111700 - 31 Oct 2024
Viewed by 446
Abstract
Aglycone soy isoflavones have notable immune-regulatory bioactivity, while glycosidic forms in soybean meal pose challenges for absorption. β-Glucosidase (EC 3.2.1.21) catalyzes the non-reducing terminal β-d-glucosidic bonds, releasing β-d-glucan and aglycones. This study evaluated the impact of enzymatically hydrolyzed soybean [...] Read more.
Aglycone soy isoflavones have notable immune-regulatory bioactivity, while glycosidic forms in soybean meal pose challenges for absorption. β-Glucosidase (EC 3.2.1.21) catalyzes the non-reducing terminal β-d-glucosidic bonds, releasing β-d-glucan and aglycones. This study evaluated the impact of enzymatically hydrolyzed soybean meal (ESM) using recombinant β-glucosidase from Aspergillus niger on the growth performance and intestinal immune function of broilers under Clostridium perfringens infection. Prior to the feeding trial, soybean meal was enzymatically digested with recombinant β-glucosidase, ensuring almost complete conversion of glycosides to aglycones. After a week of pre-feeding, a total 180 healthy AA broilers were randomly assigned to three groups—control, semi-replacement of ESM (50% ESM), and full-replacement of ESM (100% ESM)—with 6 replicates of 10 chickens, and the trial lasted 28 days. On the 36th day, broilers were challenged with 1 mL of 1 × 1010 CFU/mL Clostridium perfringens (Cp) via gavage for 3 days. The results showed that the substitution of ESM had no effect on the body weight gain of broilers but significantly reduced the feed consumption and feed-to-gain ratio (p < 0.01). The study revealed that Cp significantly disrupted jejunal morphology, while ESM significantly mitigated this damage (p < 0.05). Real-time PCR results demonstrated that compared to the Cp group, ESM restored Cp-induced intestinal barrier impairments (e.g., Occludin, Claudin-1, Muc2), normalized aberrant cellular proliferation (PCNA) and apoptosis (Caspase-1 and Caspase-3), and upregulated the expression of anti-inflammatory factor Il-10 while suppressing pro-inflammatory cytokines (Il-1β, Il-6, and Il-8) (p < 0.05). Moreover, flow cytometry analyses demonstrated that ESM promoted Treg cell-derived Il-10, which alleviated macrophage-derived inflammation. Substituting conventional soybean meal with β-glucosidase, enzymatically treated, significantly reduced feed consumption and alleviated the intestinal damage and immune dysfunctions induced by Clostridium perfringens infection in broilers. Full article
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Figure 1
<p>Schematic diagram of enzymatic digestion of soybean meal: (<b>A</b>) agarose gel of the β-glucosidase gene originated from <span class="html-italic">Aspergillus niger</span> and SDS-PAGE of heterologously expressed protein; (<b>B</b>) effects of temperature and pH on the activity and stability of recombinant protein; (<b>C</b>) the HPLC results of soybean isoflavones content in soybean meal before and after enzymatic digestion. The red circle indicated the content of glycoside forms of soy isoflavones, while the green circle indicated the content of aglycone forms.</p>
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<p>Enzymatic SM ameliorates the intestinal epithelial barrier function of broilers infected with <span class="html-italic">Clostridium perfringens</span>: (<b>A</b>) HE staining of jejunum (×40 magnification, scale bar = 500 μm); (<b>B</b>) the villus height (VH), crypt depth (CD), and the ratio of VH and CD (VH/CD) in duodenum, n = 16; (<b>C</b>) qRT-PCR analysis of tight junction, n = 8; (<b>D</b>) qRT-PCR analysis of mucin2, n = 8. Ctrl group, a basal diet; <span class="html-italic">Cp</span>, basal diet infected with <span class="html-italic">Cp</span>; 50% ESM, semi-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>; 100% ESM, full-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>. The differences among groups were determined via ANOVA. The results are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Enzymatic SM mitigates intestinal epithelial homeostasis in jejunum of broilers infected with <span class="html-italic">Clostridium perfringens</span>: (<b>A</b>) qRT-PCR analysis of cell proliferation and apoptosis genes; (<b>B</b>) qRT-PCR analysis of toll-like receptor pathway-related genes; (<b>C</b>) qRT-PCR analysis of <span class="html-italic">NFκB</span> and <span class="html-italic">iNOS</span>. Ctrl group, a basal diet; <span class="html-italic">Cp</span>, basal diet infected with <span class="html-italic">Cp</span>; 50% ESM, semi-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>; 100% ESM, full-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>. The differences among groups were determined via ANOVA. The results are presented as mean ± SD, n = 8. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Enzymatic SM promotes the expression of T cell and IL-10 expression in jejunum of broilers infected with <span class="html-italic">Clostridium perfringens</span>: (<b>A</b>) flow cytometry analysis of CD45<sup>+</sup>, CD45<sup>+</sup>CD4<sup>+</sup>, CD4<sup>+</sup>CD25<sup>+</sup>IL-10<sup>+</sup> cell frequency in jejunum LPLs of broilers, n = 6; (<b>B</b>) qRT-PCR analysis of <span class="html-italic">Il-10</span> in jejunum, n = 8. Ctrl group, a basal diet; <span class="html-italic">Cp</span>, basal diet infected with <span class="html-italic">Cp</span>; 50% ESM, semi-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>; 100% ESM, full-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>. The differences among groups were determined via ANOVA. The results are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Enzymatic SM alleviates the expression of macrophages and related cytokines in jejunum of broilers infected with <span class="html-italic">Clostridium perfringens</span>: (<b>A</b>) flow cytometry analysis of CD45<sup>+</sup>Mac<sup>+</sup>IL-6<sup>+</sup> cell frequency in jejunum LPLs of broilers, n = 6; (<b>B</b>) qRT-PCR analysis of cytokines in jejunum, n = 8. Ctrl group, a basal diet; <span class="html-italic">Cp</span>, basal diet infected with <span class="html-italic">Cp</span>; 50% ESM, semi-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>; 100% ESM, full-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>. The differences among groups were determined via ANOVA. The results are presented as mean ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>The effects of enzymatic SM on intestinal lamina plasma-cells-mediated humoral immunity of broilers infected with <span class="html-italic">Clostridium perfringens</span>. Flow cytometry analysis of Bu-1<sup>+</sup>, Bu-1<sup>+</sup>IgY<sup>+</sup>, Bu-1<sup>+</sup>IgA<sup>+</sup>, and Bu-1<sup>+</sup>IgM<sup>+</sup> cell frequency in jejunum LPLs of broilers. Ctrl group, a basal diet; <span class="html-italic">Cp</span>, basal diet infected with <span class="html-italic">Cp</span>; 50% ESM, semi-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>; 100% ESM, full-replacement of enzymatic soybean meal diet infected with <span class="html-italic">Cp</span>. The differences among groups were determined via ANOVA. The results are presented as mean ± SD, n = 6. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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12 pages, 1207 KiB  
Article
Circulating Blood-Brain Barrier Proteins for Differentiating Ischaemic Stroke Patients from Stroke Mimics
by Pragati Kakkar, Meaad Almusined, Tarun Kakkar, Theresa Munyombwe, Linetty Makawa, Kirti Kain, Ahamad Hassan and Sikha Saha
Biomolecules 2024, 14(11), 1344; https://doi.org/10.3390/biom14111344 - 22 Oct 2024
Viewed by 731
Abstract
Background: Stroke is one of the leading causes of death and disability worldwide. The diagnosis of stroke remains largely clinical, yet widely used stroke scoring systems and brain imaging do not satisfactorily allow the distinction of ischaemic stroke (IS) patients from stroke mimics [...] Read more.
Background: Stroke is one of the leading causes of death and disability worldwide. The diagnosis of stroke remains largely clinical, yet widely used stroke scoring systems and brain imaging do not satisfactorily allow the distinction of ischaemic stroke (IS) patients from stroke mimics (SMs). Blood biomarkers are promising tools that could facilitate clinical triage. Methods: This study recruited 66 patients with IS and 24 SMs. The levels of Glial fibrillary acidic protein (GFAP), Neuron-specific enolase (NSE), Neurofilament light chain (NfL) and blood-brain barrier (BBB) proteins [Occludin (OCLN), Zonula occludens 1 (ZO-1), Claudin-5] in blood serum were measured by enzyme-linked immunosorbent assay technique. Biomarker levels in IS patients and SMs were compared using the Mann–Whitney U test. Multivariable logistic regression analysis was used to evaluate the diagnostic performance of biomarkers in combination with the National Institutes of Health Stroke Scale (NIHSS) score. Results: More significant differences in circulating GFAP, NfL, OCLN, ZO-1, and Claudin-5 but not NSE were found in IS patients compared to SMs. A combination of circulating ZO-1, Claudin-5, and OCLN with NIHSS score gives the highest diagnostic accuracy, sensitivity, and specificity. Conclusions: A prediction model with circulating BBB proteins in combination with NIHSS score differentiates between IS patients and SMs. Full article
(This article belongs to the Special Issue Biomarkers of Cardiovascular and Cerebrovascular Diseases)
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<p>Biomarker levels in serum. (<b>A</b>) GFAP, (<b>B</b>) NSE, (<b>C</b>) NfL, (<b>D</b>) Occludin, (<b>E</b>) Claudin-5, and (<b>F</b>) ZO-1 in ischaemic patients (N = 66) compared to mimics (N = 24). There was a significant increase in the concentration of biomarkers in an ischaemic group compared to mimics except NSE (46 ischaemic patients, 17 mimics). (Mann–Whitney U test, <span class="html-italic">p</span> &gt; 0.05: ns, <span class="html-italic">p</span> ≤ 0.0001: ****).</p>
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<p>Serum biomarker levels based on NIHSS score. (<b>A</b>) GFAP, (<b>B</b>) NSE, (<b>C</b>) NFL, (<b>D</b>) Occludin (<b>E</b>) Claudin-5, and (<b>F</b>) ZO-1 (Mann–Whitney U test, <span class="html-italic">p</span> &gt; 0.05: ns, <span class="html-italic">p</span> ≤ 0.01: **, <span class="html-italic">p</span> ≤ 0.001: ***, <span class="html-italic">p</span> ≤ 0.0001: ****), (NIHSS &gt; 7: N = 10, 8 for NSE, NIHSS ≤ 7: N = 56, 38 for NSE and mimics: N = 24, 17 for NSE).</p>
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15 pages, 5949 KiB  
Article
Immunomodulatory Effects of a Prebiotic Formula with 2′-Fucosyllactose and Galacto- and Fructo-Oligosaccharides on Cyclophosphamide (CTX)-Induced Immunosuppressed BALB/c Mice via the Gut–Immune Axis
by Wanyun Ye, Hanxu Shi, Wentao Qian, Liping Meng, Meihua Wang, Yalin Zhou, Zhang Wen, Muke Han, Yile Peng, Hongliang Li and Yajun Xu
Nutrients 2024, 16(20), 3552; https://doi.org/10.3390/nu16203552 - 19 Oct 2024
Viewed by 1204
Abstract
Obejectives: This study explored the immunomodulatory effects of a prebiotic formula consisting of 2′-fucosyllactose (2′-FL), galacto-oligosaccharides (GOSs), and fructo-oligosaccharides (FOSs) (hereinafter referred to as 2FGF) in cyclophosphamide (CTX)-induced immunosuppressed BALB/c mice and its underlying mechanisms. Methods: Sixty healthy female BALB/c mice were randomly [...] Read more.
Obejectives: This study explored the immunomodulatory effects of a prebiotic formula consisting of 2′-fucosyllactose (2′-FL), galacto-oligosaccharides (GOSs), and fructo-oligosaccharides (FOSs) (hereinafter referred to as 2FGF) in cyclophosphamide (CTX)-induced immunosuppressed BALB/c mice and its underlying mechanisms. Methods: Sixty healthy female BALB/c mice were randomly divided into the following groups: normal control (NC) group; CTX treatment (CTX) group; 2FGF low-dose (2FGF-L) group; 2FGF medium-dose (2FGF-M) group; and 2FGF high-dose (2FGF-H) group. An immunosuppressed model was established in the 2FGF-H group by intraperitoneal injection of 80 mg/kg CTX. After 30 days of 2FGF intervention, peripheral blood, spleen tissue, thymus tissue, and intestinal tissue from the mice were collected and analyzed. The changes in weight and food intake of the mice were recorded weekly. Hematoxylin-eosin (HE) staining was used to observe the histological change of the spleen tissue. Enzyme-linked immunosorbent assay (ELISA) was employed to detect cytokine levels in peripheral blood. Flow cytometry was used to analyze T lymphocyte subgroup ratio of splenic lymphocytes. Western blot analysis was conducted on intestinal tissues to assess the expression of proteins involved in the tight junction, toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathways. Additionally, molecular techniques were used to analyze the intestinal microbiota. Results: The results showed that 2FGF restored CTX-induced splenic injury, increased the number of splenic T lymphocytes, and elevated serum cytokines such as interleukin-4 (IL-4) and IL-10. In the intestine, 2FGF upregulated the expression of intestinal epithelial tight junction proteins such as Claudin-1 and zonula occludens 1 (ZO-1), thereby enhancing intestinal barrier function and activating the MAPK and NF-κB pathways via TLR4. Furthermore, 2FGF elevated the α-diversity (Shannon and Simpson indices) of the gut microbiota in CTX-induced immunosuppressed mice, enriching bacteria species positively correlated with anti-inflammatory cytokines (e.g., IL-4) such as g_Streptomyces and g_Bacillus and negatively correlated with pro-inflammatory cytokines (e.g., IL-1β) such as g_Saccharomyces. The results suggest that 2FGF may enhance immunity via the gut–immune axis. Conclusions: The 2FGF prebiotic formula showed an immunomodulatory effect in CTX-induced immunosuppressed mice, and the mechanism of which might involve optimizing the gut flora, enhancing intestinal homeostasis, strengthening the intestinal barrier, and promoting the expression of immune factors by regulating the TLR-4/MAPK/NF-κB pathway. Full article
(This article belongs to the Section Nutritional Immunology)
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<p>The effect of 2FGF on body weight (<b>A</b>), food intake (<b>B</b>), immune organ indices (<b>C</b>), histological observation of the spleen (original magnification: ×40) (<b>D</b>), and splenic T lymphocyte subgroups in CTX-induced immunosuppressed mice (<b>E</b>). NC, normal control group; CTX, CTX-induced immunosuppressed model group; 2FGF-L, 2FGF (2′-FL/GOS/FOS = 1:3:3) administered at 1 g/kg bw; 2FGF-M, 2FGF administered at 2 g/kg bw; 2FGF-H, 2FGF administered at 4 g/kg bw. The yellow arrows in (<b>D</b>) indicate the boundaries of the red and white pulp of the spleen. Data are presented as means ± standard deviations (n = 12). Significant differences compared to the NC group are indicated by * (<span class="html-italic">p</span> &lt; 0.05), and significant differences compared to the CTX group are indicated by # (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>The effect of 2FGF on the protein expression of Claudin-1 and ZO-1 (<b>A</b>), TLR-4, <span class="html-italic">p</span>-p38, p38, <span class="html-italic">p</span>-ERK, ERK, <span class="html-italic">p</span>-JNK, JNK, <span class="html-italic">p</span>-p65, and p65 in the large intestines (ascending colon) of CTX-induced immunosuppressed mice (<b>B</b>), with representative Western blot images (<b>C</b>). Data are presented as means ± standard deviations (n = 3). Significant differences compared to the NC group are indicated by * (<span class="html-italic">p</span> &lt; 0.05) and ** (<span class="html-italic">p</span> &lt; 0.01), and significant differences compared to the CTX group are indicated by # (<span class="html-italic">p</span> &lt; 0.05) and ## (<span class="html-italic">p</span> &lt; 0.01).</p>
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<p>The effect of 2FGF on the Shannon index (<b>A</b>), Simpson index (<b>B</b>), PCoA analysis (<b>C</b>), phylum-level relative abundance (<b>D</b>), and species-level relative abundance (<b>E</b>) of the gut microbiota in CTX-induced immunosuppressed mice. Data are presented as means ± standard deviations (n = 8). Significant differences compared to the NC group are indicated by * (<span class="html-italic">p</span> &lt; 0.05) and ** (<span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Histogram of LDA scores (<b>A</b>). Correlation analysis between differential genera and cytokines (<b>B</b>). Red indicates a positive correlation, and blue indicates a negative correlation. Significant differences are indicated by * (<span class="html-italic">p</span> &lt; 0.05) and ** (<span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Histogram of LDA scores (<b>A</b>). Correlation analysis between differential genera and cytokines (<b>B</b>). Red indicates a positive correlation, and blue indicates a negative correlation. Significant differences are indicated by * (<span class="html-italic">p</span> &lt; 0.05) and ** (<span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Possible immunomodulatory mechanism of the 2FGF prebiotic formula in CTX-induced immunosuppressed mice.</p>
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15 pages, 1184 KiB  
Article
The Addition of Hot Water Extract of Juncao-Substrate Ganoderma lucidum Residue to Diets Enhances Growth Performance, Immune Function, and Intestinal Health in Broilers
by Yu-Yun Gao, Xiao-Ping Liu, Ying-Huan Zhou, Jia-Yi He, Bin Di, Xian-Yue Zheng, Ping-Ting Guo, Jing Zhang, Chang-Kang Wang and Ling Jin
Animals 2024, 14(20), 2926; https://doi.org/10.3390/ani14202926 - 11 Oct 2024
Viewed by 564
Abstract
The purpose of this experiment was to investigate the effects of Hot Water Extract of Juncao-substrate Ganoderma lucidum Residue (HWE-JGLR) on the immune function and intestinal health of yellow-feather broilers. In an animal feeding experiment, 288 male yellow-feather broilers (1 day old) were [...] Read more.
The purpose of this experiment was to investigate the effects of Hot Water Extract of Juncao-substrate Ganoderma lucidum Residue (HWE-JGLR) on the immune function and intestinal health of yellow-feather broilers. In an animal feeding experiment, 288 male yellow-feather broilers (1 day old) were randomly allocated to four treatment groups with six replicates of 12 birds each. The control (CON) group was fed a basal diet. HJ-1, HJ-2, and HJ-3 were fed a basal diet supplemented with 0.25%, 0.50%, and 1.00% HWE-JGLR, respectively. The feeding trial lasted for 63 d. The results showed increased ADFI (p = 0.033) and ADG (p = 0.045) of broilers in HJ-3, compared with the CON group. Moreover, higher contents of serum IL-4 and IL-10 and gene expression of IL-4 and IL-10 in jejunum mucosa and lower contents of serum IL-1β and gene expression of IL-1β in jejunum mucosa in HJ-3 were observed (p < 0.05). Additionally, the jejunal mucosal gene expression of Claudin-1 and ZO-1 in HJ-2 and HJ-3 was higher than that in the CON group (p < 0.05). As for the microbial community, compared with the CON group, the ACE index, Shannon index, and Shannoneven index of cecal microorganisms in HJ-2 and HJ-3 were elevated (p < 0.05). PCoA analysis showed that the cecal microbial structure of broilers in HJ-2 and HJ-3 was different from the CON group (p < 0.05). In contrast with the CON group, the broilers in HJ-2 and HJ-3 possessed more abundant Desulfobacterota at the phylum level and unclassified Lachnospiraceae, norank Clostridia vadinBB60 group and Blautia spp. at the genus level, while Turicibacter spp. and Romboutsia spp. were less (p < 0.05). In conclusion, dietary supplementation with HWE-JGLR can improve growth performance, enhance body immunity and intestinal development, and maintain the cecum microflora balance of yellow-feather broilers. Full article
(This article belongs to the Section Animal Nutrition)
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<p>Effects of HWE-JGLR on serum cytokines (<b>A</b>) and immunoglobulins (<b>B</b>) contents of broilers. Values are presented as a mean ± SD. Letters in the case of each cytokine describe significant differences between groups at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effects of HWE-JGLR on gene expression of jejunal mucosal cytokine (<b>A</b>) and jejunal tight-junction protein (<b>B</b>) in broilers. Values are presented as a mean ± SD. Letters in the case of each cytokine or jejunal tight-junction protein describe significant differences between groups at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Rarefaction curves of sequencing read (<b>A</b>) and Venn diagram (<b>B</b>) of ASVs in broilers. PCoA analysis based on the Bray-Curtis distance (<b>C</b>). Relative abundance at phylum level (<b>D</b>) and genus level (<b>E</b>) in cecum microbiota. In (<b>B</b>–<b>D</b>) A represents HJ-1, B represents HJ-2, and C represents HJ-3.</p>
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21 pages, 6048 KiB  
Article
Enhanced Effect of β-Lactoglobulin Immunization in Mice with Mild Intestinal Deterioration Caused by Low-Dose Dextran Sulphate Sodium: A New Experimental Approach to Allergy Studies
by Dagmara Złotkowska, Lidia Hanna Markiewicz, Anna Maria Ogrodowczyk, Barbara Wróblewska and Ewa Wasilewska
Nutrients 2024, 16(20), 3430; https://doi.org/10.3390/nu16203430 - 10 Oct 2024
Viewed by 851
Abstract
Background/Objectives: Cow’s milk allergy is one of the most common food allergies in children, and its pathomechanism is still under investigation. Recently, an increasing number of studies have linked food allergy to intestinal barrier dysfunction. The present study aimed to investigate changes in [...] Read more.
Background/Objectives: Cow’s milk allergy is one of the most common food allergies in children, and its pathomechanism is still under investigation. Recently, an increasing number of studies have linked food allergy to intestinal barrier dysfunction. The present study aimed to investigate changes in the intestinal microenvironment during the development of β-lactoglobulin (β-lg) allergy under conditions of early intestinal dysfunction. Methods: BALB/c mice received intraperitoneal β-lg with Freund’s adjuvant, followed by oral β-lg while receiving dextran sulphate sodium salt (DSS) in their drinking water (0.2% w/v). The immunized group without DSS and the groups receiving saline, oral β-lg, or DSS served as controls. Results: The study showed that the immunization effect was greater in mice with mild intestinal barrier dysfunction. Although DSS did not affect the mice’s humoral response to β-lg, in combination with β-lg, it significantly altered their cellular response, affecting the induction and distribution of T cells in the inductive and peripheral tissues and the activation of immune mediators. Administration of β-lg to sensitized mice receiving DSS increased disease activity index (DAI) scores and pro-inflammatory cytokine activity, altered the distribution of claudins and zonulin 1 (ZO-1) in the colonic tissue, and negatively affected the balance and activity of the gut microbiota. Conclusions: The research model used appears attractive for studying food allergen sensitization, particularly in relation to the initial events leading to mucosal inflammation and the development of food hypersensitivity. Full article
(This article belongs to the Special Issue Relationship Between Food Allergy and Human Health)
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<p>Experimental design. Abbreviations used: <span class="html-italic">β</span>-lg—<span class="html-italic">β</span>-lactoglobulin; cFA—complete Freund adjuvant; iFA—incomplete Freund adjuvant.</p>
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<p>Changes in disease activity index (DAI) (<b>A</b>) and body weight (<b>B</b>) during challenge of mice with oral <span class="html-italic">β</span>-lg and DSS on days 21–31. Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as group means ± SD. Means differ as follows: * at <span class="html-italic">p</span> ≤ 0.05, ** at <span class="html-italic">p ≤</span> 0.01.</p>
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<p>Terminal antibody endpoint titers (EpT; for IgG, IgA) and concentrations (for IgE) in blood serum (<b>A</b>,<b>B</b>,<b>D</b>) and fecal extracts (<b>C</b>) (secretory IgA). Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as group means ± SD. Means with different superscripts (letters) are different at <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>The distribution of CD4<sup>+</sup> (<b>A</b>), CD4<sup>+</sup>CD25<sup>+</sup> (<b>B</b>), CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup> (<b>C</b>), and CD4<sup>+</sup>IL-10<sup>+</sup> (<b>D</b>) T cells in Payer’s patches (PPs), mesenteric lymph nodes (MLNs), spleens (SPLs), head and neck lymph nodes (HNLNs), and peripheral blood mononuclear cells (PBMCs). Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as the mean of the group ± SD. Means with different superscripts (letters) are different at <span class="html-italic">p</span> ≤ 0.05. Differences within the same tissue are shown.</p>
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<p>Distribution of CD8<sup>+</sup> (<b>A</b>) and CD8<sup>+</sup>IL-10<sup>+</sup> (<b>B</b>) T cells in Payer’s patches (PPs), mesenteric lymph nodes (MLNs), spleens (SPLs), head and neck lymph nodes (HNLNs), and peripheral blood mononuclear cells (PBMCs). Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as the mean of the group ± SD. Means with different superscripts (letters) are different at <span class="html-italic">p</span> ≤ 0.05. Differences within the same tissue are shown.</p>
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<p>Cytokine levels in unstimulated (control) and <span class="html-italic">β</span>-lg-stimulated (<span class="html-italic">β</span>-lg-treated) mesenteric lymph node (MLN) cell cultures. Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as the mean of the group ± SD. Means with different letters (12 groups were compared with each other) differ at <span class="html-italic">p</span> ≤ 0.05 (two-way ANOVA).</p>
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<p>Effect of immunization method on the expression of interleukin-1beta (IL-1<span class="html-italic">β</span>), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), toll-like receptor-2 (TLR-2), toll-like receptor-4 (TLR-4), claudin-2 (CLDN-2), claudin-12 (CLDN-12), occludin (OCC) and zonulin-1 (ZO-1) genes (<b>A</b>), and protein content (<b>B</b>) in the small intestinal tissue. Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as the mean of the group ± SD. Means with different letters differ at <span class="html-italic">p</span> ≤ 0.05, with * differ at at <span class="html-italic">p</span> ≤ 0.05 and with ** differ at <span class="html-italic">p ≤</span> 0.01.</p>
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<p>Effect of immunization method on the expression of interleukin-1beta (IL-1<span class="html-italic">β</span>), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), toll-like receptor-2 (TLR-2), toll-like receptor-4 (TLR-4), claudin-2 (CLDN-2), claudin-12 (CLDN-12), occludin (OCC) and zonulin-1 (ZO-1) genes (<b>A</b>), and protein content (<b>B</b>) in the colonic tissue. Groups of mice: saline—healthy control group, saline administration at all stages; DSS—oral administration of DSS; o.<span class="html-italic">β</span>-lg—oral administration of <span class="html-italic">β</span>-lg; o.<span class="html-italic">β</span>-lg/DSS—oral administration of <span class="html-italic">β</span>-lg and DSS; ip.o.<span class="html-italic">β</span>-lg—intraperitoneal injection and oral administration of <span class="html-italic">β</span>-lg; ip.o.<span class="html-italic">β</span>-lg/DSS—intraperitoneal injection of <span class="html-italic">β</span>-lg and oral administration of <span class="html-italic">β</span>-lg and DSS. Data are expressed as the mean of the group ± SD. Means with different letters differ at <span class="html-italic">p</span> ≤ 0.05, with * differ at at <span class="html-italic">p</span> ≤ 0.05 and with ** differ at <span class="html-italic">p ≤</span> 0.01.</p>
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15 pages, 1375 KiB  
Article
Effect of Fermented Mulberry Leaves on Gut Health of Finishing Pigs
by Su Peng, Yiyan Cui, Miao Yu, Min Song, Zhimei Tian, Dun Deng, Zhichang Liu and Xianyong Ma
Animals 2024, 14(19), 2911; https://doi.org/10.3390/ani14192911 - 9 Oct 2024
Viewed by 786
Abstract
This study was conducted to investigate the effects of supplementing fermented mulberry leaves (FML) on intestinal morphology, antioxidant capacity, and immune function in the gut of finishing pigs. Eighteen 132-day-old healthy crossbred (Duroc × Landrace × Yorkshire) male castrated pigs were randomly divided [...] Read more.
This study was conducted to investigate the effects of supplementing fermented mulberry leaves (FML) on intestinal morphology, antioxidant capacity, and immune function in the gut of finishing pigs. Eighteen 132-day-old healthy crossbred (Duroc × Landrace × Yorkshire) male castrated pigs were randomly divided into two treatment groups with nine replicates per group. The control (CON) group was fed the basal diet, and the FML group was fed the basal diet supplemented with 10% FML. The experiment lasted 69 days. The results showed that 10% FML improved gut health. The apparent total tract digestibility in dry matter, crude protein, crude fiber, neutral detergent fiber, acidic detergent fiber, ether extract, and crude ash increased in the 10% FML group of finishing pigs compared to the CON group (p < 0.05). Duodenal, jejunal, and ileal intestinal morphology, such as villus height and villus-height-to-crypt-depth ratio, increased in the 10% FML group compared to the CON group, whereas crypt depth decreased in the duodenum, jejunum, and ileum (p < 0.05). Total antioxidant capacity increased in the ileum of the 10% FML group compared with the CON group (p < 0.05). The FML supplementation improved the contents of duodenal immunoglobulin A, jejunal interleukin-1β, interleukin-8, ileal interleukin-1β, interleukin-6, interferon-γ, and immunoglobulins A and M compared to the control group (p < 0.05). Moreover, FML downregulated the mRNA expression levels of tumor necrosis factor-α in the duodenum, Toll-like receptor 4, nuclear factor-κ B-P65, and myeloid differentiation factor 88 in the jejunum, and Toll-like receptor 4 and nuclear factor-κ B-P65 in the ileum (p < 0.05). The FML also upregulated Montrose uniting church 1 in the duodenum and claudin 2 in the ileum (p < 0.05). In conclusion, dietary supplementation with 10% FML improved the gut health of finishing pigs and FML is a potential feed ingredient for pig breeding. Full article
(This article belongs to the Section Pigs)
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<p>The small intestinal morphology in finishing pigs. CON, fed with the basal diet; FML, fed with the basal diet supplemented with 10% fermented mulberry leaves.</p>
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18 pages, 2459 KiB  
Article
Effects of Taurine and Enzymatic Cottonseed Protein Concentrate Supplementation in Low-Fishmeal Diet on Growth, Liver Antioxidant Capacity, and Intestinal Health of Golden Pompano (Trachinotus ovatus)
by Zhanzhan Wang, Shuling Liao, Zhong Huang, Jun Wang, Yun Wang, Wei Yu, Heizhao Lin, Zhenhua Ma, Zhenyan Cheng and Chuanpeng Zhou
Fishes 2024, 9(10), 405; https://doi.org/10.3390/fishes9100405 - 9 Oct 2024
Viewed by 955
Abstract
This study was conducted to investigate the impacts of the dietary addition of taurine and enzymatic cottonseed protein concentrate (ECPC) in low-fishmeal diet on the growth performance, plasma biochemical indices, hepatic antioxidant capacity, intestinal anti-inflammatory capacity, intestinal microflora, and muscle quality of golden [...] Read more.
This study was conducted to investigate the impacts of the dietary addition of taurine and enzymatic cottonseed protein concentrate (ECPC) in low-fishmeal diet on the growth performance, plasma biochemical indices, hepatic antioxidant capacity, intestinal anti-inflammatory capacity, intestinal microflora, and muscle quality of golden pompano (Trachinotus ovatus). A total of three isonitrogenous diets were given to 225 golden pompanos (5.6 ± 0.14 g). They were randomly divided into nine cages (1.0 m × 1.0 m × 1.5 m; three cages per treatment) with equal stocking numbers of twenty-five fish per cage. The results indicated that the CSM-TC group significantly increased the growth performance of juvenile T. ovatus (p < 0.05). The results indicated that compared with other groups, the addition of 1% ECPC and 0.25% taurine has been found to enhance the WGR (weight gain rate), SGR (specific growth rate), and CF (condition factor). Compared with other groups, the relative expressions of GH, GHR1, GHR2, IGF1, IGF2, and MyoG were significantly higher in fish fed with CSM-TC. The results showed that CSM-TC significantly increased the activities of alkaline phosphatase, complement 3, and complement 4 enzymes (p < 0.05). The results showed that dietary CSM-TC increased the activities of hepatic superoxide dismutase and total antioxidant capacity enzymes. Compared with other groups, the hepatic relative expressions of Nrf2, HO-1, and GSH-Px were significantly higher in fish fed with CSM-TC. The results showed that dietary CSM-TC increased the activities of intestinal chymotrypsin, lipase, and α-amylase enzymes. A CSM-TC diet significantly increased the relative expressions of IL-10, ZO-1, Occludin, Claudin-3, and Claudin-15 (p < 0.05). The results showed that CSM-C significantly increased the index of Ace and Chao1 (p < 0.05). In conclusion, a high-fermented cottonseed meal diet can have detrimental effects on physiological health in golden pompano, while adding 1% ECPC and 0.25% taurine can improve hepatic and intestinal health via attenuating inflammation and oxidative stress. Full article
(This article belongs to the Section Nutrition and Feeding)
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<p>Relative mRNA expressions of golden pompano fed with the experimental diets in muscle. The data include triplicate means. Means in the same row that have distinct superscript letters are substantially different, as determined by Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Relative mRNA expressions of antioxidant-related genes of golden pompano fed with the experimental diets in hepatic. The data include triplicate means. Means in the same row that have distinct superscript letters are substantially different, as determined by Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Relative mRNA expressions of immune-related and physical barrier-related genes of golden pompano fed with the experimental diets in intestinal. The data include triplicate means. Means in the same row that have distinct superscript letters are substantially different, as determined by Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Venus map of OTUs of the intestinal flora of golden pompano fed with the experimental diets. The data include triplicate means. Means in the same row that have distinct superscript letters are substantially different, as determined by Duncan’s test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Heatmap of phylum of the intestinal flora of golden pompano fed with the experimental diets.</p>
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<p>Heatmap of the genera of the intestinal flora of golden pompano fed with the experimental diets.</p>
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19 pages, 6264 KiB  
Article
The Role of Claudins in the Pathogenesis of Dextran Sulfate Sodium-Induced Experimental Colitis: The Effects of Nobiletin
by Asmaa Al-Failakawi, Aishah Al-Jarallah, Muddanna Rao and Islam Khan
Biomolecules 2024, 14(9), 1122; https://doi.org/10.3390/biom14091122 - 4 Sep 2024
Viewed by 812
Abstract
Background: The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic [...] Read more.
Background: The pathogenesis of inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease is not well understood. This study investigated the roles and regulation of the claudin-1, -2, -3, and -4 isoforms in the pathogenesis of ulcerative colitis, and the potential therapeutic effects of nobiletin. Methods: Colitis was induced in rats by administering dextran sulfate sodium [DSS] in drinking water for seven days. Animals were treated daily with nobiletin [oral, 60 mg/Kg body weight] and studied in four groups, C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. On day seven, the animals were sacrificed, and colonic tissues were collected and analyzed. Results: Both macroscopic and microscopic findings suggest the progression of colitis. In the inflamed colon, claudin-1 and -4 proteins were decreased, claudin-2 increased, while the claudin-3 protein remained unchanged. Except for claudin-1, these changes were not paralleled by mRNA expression, indicating a complex regulatory mechanism. Uniform β-actin expression along with consistent quality and yield of total RNA indicated selectivity of these changes. Nobiletin treatment reversed these changes. Conclusions: Altered expression of the claudin isoforms -1, -2, and -4 disrupts tight junctions, exposing the lamina propria to microflora, leading to electrolyte disturbance and the development of ulcerative colitis. Nobiletin with its anti-inflammatory properties may be useful in IBD. Full article
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<p>(<b>A</b>) Body weight (Wt) change [percentage of day 0 body weight] and (<b>B</b>) the food efficiency ratios of the animals from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated pair of groups.</p>
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<p>(<b>A</b>) Colon weights [g/cm colon length], (<b>B</b>) the disease activity score, (<b>C</b>) a representative histology of hematoxylin and eosin-stained [left panel] and alcian blue-stained [right panel] colonic tissue sections, and (<b>D</b>) the histological score of the animals from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated pair of groups. Magnification: 10×. Scalebar = 100 μm.</p>
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<p>A bar diagram showing MPO activity units/min-mg in the colonic segments from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated groups.</p>
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<p>(<b>A</b>) Representative photomicrographs showing immunofluorescence staining, and (<b>B</b>–<b>E</b>) a bar diagram showing the expression levels of the indicated claudin isoforms measured as mean fluorescence intensity [MFI, arbitrary units] using a specific primary antibody and an FITC-labeled secondary antibody in the tissue sections of colon from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The tissue sections were counter-stained with DAPI. Claudins are expressed in the surface lining epithelium [arrows] and in the epithelial cells [arrow heads] lining the glands [G]. L—lumen of the colon. Magnification = 20×, Scalebars = 100 μm. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated pair of groups.</p>
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<p>(<b>A</b>) A bar diagram showing the protein expression levels relative to β-actin [ratios] of claudin-1, (<b>B</b>) claudin-2, (<b>C</b>) claudin-3, and (<b>D</b>) claudin-4 isoforms in the colon from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated pair of groups. <span class="html-italic">p</span> &lt; 0.05 with respect to C. The insets show the representative ECL Western blot analysis of the indicated claudin isoform and β-actin.</p>
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<p>A bar diagram showing the mRNA expression levels as ratios relative to β-actin mRNA of the indicated claudin mRNA isoforms -1, -2, and -4 using (<b>A</b>) the endpoint RT-PCR method and (<b>B</b>) SYBR green RT-PCR-based C<sub>T</sub> calculations for claudin isoforms -1 and -2 in the LiCl-purified colonic RNA samples from the test conditions: C [non-colitis control], D [DSS-induced colitis], CN [nobiletin-treated non-colitis control], and DN [nobiletin-treated DSS-induced colitis]. The data are the mean ± SE [n = 10]. *, <sup>#</sup> indicate significance at <span class="html-italic">p</span> &lt; 0.05 between the indicated pair of groups.</p>
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<p>A proposed mechanism of the pathogenesis of DSS-induced ulcerative colitis and its reversal by nobiletin treatment in colitis rats. The figure was created using BioRender with permission.</p>
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14 pages, 4545 KiB  
Article
Protection of Tight Junctional Complexes between hCMEC/D3 Cells by Deep-Sea Fibrinolytic Compound FGFC1
by Xiaozhen Diao, Hui Han, Haoyu Sun, Haixing Zhang and Wenhui Wu
Mar. Drugs 2024, 22(8), 341; https://doi.org/10.3390/md22080341 - 26 Jul 2024
Cited by 1 | Viewed by 1117
Abstract
Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood–brain barrier (BBB), whose regulation closely correlates to the BBB’s integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of [...] Read more.
Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood–brain barrier (BBB), whose regulation closely correlates to the BBB’s integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of the BBB via the construction of an in vitro model. This study aims to investigate the protective effect of the deep-sea-derived fibrinolytic compound FGFC1 against H2O2-induced dysfunction of TJCs and to elucidate the underlying mechanism. The barrier function was shown to decline following exposure to 1 mM H2O2 in an in vitro model of hCMEC/D3 cells, with a decreasing temperature-corrected transendothelial electrical resistance (tcTEER) value. The decrease in the tcTEER value was significantly inhibited by 80 or 100 µM FGFC1, which suggested it efficiently protected the barrier integrity, allowing it to maintain its function against the H2O2-induced dysfunction. According to immunofluorescence microscopy (IFM) and quantitative real-time polymerase chain reaction (qRT-PCR), compared to the H2O2-treated group, 80~100 µM FGFC1 enhanced the expression of claudin-5 (CLDN-5) and VE-cadherin (VE-cad). And this enhancement was indicated to be mainly achieved by both up-regulation of CLDN-5 and inhibition of the down-regulation by H2O2 of VE-cad at the transcriptional level. Supported by FGFC1’s molecular docking to these proteins with reasonable binding energy, FGFC1 was proved to exert a positive effect on TJCs’ barrier function in hCMEC/D3 cells via targeting CLDN-5 and VE-cad. This is the first report on the protection against H2O2-induced barrier dysfunction by FGFC1 in addition to its thrombolytic effect. With CLDN-5 and VE-cad as the potential target proteins of FGFC1, this study provides evidence at the cellular and molecular levels for FGFC1’s reducing the risk of bleeding transformation following its application in thrombolytic therapy for cerebral thrombosis. Full article
(This article belongs to the Special Issue Bioactive Natural Products from the Deep-Sea-Sourced Microbes)
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<p>The construction of the hCMEC/D3-based in vitro barrier model confirmed by TEER assay. (<b>a</b>) hCMEC/D3 cells were reseeded on the PET membrane of the hanging inserts in 24-well microplates and the barrier function was measured by the probe of a Millicell ERS-2 system; (<b>b</b>) hCMEC/D3 cells successfully formed a monolayer from 6 to 8 days after the reseeding, when the tcTEER values of the model became stable around 210–265 Ω/cm<sup>2</sup> (310.15 K). Relative tcTEER values are means ± SD, <span class="html-italic">n</span> = 6.</p>
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<p>The H<sub>2</sub>O<sub>2</sub>-induced barrier dysfunction by TEER assay in hCMEC/D3 cells. (<b>a</b>) hCMEC/D3 cells treated with solutions with different concentrations of H<sub>2</sub>O<sub>2</sub> were investigated by CCK-8; (<b>b</b>) the in vitro model of hCMEC/D3 was exposed to 3.5 h incubation with different concentration H<sub>2</sub>O<sub>2</sub> solutions. Each cell viability or tcTEER value is indicated as mean ± SD, <span class="html-italic">n</span> = 6. Asterisks indicate a significant difference among solutions with various concentrations of H<sub>2</sub>O<sub>2</sub> (** <span class="html-italic">p</span> &lt; 0.025, *** <span class="html-italic">p</span> &lt; 0.01), as determined by the Kruskal–Wallis test.</p>
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<p>The protection of TJCs in hCMEC/D3 cells from barrier dysfunction (red) by FGFC1 at various concentrations (green series). (<b>a</b>) The maintenance of relative tcTEER values by FGFC1 solutions (FS_55, 100) against 1 mM H<sub>2</sub>O<sub>2</sub> treatment (NC) or not (blank). (<b>b</b>,<b>c</b>) The relationship between relative tcTEER values and the concentration of FGFC1 solution (FS_25, 55, 80, 100) against 1 mM H<sub>2</sub>O<sub>2</sub> treatment (NC). Monolayers constructed by hCMEC/D3 cells were pretreated with or without (NC) different concentrations (25, 55, 80, and 100 µM) of the FGFC1 solution for 2 h, followed by 1 mM H<sub>2</sub>O<sub>2</sub> treatment for 4 h or not (blank). Relative tcTEER values are means ± SD, <span class="html-italic">n</span> = 6. Asterisks indicate a significant difference compared to blank (** <span class="html-italic">p</span> &lt; 0.005), determined by the Mann–Whitney U test, or among various concentrations of the FGFC1 solutions (* <span class="html-italic">p</span> &lt; 0.05), determined by the Kruskal–Wallis test.</p>
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<p>The protection by FGFC1 of the expression of TJC proteins including CLDN-5, OCLN, ZO-1, and VE-cad. Monolayers constructed by hCMEC/D3 cells were pretreated with different concentrations of the FGFC1 solution (80 and 100 µM) or without (NC) for 2 h, followed by 1 mM H<sub>2</sub>O<sub>2</sub> treatment or not (blank) for 4 h. Images were observed by fluorescence microscopy. Each image is representative of 3 similar experiments. The scale bar is 200 µm.</p>
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<p>The enhancements in TJC protein expression in barrier dysfunction (red) caused by various concentrations of FGFC1 (green series). Four immunostained photomicrographs of CLDN-5 (<b>a</b>), OCLN (<b>b</b>), ZO-1 (<b>c</b>), and VE-cad (<b>d</b>) were separately randomly selected in each group and semi-quantitatively analyzed by ImageJ. Immunostained mean value is calculated by the mean integral optical density of each group relative to blank. Bars are means ± SD, <span class="html-italic">n</span> = 4. Asterisks indicate a significant difference between various concentrations of FGFC1 solutions (*** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.005), as determined by the Kruskal–Wallis test.</p>
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<p>The influence of various concentrations of FGFC1 solution on the expression of TJC proteins (CLDN-5 (<b>a</b>), OCLN (<b>b</b>), ZO-1 (<b>c</b>), and VE-cad (<b>d</b>)) at the transcriptional level. Monolayers constructed by hCMEC/D3 cells were pretreated with various concentrations of FGFC1 solution (55, 80, and 100 µM) or without (NC) for 2 h, followed by 1 mM H2O2 treatment or not for 4 h. The total RNA was extracted from each group pretreated with the FGFC1 solution (2 h) at 2.0, 3.0, 3.5 h incubation time with H2O2 to investigate the mRNA level of each TJC protein. The mRNA expression relative to the blank is shown as mean ± SD, <span class="html-italic">n</span> = 6. Asterisks indicate a significant difference among various concentrations of FGFC1 solution (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.025), as determined by the Kruskal–Wallis test.</p>
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<p>Schematic representation of the interaction between FGFC1 and the optimal conformation of CLDN-5, OCLN, and VE-cad. (<b>a-1</b>–<b>c-1</b>) The 3D model of FGFC1 docking CLDN-5, OCLN, and VE-cad (yellow: FGFC1, brown: CLDN-5, green: OCLN, purple: VE-cad). (<b>a-2</b>–<b>c-2</b>) Planar model of the binding sites of CLDN-5, OCLN, and VE-cad by FGFC1.</p>
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<p>Schematic representation of the interaction between FGFC1 and the optimal conformation of ZO-1. (<b>a-1</b>–<b>c-1</b>) The 3D model of FGFC1 docking three domains of ZO-1 (yellow: FGFC1, blue: PDZ1, pink: PDZ2, red: PDZ3). (<b>a-2</b>–<b>c-2</b>) Planar model of the binding sites of PDZ1/2/3 of ZO-1 by FGFC1.</p>
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<p>The optimization of the concentrations of FGFC1 solutions. (<b>a</b>) hCMEC/D3 cells pretreated with different concentrations (25, 55, 80, 100 µM) of FGFC1 (FS_25/55/80/100) followed by 2 and 4 h incubation with H<sub>2</sub>O<sub>2</sub> were investigated by CCK-8. Each cell viability value is indicated as mean ± SD, <span class="html-italic">n</span> = 6. (<b>b</b>) The barriers constructed by hCMEC/D3 cells treated with various concentrations of FGFC1 solution, whose morphology characteristics were observed under the microscopy. Red arrows refer to the enlarged interval induced by the high concentrations of FGFC1 solutions. Each image is representative of 3 similar experiments. The scale bar is 100 µm.</p>
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24 pages, 4736 KiB  
Article
Biophysical Analysis of a Minimalistic Kidney Model Expressing SGLT1 Reveals Crosstalk between Luminal and Lateral Membranes and a Plausible Mechanism of Isosmotic Transport
by Erik Hviid Larsen and Jens Nørkær Sørensen
Biomolecules 2024, 14(8), 889; https://doi.org/10.3390/biom14080889 - 23 Jul 2024
Viewed by 709
Abstract
We extended our model of the S1 tubular segment to address the mechanisms by which SGLT1 interacts with lateral Na/K pumps and tight junctional complexes to generate isosmotic fluid reabsorption via tubular segment S3. The strategy applied allowed for simulation of laboratory experiments. [...] Read more.
We extended our model of the S1 tubular segment to address the mechanisms by which SGLT1 interacts with lateral Na/K pumps and tight junctional complexes to generate isosmotic fluid reabsorption via tubular segment S3. The strategy applied allowed for simulation of laboratory experiments. Reproducing known experimental results constrained the range of acceptable model outputs and contributed to minimizing the free parameter space. (1) In experimental conditions, published Na and K concentrations of proximal kidney cells were found to deviate substantially from their normal physiological levels. Analysis of the mechanisms involved suggested insufficient oxygen supply as the cause and, indirectly, that a main function of the Na/H exchanger (NHE3) is to extrude protons stemming from mitochondrial energy metabolism. (2) The water path from the lumen to the peritubular space passed through aquaporins on the cell membrane and claudin-2 at paracellular tight junctions, with an additional contribution to water transport by the coupling of 1 glucose:2 Na:400 H2O in SGLT1. (3) A Na-uptake component passed through paracellular junctions via solvent drag in Na- and water-permeable claudin-2, thus bypassing the Na/K pump, in agreement with the findings of early studies. (4) Electrical crosstalk between apical rheogenic SGLT1 and lateral rheogenic Na/K pumps resulted in tight coupling of luminal glucose uptake and transepithelial water flow. (5) Isosmotic transport was achieved by Na-mediated ion recirculation at the peritubular membrane. Full article
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Figure 1

Figure 1
<p>(<b>A</b>) Membrane organization of the kidney tubule epithelium. The apical (<span class="html-italic">am</span>) and lateral (<span class="html-italic">lm</span>) cell membranes have similar relatively large areas compared with the contra-luminal (peritubular) cell membrane (<span class="html-italic">pm</span>) contacting the basement membrane (<span class="html-italic">bm</span>). Tight junctions (<span class="html-italic">tj</span>) provide entrance to the lateral intercellular space (<span class="html-italic">lis</span>). The so-called ‘basal infoldings’ are continuous with the lateral membranes; the interspace basement membrane (<span class="html-italic">ibm</span>) providing an exit from <span class="html-italic">lis</span> is approximately 10% of the area of the entire basement membrane. Sodium pumps are confined to the lateral membranes and to the complex of lateral membrane infoldings at the cell base. Thus, lateral and peritubular membranes represent different functional domains, implying that sodium ions transported into the cell through the <span class="html-italic">am</span> are actively transported into the <span class="html-italic">lis</span> in order to exit the epithelium through the <span class="html-italic">ibm</span>. It follows that the so-called ‘basolateral’ osmotic permeability conferred by AQP1 is confined to lateral membranes, with the lateral intercellular space constituting a compartment of its own. (<b>B</b>) Functional membrane organization of the proximal tubule S3 segment. SGLT1 (yellow) couples 1 glucose with 2 Na and 400 H<sub>2</sub>O. The driving force for glucose is given by the transmembrane concentration differences of sodium and glucose and the apical membrane potential, according to Equation (3b). Together with paracellular solvent drag, the apical 1Na–2Cl–1K cotransporter ensures the reabsorption of filtered K. By being water-permeable, the junctional ion and glucose pathways play important roles in preventing the loss of glucose via urine. In modeling isosmotic transport, the serosal Na–Cl cotransporter was activated iteratively to achieve overall osmotic equilibrium between the peritubular and reabsorbed fluid. (<b>C</b>) ‘Saturation’ of the glucose flux across the apical membrane (pmol/cm<sup>2</sup>/s) with the luminal glucose concentration (mM), which, in our mathematical analysis, was due to glucose concentration-dependent SGLT1 permeability according to Equation (3a) [<a href="#B16-biomolecules-14-00889" class="html-bibr">16</a>]. (<b>D</b>) Instantaneous relationship between the Na flux carried by SGLT1 and apical membrane potential according to Equation (3b). The constant electrical field in the membrane and the fixed stoichiometry of 2 Na–1 glucose predict strong inward rectification of SGLT1 fluxes. The reversal potential, <math display="inline"><semantics> <mrow> <msubsup> <mi>E</mi> <mrow> <mi>r</mi> <mi>e</mi> <mi>v</mi> </mrow> <mrow> <mi>S</mi> <mi>G</mi> <mi>L</mi> <mi>T</mi> <mn>1</mn> </mrow> </msubsup> </mrow> </semantics></math>, is +60 mV at physiological concentrations and 37 °C, i.e., ~125 mV above the apical membrane potential of approximately −65 mV.</p>
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<p>Computations predict that mammalian kidney late proximal straight tubules spontaneously generate a hyperosmotic transportate. Model epithelium ‘perfused on both sides’ with simulated saline containing (mM), 146 Na, 4 K, 150 Cl and 5 glucose, as indicated on the left-hand top and left-hand bottom panels, respectively. Notably, the epithelium generates significant transepithelial water flow at transepithelial osmotic equilibrium. Note that the space between the cells, <span class="html-italic">lis</span>, is slightly hyperosmotic (307 mosM) relative to bilateral bathing solutions of similar composition (305 mosM), while the fluid flowing into the peritubular space across <span class="html-italic">ibm</span> is significantly hyperosmotic (362 mosM).</p>
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<p>Computed trans- and intraepithelial biophysical variables following a step change in bilateral glucose from 0.5 to 5 mM at <span class="html-italic">t</span> = 20 s. (<b>A</b>) The evoked fast increase in rheogenic SGLT1 flux passing a peak of 628 pmol·cm<sup>−2</sup>·s<sup>−1</sup> (red) leads to fast cell depolarization from −77.1 to −60.7 mV (black). The subsequently slower relaxation to −65.7 mV follows the transient decrease in rheogenic 2Na–1glucose uptake, while the cellular glucose concentration increases to 8 mM (green). (<b>B</b>) The fast increase in the depolarization-generated rheogenic active Na flux across <span class="html-italic">lm</span> proceeds through a slower increase caused by the increase in the cellular Na concentration, as indicated in the red graph. (<b>C</b>) Uptake by <span class="html-italic">lis</span> of NaCl results in an increase in the osmolarity (LIS<sup>osm</sup>) and volume (LIS<sup>vol</sup>) of the lateral intercellular space. (<b>D</b>) The increase in <span class="html-italic">lis</span> volume results in an increase in hydrostatic pressure (LIS<sup>hp</sup>), which drives fluid out of <span class="html-italic">lis</span> into the peritubular space, resulting in a steady state transepithelial water uptake of 10.8 nL·cm<sup>−2</sup>·s<sup>−1</sup> (blue).</p>
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<p>(<b>A</b>) Computed water flows in the three entrance pathways of the proximal tubule S3 segment, continued from <a href="#biomolecules-14-00889-f003" class="html-fig">Figure 3</a>A–D. At 20 s, the glucose concentration increased bilaterally from 0.5 to 5 mM. The time course of <span class="html-italic">inward</span> water transport through two pathways in the apical membrane, AQP1 and SGLT1, was compared with that of junctional water transport through cation-permeable claudin-2. The dotted blue graph shows the time course of fluid <span class="html-italic">exit</span> across the interspace basement membrane (<span class="html-italic">ibm</span>) approaching a steady-state flow of 10.8 nL·cm<sup>−2</sup>·s<sup>−1</sup>. This is the sum of the stationary flows in the three luminal entrance pathways, AQP = 3.20 nL·cm<sup>−2</sup>·s<sup>−1</sup>, claudin-2 = 3.30 nL·cm<sup>−2</sup>·s<sup>−1</sup>, and SGLT1 = 4.32 nL·cm<sup>−2</sup>·s<sup>−1</sup>. (<b>B</b>) Computed water flows in the entrance pathways of the proximal tubule S3 segment, continued from (<b>A</b>). The protocol of (<b>A</b>) was applied, however, eliminating the water channel in SGLT1 (green graph) by replacing Equation (3b) in <a href="#sec3-biomolecules-14-00889" class="html-sec">Section 3</a> with <math display="inline"><semantics> <mrow> <msubsup> <mi>J</mi> <mrow> <msub> <mi>H</mi> <mn>2</mn> </msub> <mi>O</mi> </mrow> <mrow> <mi>S</mi> <mi>G</mi> <mi>L</mi> <mi>T</mi> <mn>1</mn> </mrow> </msubsup> <mo>=</mo> <mn>0</mn> <mo>×</mo> <msubsup> <mi>J</mi> <mrow> <mi>G</mi> <mi>l</mi> <mi>u</mi> <mi>c</mi> </mrow> <mrow> <mi>S</mi> <mi>G</mi> <mi>L</mi> <mi>T</mi> <mn>1</mn> </mrow> </msubsup> </mrow> </semantics></math>. The time course of inward water transport through the apical membrane, AQP1 and SGLT1, respectively, is compared to that of the junctional water transport through cation-permeable claudin-2. The dotted blue graph shows the time course of fluid exit across the interspace basement membrane (<span class="html-italic">ibm</span>) approaching a steady-state flow of 11.0 nL·cm<sup>−2</sup>·s<sup>−1</sup>, which is the sum of stationary flows in the three luminal entrance pathways, AQP = 7.07 nL·cm<sup>−2</sup>·s<sup>−1</sup>, claudin-2 = 3.93 nL·cm<sup>−2</sup>·s<sup>−1</sup>, and SGLT1 = 0 nL·cm<sup>−2</sup>·s<sup>−1</sup>.</p>
Full article ">Figure 4 Cont.
<p>(<b>A</b>) Computed water flows in the three entrance pathways of the proximal tubule S3 segment, continued from <a href="#biomolecules-14-00889-f003" class="html-fig">Figure 3</a>A–D. At 20 s, the glucose concentration increased bilaterally from 0.5 to 5 mM. The time course of <span class="html-italic">inward</span> water transport through two pathways in the apical membrane, AQP1 and SGLT1, was compared with that of junctional water transport through cation-permeable claudin-2. The dotted blue graph shows the time course of fluid <span class="html-italic">exit</span> across the interspace basement membrane (<span class="html-italic">ibm</span>) approaching a steady-state flow of 10.8 nL·cm<sup>−2</sup>·s<sup>−1</sup>. This is the sum of the stationary flows in the three luminal entrance pathways, AQP = 3.20 nL·cm<sup>−2</sup>·s<sup>−1</sup>, claudin-2 = 3.30 nL·cm<sup>−2</sup>·s<sup>−1</sup>, and SGLT1 = 4.32 nL·cm<sup>−2</sup>·s<sup>−1</sup>. (<b>B</b>) Computed water flows in the entrance pathways of the proximal tubule S3 segment, continued from (<b>A</b>). The protocol of (<b>A</b>) was applied, however, eliminating the water channel in SGLT1 (green graph) by replacing Equation (3b) in <a href="#sec3-biomolecules-14-00889" class="html-sec">Section 3</a> with <math display="inline"><semantics> <mrow> <msubsup> <mi>J</mi> <mrow> <msub> <mi>H</mi> <mn>2</mn> </msub> <mi>O</mi> </mrow> <mrow> <mi>S</mi> <mi>G</mi> <mi>L</mi> <mi>T</mi> <mn>1</mn> </mrow> </msubsup> <mo>=</mo> <mn>0</mn> <mo>×</mo> <msubsup> <mi>J</mi> <mrow> <mi>G</mi> <mi>l</mi> <mi>u</mi> <mi>c</mi> </mrow> <mrow> <mi>S</mi> <mi>G</mi> <mi>L</mi> <mi>T</mi> <mn>1</mn> </mrow> </msubsup> </mrow> </semantics></math>. The time course of inward water transport through the apical membrane, AQP1 and SGLT1, respectively, is compared to that of the junctional water transport through cation-permeable claudin-2. The dotted blue graph shows the time course of fluid exit across the interspace basement membrane (<span class="html-italic">ibm</span>) approaching a steady-state flow of 11.0 nL·cm<sup>−2</sup>·s<sup>−1</sup>, which is the sum of stationary flows in the three luminal entrance pathways, AQP = 7.07 nL·cm<sup>−2</sup>·s<sup>−1</sup>, claudin-2 = 3.93 nL·cm<sup>−2</sup>·s<sup>−1</sup>, and SGLT1 = 0 nL·cm<sup>−2</sup>·s<sup>−1</sup>.</p>
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<p>Isosmotic transport was achieved by regulating the recirculation flux of small ions between the lateral intercellular space and the peritubular solution; see <a href="#biomolecules-14-00889-f001" class="html-fig">Figure 1</a>B. As in the small intestine, isosmotic transport occurs through regulation of the activity of the cotransporter of the peritubular membrane (red). Notably, significant cell swelling from 966 (<a href="#biomolecules-14-00889-f002" class="html-fig">Figure 2</a>) to 1420 nL/cm<sup>2</sup> (above) was caused by the uptake of Na and Cl via the cotransporter. The water flows in the two apical pathways are as follows. AQP-mediated 4.52 nL·cm<sup>−2</sup>·s<sup>−1</sup> and SGLT1-mediated 4.35 nL·cm<sup>−2</sup>·s<sup>−1</sup>.</p>
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<p>Increasing the lateral membrane’s Cl permeability from 0.3 × 10<sup>−8</sup> m/s (<a href="#biomolecules-14-00889-f005" class="html-fig">Figure 5</a>) to 0.6 × 10<sup>−7</sup> m/s to simulate VRAC activation decreased the epithelial cell volume back to its ‘physiological value’ as in <a href="#biomolecules-14-00889-f002" class="html-fig">Figure 2</a>. The water flow mediated by AQP was 4.73 nL·cm<sup>−2</sup>·s<sup>−1</sup>, which is about the same as the water flow mediated by SGLT1, 4.07 nL·cm<sup>−2</sup>·s<sup>−1</sup>. The water uptake by solvent drag through lateral intercellular space was 3.71 nL·cm<sup>−2</sup>·s<sup>−1</sup>.</p>
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21 pages, 4547 KiB  
Article
Dietary Protease Supplementation Improved Growth Performance and Nutrients Digestion via Modulating Intestine Barrier, Immunological Response, and Microbiota Composition in Weaned Piglets
by Tao Liu, Wen Ma, Jun Wang, Yulong Wei, Yibo Wang, Zheng Luo, Ying Zhang, Xiangfang Zeng, Wutai Guan, Dan Shao and Fang Chen
Antioxidants 2024, 13(7), 816; https://doi.org/10.3390/antiox13070816 - 8 Jul 2024
Viewed by 1300
Abstract
Despite mounting evidence for dietary protease benefits, the mechanisms beyond enhanced protein degradation are poorly understood. This study aims to thoroughly investigate the impact of protease addition on the growth performance, intestinal function, and microbial composition of weaned piglets. Ninety 28-day-old weaned pigs [...] Read more.
Despite mounting evidence for dietary protease benefits, the mechanisms beyond enhanced protein degradation are poorly understood. This study aims to thoroughly investigate the impact of protease addition on the growth performance, intestinal function, and microbial composition of weaned piglets. Ninety 28-day-old weaned pigs were randomly assigned to the following three experimental diets based on their initial body weight for a 28-day experiment: (1) control (CC), a basic diet with composite enzymes without protease; (2) negative control (NC), a diet with no enzymes; and (3) dietary protease (PR), a control diet with protease. The results show that dietary proteases significantly enhanced growth performance and boosted antioxidant capacity, increasing the total antioxidant capacity (T-AOC) levels (p < 0.05) while reducing malonaldehyde levels (p < 0.05). Additionally, protease addition reduced serum levels of inflammatory markers TNF-α, IL-1β, and IL-6 (p < 0.05), suppressed mRNA expression of pro-inflammatory factors in the jejunum (p < 0.01), and inhibited MAPK and NF-κB signaling pathways. Moreover, protease-supplemented diets improved intestinal morphology and barrier integrity, including zonula occludens protein 1(ZO-1), Occludin, and Claudin-1 (p < 0.05). Microbiota compositions were also significantly altered by protease addition with increased abundance of beneficial bacteria (Lachnospiraceae_AC2044_group and Prevotellaceae_UCG-001) (p < 0.05) and reduced harmful Terrisporobacter (p < 0.05). Further correlation analysis revealed a positive link between beneficial bacteria and growth performance and a negative association with inflammatory factors and intestinal permeability. In summary, dietary protease addition enhanced growth performance in weaned piglets, beneficial effects which were associated with improved intestinal barrier integrity, immunological response, and microbiota composition. Full article
(This article belongs to the Special Issue Role of Antioxidants Intake on Gut Microbiome)
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Figure 1

Figure 1
<p>Effects of proteases on plasma antioxidant indicators in weaned piglets. (<b>A</b>–<b>E</b>) represent the changes in plasma T-AOC, MDA, GSH, GSH-PX, and SOD. All data with error bars are mean ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>Effects of proteases on urea nitrogen, inflammation, and immunity in weaned piglets. (<b>A</b>–<b>E</b>) denote the changes in plasma levels of BUN, TNF-α, IL-1β, IL-6, and IL-12. (<b>F</b>–<b>H</b>) denote the changes in plasma levels of IgA, IgG, and IgM. All data with error bars are mean ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>The effect of proteases on the intestinal morphology of weaned piglets. (<b>A</b>) Representative images and histomorphometry analysis of duodenum, jejunum, and ileum tissue sections from weaned piglets stained with H&amp;E. (<b>B</b>–<b>D</b>) Villus height (VH), crypt depth (CD), and VH/CD ratio for the duodenum, respectively. (<b>E</b>–<b>G</b>) VH, CD, and VH/CD ratio for the jejunum, respectively. (<b>H</b>–<b>J</b>) VH, CD, and VH/CD ratio for the ileum, respectively. All data with error bars are mean ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>The effect of protease on the intestinal integrity of weaned piglets. (<b>A</b>–<b>D</b>) Protein levels of ZO-1, Claudin-1, and Occludin in the jejunum. (<b>E</b>–<b>G</b>) Changes in blood, DAO, D-Lactate, and LPS. All data with error bars are mean ± SEM (<span class="html-italic">n</span> = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>The effect of proteases on intestinal inflammation and the MAPK signaling pathway of weaned piglets. (<b>A</b>–<b>C</b>) Proinflammatory factor mRNA expression (<span class="html-italic">TNF-α</span>, <span class="html-italic">IL-1β</span>, <span class="html-italic">IL-6</span>) in intestinal inflammatory factors. (<b>D</b>–<b>H</b>) Western blotting was used to detect the protein levels of phosphorylated ERK (P-ERK), phosphorylated JNK (P-JNK), phosphorylated P38 (P-P38), and phosphorylated NF-κB (P-NF-κB). All data are shown as means ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>The effect of proteases on the diversity of gut microbiota in weaned piglets. (<b>A</b>) Venn diagram for the OTUs. (<b>B</b>) Beta diversity was assessed by PCoA at Bray–Curtis distances. (<b>C</b>–<b>E</b>) Alpha diversity analyses for Simpson, Shannon, and goods_coverage indices. (<b>F</b>) Relative abundance at level of microbial phylum. (<b>G</b>) Relative abundance at level of microbial genus. All data shown as means ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effects of protease on intestinal flora diversity and metabolism of weaned piglets. (<b>A</b>) Relative abundance of <span class="html-italic">Firmicutes</span>. (<b>B</b>) Relative abundance of <span class="html-italic">Bacteroidetes</span>. (<b>C</b>) Ratio of <span class="html-italic">Firmicutes</span>/<span class="html-italic">Bacteroidetes</span> (F/B). (<b>D</b>–<b>F</b>) <span class="html-italic">Lachnospiraceae_AC2044_group</span>, <span class="html-italic">Prevotellaceae_UCG-001</span>, and <span class="html-italic">Terrisporobacter</span> between the CC group, PR group, and the NC group of relative abundance. (<b>G</b>) The metabolic pathway information of each differential gene was obtained. (<b>H</b>) The relative values are indicated by color intensity according to the legend indicated in the right corner. All data with error bars are mean ± SEM (n = 6, 5 replicates). *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01. <sup>a,b</sup> Different letters indicate significant differences between the 2 data.</p>
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<p>Correlation analysis of intestinal microbiota with intestinal permeability and inflammation. (<b>A</b>–<b>C</b>) Linear correlation analyses were carried out to explore the correlation between levels of inflammatory factors and the abundance of <span class="html-italic">Lachnospiraceae_AC2044_group</span>, <span class="html-italic">Prevotellaceae_UCG-001</span>, and <span class="html-italic">Terrisporobacter</span>, respectively. (<b>D</b>–<b>F</b>) Linear correlation analyses were performed to examine the association between intestinal leakiness and the abundance of <span class="html-italic">Lachnospiraceae_AC2044_group</span>, <span class="html-italic">Prevotellaceae_UCG-001</span>, and <span class="html-italic">Terrisporobacter</span>, respectively.</p>
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<p>Correlation analysis of intestinal microbiota with the growth performance of weaned piglets. (<b>A</b>–<b>C</b>) Sequential linear correlation analyses were conducted to assess the relationship between growth performance and the abundance of <span class="html-italic">Lachnospiraceae_AC2044_group</span>, <span class="html-italic">Prevotellaceae_UCG-001</span>, and <span class="html-italic">Terrisporobacter</span>, respectively. (<b>D</b>) Correlation analysis of intestinal microbiota and growth performance. *, <span class="html-italic">p</span> &lt; 0.05.</p>
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17 pages, 2768 KiB  
Article
Targeting S. aureus Extracellular Vesicles: A New Putative Strategy to Counteract Their Pathogenic Potential
by Giulio Petronio Petronio, Maria Di Naro, Noemi Venditti, Antonio Guarnieri, Marco Alfio Cutuli, Irene Magnifico, Alessandro Medoro, Emanuele Foderà, Daniela Passarella, Daria Nicolosi and Roberto Di Marco
Pharmaceutics 2024, 16(6), 789; https://doi.org/10.3390/pharmaceutics16060789 - 11 Jun 2024
Viewed by 1277
Abstract
Long-term inflammatory skin disease atopic dermatitis is characterized by dry skin, itching, and eczematous lesions. During inflammation skin barrier protein impairment promotes S. aureus colonisation in the inflamed skin, worsening AD patient’s clinical condition. Proteomic analysis revealed the presence of several immune evasion [...] Read more.
Long-term inflammatory skin disease atopic dermatitis is characterized by dry skin, itching, and eczematous lesions. During inflammation skin barrier protein impairment promotes S. aureus colonisation in the inflamed skin, worsening AD patient’s clinical condition. Proteomic analysis revealed the presence of several immune evasion proteins and virulence factors in S. aureus extracellular vesicles (EVs), suggesting a possible role for these proteins in the pathophysiology of atopic dermatitis. The objective of this study is to assess the efficacy of a wall fragment obtained from a patented strain of C. acnes DSM28251 (c40) and its combination with a mucopolysaccharide carrier (HAc40) in counteract the pathogenic potential of EVs produced by S. aureus ATCC 14458. Results obtained from in vitro studies on HaCaT keratinocyte cells showed that HAc40 and c40 treatment significantly altered the size and pathogenicity of S. aureus EVs. Specifically, EVs grew larger, potentially reducing their ability to interact with the target cells and decreasing cytotoxicity. Additionally, the overexpression of the tight junctions mRNA zona occludens 1 (ZO1) and claudin 1 (CLDN1) following EVs exposure was decreased by HAc40 and c40 treatment, indicating a protective effect on the epidermal barrier’s function. These findings demonstrate how Hac40 and c40 may mitigate the harmful effects of S. aureus EVs. Further investigation is needed to elucidate the exact mechanisms underlying this interaction and explore the potential clinical utility of c40 and its mucopolysaccharide carrier conjugate HAc40 in managing atopic dermatitis. Full article
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Figure 1

Figure 1
<p>(<b>A</b>) SDS-PAGE with Coomassie blue staining (kDa), ladder: Opti-Protein XL Marker, BSA: bovine serum albumin, BEV: PL extracted <span class="html-italic">S. aureus</span> BEV, BEV-HAc40: BEV treated with HAc40, BEV-c40: BEV treated with c40, α toxin: <span class="html-italic">S. aureus</span> α-hemolysin, C−: growth media devoid of bacterial growth, PBS: phosphate buffer loaded and BEV-HA: BEV treated with HA only; (<b>B</b>) spectrophotometric analysis of BEV bands; blue columns on the graph from 1 to 8 correspond to the maximum intensity peaks detected (in pixel) for the ladder (subfigure <b>A</b>), 1: 135 kDa, 2: 100 kDa, 3: 75 kDa, 4: 63 kDA, 5: 48 kDa, 6: 35 kDa, 7: 25 kDa, 8: 20 kDa. (<b>C</b>) Standard calibration curve.</p>
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<p>(<b>A</b>) Analysis using Zetasizer Nano ZS of BEVs isolated from <span class="html-italic">S. aureus</span>; (<b>B</b>) Analysis using Zetasizer Nano ZS of HA, HAc40, c40; (<b>C</b>) Analysis using Zetasizer Nano ZS of BEV-HA, BEV-HAc40, BEV-c40.</p>
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<p>Assessment of adherent cells by microscopic evaluation and image analysis. Mean area (pixel<sup>2</sup>) with standard deviation (*** <span class="html-italic">p</span>-value &lt; 0.001).</p>
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<p>Microscopic images of in vitro treated HaCaT cells stained with eosin (40× magnification). Column A cells not exposed to BEV: PBS Control, HA treatment, HAc40 and c40 treatment. Column B cells exposed to BEV: BEV, BEV exposed to HA (BEV-HA), BEV exposed to HAc40 (BEV-HAc40), BEV exposed to c40 (BEV-c40).</p>
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<p>MTT assay results show significant differences in cell viability among treatment groups. BEV-treated cells exhibit reduced viability compared to untreated cells (q = 9.873, <span class="html-italic">p</span> ≤ 0.001, ***), as well as HA-treated cells (q = 7.988, <span class="html-italic">p</span> ≤ 0.001 ***). Significant differences are observed in comparisons with BEV-HA, BEV-Hac40 and BEV-c40 treated cells (<span class="html-italic">p</span> ≤ 0.01 ** or ≤0.001 ***). No significant differences in cell viability are noted within pairs of treatments such as HA vs. BEV-HA, HAc40 vs. BEV-Hac40, and c40 vs. BEV-c40 (<span class="html-italic">p</span> &gt; 0.05, ns).</p>
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<p>RT-qPCR Tight Junction expression, normalized by GAPDH as housekeeping gene and analyzed with delta delta Ct method. (<b>A</b>) ZO1 gene fold expression for HAc40 HA and c40 treated HaCaT cell line. (<b>B</b>) ZO1 gene fold expression for BEV BEV-HAc40 BEV-HA and BEV-c40 treated HaCaT cell line. (<b>C</b>) CLDN1 gene fold expression for HAc40 HA and c40 treated HaCaT cell line. (<b>D</b>) CLDN1 gene fold expression for BEV BEV-HAc40 BEV-HA and BEV-c40 treated HaCaT cell line. Statistical analysis significance results (<span class="html-italic">p</span>-value) obtained by oneway ANOVA comparing gene expression levels between BEV alone and BEV-HAc40 BEV-HA and BEV-c40 treated for both CLDN1 and ZO1 was included as follows: *** <span class="html-italic">p</span> ≤ 0.001, **** <span class="html-italic">p</span> ≤ 0.0001.</p>
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13 pages, 2397 KiB  
Communication
Protective Effect of Red Light-Emitting Diode against UV-B Radiation-Induced Skin Damage in SKH:HR-2 Hairless Mice
by Eun-Chae Cho, Surin Ahn, Kyung-Ok Shin, Joon Byeong Lee, Hyo-Jeong Hwang and Yean-Jung Choi
Curr. Issues Mol. Biol. 2024, 46(6), 5655-5667; https://doi.org/10.3390/cimb46060338 - 6 Jun 2024
Viewed by 742
Abstract
In this in vivo study on hairless mice, we examined the effects of light-emitting diode (LED) treatment applied prior to ultraviolet B (UVB) irradiation. We found that pre-treating with LED improved skin morphological and histopathological conditions compared to those only exposed to UVB [...] Read more.
In this in vivo study on hairless mice, we examined the effects of light-emitting diode (LED) treatment applied prior to ultraviolet B (UVB) irradiation. We found that pre-treating with LED improved skin morphological and histopathological conditions compared to those only exposed to UVB irradiation. In our study, histological evaluation of collagen and elastic fibers after LED treatment prior to UVB irradiation showed that this pretreatment significantly enhanced the quality of fibers, which were otherwise poor in density and irregularly arranged due to UV exposure alone. This suggests that LED treatment promotes collagen and elastin production, leading to improved skin properties. Additionally, we observed an increase in Claudin-1 expression and a reduction in nuclear factor-erythroid 2-related factor 2 (Nrf-2) and heme-oxygenase 1 (HO-1) expression within the LED-treated skin tissues, suggesting that LED therapy may modulate key skin barrier proteins and oxidative stress markers. These results demonstrate that pretreatment with LED light can enhance the skin’s resistance to UVB-induced damage by modulating gene regulation associated with skin protection. Further investigations are needed to explore the broader biological effects of LED therapy on other tissues such as blood vessels. This study underscores the potential of LED therapy as a non-invasive approach to enhance skin repair and counteract the effects of photoaging caused by UV exposure. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Figure 1
<p>(<b>A</b>) Animal experiment design and (<b>B</b>) body weight changes in hairless mice treated with test materials. (<b>C</b>) The mice were exposed to light-emitting diode (LED) light (630 nm) for 30 min twice daily for five days. All mice, except those in the control group, were irradiated with 200 mJ/cm<sup>2</sup> UVB once a day for three days before sacrifice. At the end of the experimental period, dorsal skin tissues were immediately collected for further analysis. Each value represents the mean ± SD (n = 5).</p>
Full article ">Figure 1 Cont.
<p>(<b>A</b>) Animal experiment design and (<b>B</b>) body weight changes in hairless mice treated with test materials. (<b>C</b>) The mice were exposed to light-emitting diode (LED) light (630 nm) for 30 min twice daily for five days. All mice, except those in the control group, were irradiated with 200 mJ/cm<sup>2</sup> UVB once a day for three days before sacrifice. At the end of the experimental period, dorsal skin tissues were immediately collected for further analysis. Each value represents the mean ± SD (n = 5).</p>
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<p>Effect of light emitting diode (LED) treatment on histological changes in ultraviolet (UVB)-irradiated skin of Skh:HR-1 hairless mice. Hematoxylin and eosin (H&amp;E) staining of dorsal skin in the control, UV, LED, and UV + LED groups. The mice were exposed to LED light (630 nm) for 30 min twice daily for five days. All mice, except those in the control group, were irradiated with 200 mJ/cm<sup>2</sup> UVB once a day for three days before sacrifice. Dorsal skin tissues were excised, and paraffin sections were prepared. Changes in skin tissue were measured using a corresponding staining kit. H&amp;E staining revealed that the group treated with LED + UVB exhibited reduced epidermal thickening and fewer inflammatory cells compared to the UVB-only group, indicating that both epidermal and dermal damage were alleviated (red arrows). Scale bars, 100 μm.</p>
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<p>Effect of light emitting diode (LED) treatment on collagen fibers and elastin fibers in ultraviolet (UV)B-irradiated skin of Skh:HR-1 hairless mice. Masson’s trichrome staining of the dorsal skin reveals collagen deposition (blue). Elastic staining of the dorsal skin revealed elastin fibers (blue-black). The mice were exposed to LED light (630 nm) for 30 min twice daily for five days. All mice, except those in the control group, were irradiated with 200 mJ/cm<sup>2</sup> UVB once a day for three days before sacrifice. Dorsal skin tissues were excised, and paraffin sections were prepared. Changes in skin tissue were measured using a relevant staining kit. Masson’s trichrome staining demonstrated that in the LED + UVB treated group, regeneration of damaged dermal tissue was evident, with marked formation of collagen fibers and improved dermal tissue morphology (red arrows). Elastic staining indicated that the elastin content in the LED + UVB treated group was comparable to that of the control group, as highlighted by red arrows. Scale bars, 100 μm.</p>
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<p>Effect of light-emitting diode (LED) treatment on Claudin-1 and Nrf2/HO-1 signaling pathway in ultraviolet (UV)B-irradiated skin of Skh:HR-1 hairless mice. The mice were exposed to LED light (630 nm) for 30 min twice daily for five days. All mice, except those in the control group, were irradiated with 200 mJ/cm<sup>2</sup> UVB once a day for three days before sacrifice. Dorsal skin tissues were excised and homogenized. Total lysates of dorsal skin tissues were prepared and analyzed by Western blot using the indicated antibodies. The Western blot images are representative of three independent experiments. Quantitative analysis of the Western blot results. Protein expression was normalized to β that of-actin and presented relative to the control group. Each value represents the mean ± SD (n = 5). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 significantly different from the control group.</p>
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18 pages, 2585 KiB  
Article
Unveiling the Novel Benefits of Co-Administering Butyrate and Active Vitamin D3 in Mice Subjected to Chemotherapy-Induced Gut-Derived Pseudomonas aeruginosa Sepsis
by Fu-Chen Huang and Shun-Chen Huang
Biomedicines 2024, 12(5), 1026; https://doi.org/10.3390/biomedicines12051026 - 7 May 2024
Viewed by 1154
Abstract
Cancer patients face increased susceptibility to invasive infections, primarily due to ulcerative lesions on mucosal surfaces and immune suppression resulting from chemotherapy. Pseudomonas aeruginosa (P. aeruginosa) bacteremia is notorious for its rapid progression into fatal sepsis, posing a significant threat to [...] Read more.
Cancer patients face increased susceptibility to invasive infections, primarily due to ulcerative lesions on mucosal surfaces and immune suppression resulting from chemotherapy. Pseudomonas aeruginosa (P. aeruginosa) bacteremia is notorious for its rapid progression into fatal sepsis, posing a significant threat to cancer patients, particularly those experiencing chemotherapy-induced neutropenia. This bacterial infection contributes significantly to morbidity and mortality rates among such individuals. Our latest report showed the mutually beneficial effects of postbiotic butyrate on 1,25-dihydroxyvitamin D3 (1,25D3)-controlled innate immunity during Salmonella colitis. Hence, we investigated the impact of butyrate and 1,25D3 on chemotherapy-induced gut-derived P. aeruginosa sepsis in mice. The chemotherapy-induced gut-derived P. aeruginosa sepsis model was established through oral administration of 1 × 107 CFU of the P. aeruginosa wild-type strain PAO1 in C57BL/6 mice undergoing chemotherapy. Throughout the infection process, mice were orally administered butyrate and/or 1,25D3. Our observations revealed that the combined action of butyrate and 1,25D3 led to a reduction in the severity of colitis and the invasion of P. aeruginosa into the liver and spleen of the mice. This reduction was attributed to an enhancement in the expression of defensive cytokines and antimicrobial peptides within the cecum, coupled with decreased levels of zonulin and claudin-2 proteins in the mucosal lining. These effects were notably more pronounced when compared to treatments administered individually. This study unveils a promising alternative therapy that involves combining postbiotics and 1,25D3 for treating chemotherapy-induced gut-derived P. aeruginosa sepsis. Full article
(This article belongs to the Special Issue Aryl Hydrocarbon Receptor in Human Diseases)
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Graphical abstract

Graphical abstract
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<p>The combined administration of butyrate and 1,25D3 effectively mitigates colitis in mice that have undergone chemotherapy and subsequently developed gut-derived <span class="html-italic">P. aeruginosa</span> sepsis. Female C57BL/6 mice aged 6–8 weeks sourced from Charles River, USA, were bred and maintained under specific-pathogen-free conditions at the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. These mice were infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU (suspended in 100 μL PBS). An open control group was administered 100 μL of sterile 1xPBS buffer. Prior to and following infection, the mice were orally administered either a vehicle control (5% dimethyl sulfoxide), 1,25D3 at a dosage of 0.2 μg/25g mice/day (VD group), butyrate (BU group), or a combination of 1,25D3 and butyrate (VD + BU group) on a daily basis, as described in the Materials and Methods section. Diarrhea situation scores (<b>a</b>) of mice were recorded daily. The cecum specimens were surgically removed, fixed in formaldehyde, and subsequently processed for staining with hematoxylin and eosin (H&amp;E). (<b>b</b>) Representative histological images of the cecum from various experimental groups were captured at magnifications of 2×, 20× and 50×. (<b>c</b>) Pathological scoring for colitis was conducted based on the assessment of cecum sections obtained from mice in different experimental groups. The data shown are means ± SEM (<span class="html-italic">n</span> = 6 mice/group). *, <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>The immunoregulatory effects of combined administration of 1,25D3 and butyrate on cecal cytokines and antimicrobial peptides in mice that had undergone chemotherapy and subsequently developed gut-derived <span class="html-italic">P. aeruginosa</span> sepsis. Female C57BL/6 mice, aged 6 to 8 weeks and obtained from Charles River, USA, were bred and housed under specific-pathogen-free conditions at the animal facility within the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. Mice were either infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU suspended in 100 μL PBS or given 100 μL of sterile 1xPBS buffer as an open control. Before and after infection, mice received daily oral gavage of vehicle control (5% dimethyl sulfoxide), vitamin D3 at a dose of 0.2 μg/25 g mice/day (VD group), or butyrate (BU group), or a combination of both 1,25D3 and butyrate (VD + BU group), as described in <a href="#sec2-biomedicines-12-01026" class="html-sec">Section 2</a>. Total RNA was isolated from the cecal tissues of the mice. Subsequently, the gene expressions of various markers including IL-6 (<b>a</b>), IL-1β (<b>b</b>), TNF-α (<b>c</b>), IL-17A (<b>d</b>), IL-22 (<b>e</b>), and CRAMP (a homolog of human cathelicidin LL-37) (<b>f</b>), as well as ATG16L1 (<b>g</b>) and AhR (<b>h</b>) mRNA, were assessed using real-time quantitative PCR. The values obtained were determined as fold increases relative to the levels observed in mice solely infected with <span class="html-italic">Salmonella</span> for comparison purposes. The presented data are displayed as means ± the standard error of the mean (SEM) with a sample size of 7 mice per group (<span class="html-italic">n</span> = 6 mice/group). An asterisk (*) denotes significant differences observed among the groups, as determined by one-way analysis of variance (ANOVA). *, <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 2 Cont.
<p>The immunoregulatory effects of combined administration of 1,25D3 and butyrate on cecal cytokines and antimicrobial peptides in mice that had undergone chemotherapy and subsequently developed gut-derived <span class="html-italic">P. aeruginosa</span> sepsis. Female C57BL/6 mice, aged 6 to 8 weeks and obtained from Charles River, USA, were bred and housed under specific-pathogen-free conditions at the animal facility within the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. Mice were either infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU suspended in 100 μL PBS or given 100 μL of sterile 1xPBS buffer as an open control. Before and after infection, mice received daily oral gavage of vehicle control (5% dimethyl sulfoxide), vitamin D3 at a dose of 0.2 μg/25 g mice/day (VD group), or butyrate (BU group), or a combination of both 1,25D3 and butyrate (VD + BU group), as described in <a href="#sec2-biomedicines-12-01026" class="html-sec">Section 2</a>. Total RNA was isolated from the cecal tissues of the mice. Subsequently, the gene expressions of various markers including IL-6 (<b>a</b>), IL-1β (<b>b</b>), TNF-α (<b>c</b>), IL-17A (<b>d</b>), IL-22 (<b>e</b>), and CRAMP (a homolog of human cathelicidin LL-37) (<b>f</b>), as well as ATG16L1 (<b>g</b>) and AhR (<b>h</b>) mRNA, were assessed using real-time quantitative PCR. The values obtained were determined as fold increases relative to the levels observed in mice solely infected with <span class="html-italic">Salmonella</span> for comparison purposes. The presented data are displayed as means ± the standard error of the mean (SEM) with a sample size of 7 mice per group (<span class="html-italic">n</span> = 6 mice/group). An asterisk (*) denotes significant differences observed among the groups, as determined by one-way analysis of variance (ANOVA). *, <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Combination of postbiotic butyrate and active 1,25D3 attenuates systemic translocation of gut-derived <span class="html-italic">P. aeruginosa</span> in mice. Female C57BL/6 mice, aged 6 to 8 weeks and obtained from Charles River, USA, were bred and housed under specific-pathogen-free conditions at the animal facility within the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. Mice were either infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU suspended in 100 μL PBS or given 100 μL of sterile 1xPBS buffer as an open control. Before and after infection, mice received daily oral gavage of vehicle control (5% dimethyl sulfoxide), vitamin D3 at a dose of 0.2 μg/25 g mice/day (VD group), or butyrate (BU group), or a combination of both 1,25D3 and butyrate (VD + BU group). The quantities of bacteria retrieved from liver (<b>a</b>) and spleen (<b>b</b>) homogenates of infected and treated mice were measured. The presented data are displayed as means ± SEM (<span class="html-italic">n</span> = 6 mice/group). An asterisk (*) denotes significant differences observed among the groups, as determined by one-way analysis of variance (ANOVA). *, <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 4
<p>The combined use of butyrate and active 1,25D3 reduced the expression of zonulin and claudin-2 proteins in the cecal tissue of mice that had undergone chemotherapy and subsequently developed gut-derived <span class="html-italic">P. aeruginosa</span> sepsis. Female C57BL/6 mice, aged 6 to 8 weeks and obtained from Charles River, USA, were bred and housed under specific-pathogen-free conditions at the animal facility within the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. Mice were either infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU suspended in 100 μL PBS or given 100 μL of sterile 1xPBS buffer as an open control. Prior to and after infection, the mice were orally administered either a vehicle control (5% dimethyl sulfoxide), treated with 0.2 μg of 1,25D3 per 25 g of mice per day (VD group), given butyrate (BU group), or received both 1,25D3 and butyrate (VD + BU group) on a daily basis, as described in the Material and Methods section. The detection of zonulin (<b>a</b>), claudin-2 (<b>b</b>) and AhR (<b>c</b>) expression in these groups was performed through immunohistochemistry (IHC) staining (original magnification, 400×; scale bar, 25 µm; <span class="html-italic">n</span> = 3). Zonulin (<b>d</b>), claudin-2 (<b>e</b>) and AhR (<b>f</b>) protein expressions in the IHC images were analyzed using ImageJ software (Java 1.8.0_345). ** <span class="html-italic">p</span>&lt; 0.01, *** <span class="html-italic">p</span>&lt; 0.001.</p>
Full article ">Figure 4 Cont.
<p>The combined use of butyrate and active 1,25D3 reduced the expression of zonulin and claudin-2 proteins in the cecal tissue of mice that had undergone chemotherapy and subsequently developed gut-derived <span class="html-italic">P. aeruginosa</span> sepsis. Female C57BL/6 mice, aged 6 to 8 weeks and obtained from Charles River, USA, were bred and housed under specific-pathogen-free conditions at the animal facility within the Center for Cellular and Biomolecular Research in Kaohsiung, Taiwan. Mice were either infected with <span class="html-italic">P. aeruginosa</span> PAO1-LAC at a concentration of 10<sup>7</sup> CFU suspended in 100 μL PBS or given 100 μL of sterile 1xPBS buffer as an open control. Prior to and after infection, the mice were orally administered either a vehicle control (5% dimethyl sulfoxide), treated with 0.2 μg of 1,25D3 per 25 g of mice per day (VD group), given butyrate (BU group), or received both 1,25D3 and butyrate (VD + BU group) on a daily basis, as described in the Material and Methods section. The detection of zonulin (<b>a</b>), claudin-2 (<b>b</b>) and AhR (<b>c</b>) expression in these groups was performed through immunohistochemistry (IHC) staining (original magnification, 400×; scale bar, 25 µm; <span class="html-italic">n</span> = 3). Zonulin (<b>d</b>), claudin-2 (<b>e</b>) and AhR (<b>f</b>) protein expressions in the IHC images were analyzed using ImageJ software (Java 1.8.0_345). ** <span class="html-italic">p</span>&lt; 0.01, *** <span class="html-italic">p</span>&lt; 0.001.</p>
Full article ">
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