Effects of Phycocyanin On Pulmonary and Gut Microbiota in A Radiation-Induced Pulmonary Fibrosis Model
Effects of Phycocyanin On Pulmonary and Gut Microbiota in A Radiation-Induced Pulmonary Fibrosis Model
Effects of Phycocyanin On Pulmonary and Gut Microbiota in A Radiation-Induced Pulmonary Fibrosis Model
Original article
A R T I C L E I N F O A B S T R A C T
Keywords: Objective: Radiation pneumonia and fibrosis are major clinical complications of radiotherapy for thoracic tumor
Thorax irradiation patients, and may significantly reduce survival and quality of life. At present, no safe and effective radiation
Pulmonary fibrosis protection measures have been approved for clinical use. Phycocyanin, a protein responsible for photosynthesis
Microbiota
from Spirulina, has been shown to have a variety of biological activities and to be beneficial for a variety of
Phycocyanin
diseases, including pulmonary fibrosis. However, the preventive and protective effects of phycocyanin on
radiation-induced pulmonary fibrosis have not been studied.
Design: X-ray single dose irradiation was used on the chest of mice to prepare a mouse model of pulmonary
fibrosis, from which the effect of phycocyanin on pulmonary histopathologic change, pulmonary fibrosis, the
microbiota in lung and gut, LPS, TNF-α, and IL-6 at different time after irradiation were evaluated.
Results: Phycocyanin alleviated the radiation-induced lung injury and reduced the level of inflammatory factors.
Thorax irradiation led to the disorder in microbiota of the lung and gut. The variation trend of the diversity of the
two tissues was opposite, but that of the microbiota composition was similar. The phycocyanin intervention
regulated the composition of the lung and gut microbiota, transformed them into normal state, and reduced the
level of LPS, which significantly reduced the abundance of inflammation-related bacteria, and increased the
abundance of probiotics that produce short-chain fatty acids.
Conclusion: Phycocyanin could regulate the radiation-induced disorder in lung and gut microbiota of mice, and
reduce the radiation-induced lung inflammation and fibrosis.
1. Introduction large amount of oxidative damage and the release of fibrotic cytokines.
These processes eventually lead to excessive deposition of extracellular
Radiation therapy for esophageal cancer, lung cancer, or other matrix ECM and the formation of lung fibrosis. In recent years, with the
thoracic spine cancer often causes acute radiation pneumonia or chronic in-depth research on lung diseases, more and more studies have proved
radiation pneumonia. The radiation-induced lung injury (RILI) can even that there is an important crosstalk between gut microbes and the lungs,
further lead to lung fibrosis. The incidence of RILI in patients receiving which plays an important role in maintaining the health of the host.
thoracic radiotherapy and lung cancer patients receiving high-dose Studies have shown that gut microbes can affect lung immunity, and
radiotherapy can reach 30 %–40 %, respectively [1,2] Patients with many lung diseases are often accompanied by changes in gut microbes
RILI often present with cough, fever and chest pain. The consequences of [3–5]. However, reports on the changes of lung microbiota in the lung
RILI can even lead to respiratory failure and even death of patients. As it and gut after RILI. In addition, facing the global pandemic of Covid-19, it
involves a variety of cells, the mechanism of RILI causing pulmonary is urgently needed to find active substances that can slow down the
fibrosis is more complicated, including complex intermolecular in pneumonia and enteritis. Covid-19 can causes serious inflammation of
teractions. Radiation can cause apoptosis of epithelial cells, as well as a human lungs, causing difficulty in breathing and pulmonary fibrosis,
* Corresponding authors.
E-mail addresses: binliu0736@126.com (B. Liu), sqin@yic.an.cn (S. Qin).
1
Wenjun Li and Lina Lu contributed equally to this study.
https://doi.org/10.1016/j.biopha.2020.110826
Received 25 August 2020; Received in revised form 18 September 2020; Accepted 25 September 2020
Available online 14 October 2020
0753-3322/© 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
W. Li et al. Biomedicine & Pharmacotherapy 132 (2020) 110826
and eventually lung failure. ACE2 in lungs and gastrointestinal tract may 2.5. Histopathological analysis
be the transmission route of Covid-19.
Phycocyanin (PC) is a light-harvesting pigment protein isolated from The tissue sections were stained with HE and Masson and three
cyanobacteria and can participate in the photosynthesis of algae. In the C57BL/6 mice were used in each group. A Pannoramic MIDI scanner was
past 20 years, the role of PC in promoting health and treating diseases used to take pictures of the stained sections. Lung injury scores were
has been widely studied and reported. Previous studies have shown that assessed according to previous methods. In brief, a score of 0, 1, 2, 3 and
PC has multiple activities such as antioxidant and anti-inflammatory [6, 4 represents no damage, mild damage, moderate damage, severe dam
7]. It has been reported that PC can alleviate paraquat-induced lung age and very severe histologic changes, respectively [55]. In addition,
injury in rats [8]. Research found that in the bleomycin-induced mouse fibrosis was quantified using the modified Ashcroft scoring [13].
lung fibrosis model, PC could regulate TLR2-MyD88-NF-κB signaling
pathways alleviate pulmonary fibrosis [9], and in addition, could 2.6. Lung and intestinal microbiota analysis
significantly reduce bacteria that associated with inflammation, thereby
alleviating pulmonary fibrosis [10–12]. Wash the lungs of the mice with 0.5 mL of phosphate buffer. The
Thus, we hypothesized that, before or after RILI, PC intervention lavage fluid is then collected and used for genetic testing [12,14]. The
could change the colonizing pattern of the gut microbiota and affect the extraction of total DNA and the evaluation of bacterial diversity used
composition of lung microbiota, thereby alleviating the lung injury. PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, USA, Cali
Therefore, we induced pulmonary fibrosis in mice by X-ray single-dose fornia) and Illumina Hiseq platform.
irradiation on thorax and studied the effect of PC on radiation induced
pulmonary fibrosis by evaluating the changes in lung histopathology, 2.7. Enzyme-linked immunosorbent assay (ELISA)
inflammatory factor levels, and microbiota in the lung and gut.
ELISA kits (Shanghai Enzyme Link Biotechnology Co., Ltd., China)
2. Materials and methods were used to detect the contents of interleukin (IL)-6, lipopolysaccharide
(LPS) and tumor necrosis factor alpha (TNF-α) in the samples.
2.1. Ethics statement
2.8. Statistical analysis
Our experimental protocol was formulated in accordance with the
"Laboratory Animal Care Guidelines". The animal experiment ethics Flora analysis used Wilcox rank sum test to determine the differences
committee of Lanzhou University approved the experimental protocol. among groups. We use SPSS 19.0 software to analyze the results. If P <
0.05, the result was considered statistically significant.
2.2. Materials and animals
3. Results
PC was extracted from Spirulina. King Dnarmse Spirulina Company
(Fuqing, China) provided a medical grade PC for this experiment. Male 3.1. PC intervention reduced lung tissue damage and fibrosis caused by
C57BL/6 mice were purchased from Lanzhou Medical College in China. thoracic irradiation
All animals were raised at 22 ± 2 ◦ C and were given standard food and
water. During the experiment, we found that after irradiation at the chest
cavity, the fur around the mouse’s chest was shed and turned pale with
time. One month after irradiation, the fur was basically the same as that
2.3. Experimental design of the normal control group. However, after 3 and 5 months after irra
diation, the fur was obviously whitened. From the appearance of fur, the
4 to 6-week-old and male C57BL/6 mice were randomly divided into whitening phenomenon of the PC intervention group was significantly
4 groups in body weight, 15 mice in each group, i.e., the normal control less than that of the irradiation group (Fig. 1).
group (C), radiotherapy only (RT), PC pre-administration plus radio HE staining and Masson staining were used to analyze lung injury
therapy group (PC + RT group), and radiotherapy plus PC administra and fibrosis, and the results were compared 1 month after thoracic
tion group (RT + PC group). RT group received chest RT alone. PC + RT irradiation with that of group C, showing that the mouse lung tissue had
group received RT after 30 days with PC (50 mg/kg/d), no further obvious lymphocyte and neutrophil infiltration (green arrow in Fig. 2),
administration after irradiation, only the same volume of double alveolar tissue edema, and a small amount of blue collagen fibers were
distilled water was given. RT + PC group was treated with PC (50 mg/ deposited. Three to 5 months after thoracic irradiation, with the pro
kg/d) after RT. Animals in groups C and RT were given equal volume of longation of time, the lung tissue damage in mice increased, inflam
distilled water. At the time points of 1, 3, and 5 months after RT, five matory cell infiltration increased, and eosinophilic mucus secretion
animals in each group were given general anesthesia, blood samples, appeared in the bronchial cavity (yellow arrow in Fig. 2). There is
intestine, and lung tissues were collected. The RT conditions were as epithelial cell shedding in the tracheal cavity (blue arrow in Fig. 2),
follows. All mice (C group, RT group, PC + RT group and RT + PC group) edema around the blood vessels, and loose connective tissue (red arrow
were under general anesthesia. Part of the mice (RT group, PC + RT Fig. 2). The alveolar wall thickens, collagen fibers proliferate, and fi
group and RT + PC group) were irradiated to the entire chest by the X- broblasts increase (black arrows Fig. 2). Both prophylactic and thera
ray therapy instrument (20 Gy, 2 Gy/min). peutic administration of PC reduced the infiltration of inflammatory
cells and reduced the deposition of collagen fibers. This shows that PC
2.4. Sample collection intervention can alleviate lung injury caused by chest irradiation.
One month after the mice were irradiated, the feces from the cecum 3.2. PC intervention reduces the increase of inflammatory factors and LPS
of the animals were collected and stored in liquid nitrogen at − 80 ◦ C for induced by thoracic irradiation
detection of bacterial 16S ribosomal RNA (rRNA) V3 and V4. At 1, 3 and
5 months after the mice were irradiated, 5 mice from each experimental Using the ELISA method, we detected the content of TNF-α, LPS and
group were selected and sacrificed. Animal serum, intestinal tissue and IL-6 in lung tissue, intestine, and serum. The results show that, the
lung tissue were collected and stored in a refrigerator at − 80 ◦ C. The content of TNF-α and IL-6 in lung tissue or serum increased significantly
lung tissue was stained with 4 % hematoxylin-eosin (HE) and MASSON. at the three time points (1, 3, and 5 months) after thoracic irradiation
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compared with the normal control group, while the PC medication matter how the lung flora or intestinal flora varied, the difference in
group the contents of TNF-α and IL-6 were decreased. Although flora between groups C and RT was remarkable, and the bacterial
compared with lung tissue and serum, the contents of IL-6 and TNF-α in structure after PC intervention was similar to that of group C (Fig. 5). In
intestinal tissue did not change significantly, but at the 5-month time terms of the regulation of lung flora, the flora was more similar to that of
point, the content of IL-6 and TNF-αin intestinal tissue was significantly group C. For the regulation of intestinal flora, the flora structure of PC +
higher than that of normal control group (Fig. 3 a–b). Thoracic irradi RT group was closer to that of group C (Figs. 4–6).
ation significantly increased the content of LPS in lung tissue, serum, After analyzing the flora composition of each group, we found that at
and feces, and PC intervention also reduced its content to varying de portal level, the main bacteria of normal lung flora are Fimicutes, Bac
grees (Fig. 3 c). teroidetes, and Proteobacteria. Meanwhile, the intestinal flora included
mainly Bacteroidetes and Fimicutes. Before irradiation, the content of
3.3. PC intervention regulates lung and intestinal flora Bacteroidetes in the lung tissue was only one-fourth of that of Fimicutes.
After irradiation, the proportion of Bacteroidetes doubled that of the
We measured the lung and intestinal flora using 16srRNA technol original, while the content of Fimuteutes decreased by about 15 %.
ogy. The results of α diversity analysis show that the diversity index of Although the change of intestinal flora was not as great as that of
lung flora increased after irradiation compared to the normal control abdominal flora, the variation trends of Bacteroidetes and Fimutes were
group. In addition, although the diversity of intestinal flora decreased the same to that of the lung flora for showing also the increase of Bac
but not as significantly as the lung flora did, the trend was opposite. Both teroidetes and the decrease of Fimuteutes. The PC intervention stabi
pre-administration and therapeutic administration have reduced the lized the normal flora to a certain extent. At the same time, in the PC-
change of the flora diversity to a certain extent (Fig. 4), which showed administered group, the content of Actinobacteria increased in both
that PC had a certain ability to regulate the disturbance of flora caused lung and intestinal flora (Fig. 6).
by thoracic irradiation. Additional analysis of the flora composition at genus level showed
After principal component analysis of each group, we found that no that the abundances of Alisipes, Lachnoclostridium and Bacteroides in the
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lung flora increased after irradiation, while those of Lactococcus, Dubo correlated with the above indicators; while Bifidobacterium, Lactococcus,
siella, Lactobacillus, Turicibacter, Candidatus-Saccharimonas, Romboutsia, and Lactobacillus were negatively correlated with the above indicators
and Bifidobacterium decreased. In the intestinal flora after irradiation, (Fig. 8b).
the contents of Alisipes, Mucispirillum, Helicobacter, Turibacter, Para
bacteroides, Lachnoclostridium, and Intestinimonas increased, while those 4. Discussion
of Alloprevotella, Muribaculum, Anaerotruncus, Enterococcus, Bacteroides,
Ruminiclostridium, Lactococcus, and Lactobacillus decreased. After irra In this paper, we studied the preventive and protective effects of PC
diation, five genera of Alisipes, Lactococcus, Lactobacillus, Lachnoclostri on radiation-induced lung injury, and analyzed its regulatory effects on
dium, and Bifidobacterium showed the same trend in the lungs and radiation-induced disturbances of lung and intestinal flora. The results
intestines. However, PC administration alleviated the changes of genera show that the whitening phenomenon of the PC intervention group was
such as Alisipes, Lachnoclostridium, Lactococcus, Lactobacillus, Mucispir significantly less than that of the irradiation group and both PC pre-
illum, Helicobacter, Turibacter, Parabacteroides, Intestinimonas, Bifido administration and therapeutic administration reduced the levels of
bacterium etc. Although the Faecalibacterium in the lungs and intestines inflammatory factors and LPS in lung tissue, serum, and intestinal tract.
did not change significantly after irradiation, PC intervention increased In addition, chest irradiation can lead to disorders of the lung and in
the relative abundance of Faecalibacterium in the lungs and intestines testinal flora, while PC intervention significantly alleviated the disorder
(Fig. 7). of the flora. This result is very similar to our previous report on the ef
The Spearman method was used to analyze the correlation between fects of phycocyanin on bleomycin-induced pulmonary fibrosis and the
the levels of factors (TNF-α, LPS and IL-6) in the sample and the abun intestinal microbiota in C57BL/6 mice [10].
dance of different bacterial species. The results show that Lachnoclos Radiation-induced pulmonary fibrosis is a common and serious side
tridium was positively correlated with the above indicators in the lung effect of radiotherapy, which is usually inevitable and seriously affects
flora; Lactococcus, Dubosiella, Lactobacillus, Turicibacter, Candidatus- the quality of life and survival rate of patients [15]. According to the
Saccharimonas, Romboutsia, and Bifidobacterium were negatively corre different irradiation time, radiation pulmonary fibrosis includes early
lated with the above indicators, and the correlation was statistically inflammation and late fibrosis [16]. In the experiment, we used HE and
significant (Fig. 8a). In the intestinal flora, Alistopes, Parabacteroides, Masson staining methods to analyze the degree of lung tissue damage
Parasutterella, Lachnoclostridium, and Intestinimonas were positively and fibrosis. After irradiating the chest cavity with a dose of 20 Gy, we
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Fig. 3. The effect of PC on the levels of LPS and inflammatory factors after thoracic irradiation.
a: tumor necrosis factor alpha (TNF-α) content in lung tissue, serum and intestinal tissue; b: interleukin 6 (IL-6) content in lung tissue, serum and intestinal tissue; c:
lipopolysaccharide (LPS)) Content in lung tissue, serum and feces.
(C: normal control group; RT: thoracic irradiation alone group; PC + RT: thoracic irradiation group one month after PC pre-administration; RT + PC: PC admin
istration group after thoracic irradiation; data is average ± standard. Poor (n = 5) means that the difference is statistically significant: * P < 0.05, ** P < 0.01,
compared with RT group).
Fig. 4. The effect of PC on alpha diversity of lung and intestinal flora after thoracic irradiation.
a: diversity of lung flora; b: diversity of intestinal flora.
(C: normal control group; RT: thoracic irradiation alone group; PC + RT: thoracic irradiation group one month after PC pre-administration; RT + PC: PC admin
istration group after thoracic irradiation; data is average ± standard Poor (n = 5); Observed_species: with the increase of sequencing depth, the number of OTUs
actually observed; PD-whole-tree: community pedigree diversity; Shannon index: the diversity of the flora; ACE index: the abundance of the flora.).
found that the structure of the alveoli showed varying degrees of dam deposition, which indicates that PC can relieve fibrosis to a certain
age and pulmonary fibrosis. Both pre-administration and therapeutic extent.
administration of PC reduced inflammation damage and collagen fiber A large number of early inflammatory factors can directly damage
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Fig. 6. The effect of PC on β diversity of lung flora and intestinal flora after chest irradiation (UPGMA cluster analysis).
a: β diversity of lung flora; b: β diversity of intestinal flora.
(C: normal control group; RT: thoracic irradiation group only; PC + RT: thoracic irradiation group one month after PC pre-administration; RT + PC: PC adminis
tration group after thoracic irradiation).
Fig. 7. The effect of PC on the abundance of lung and intestinal flora in each group after thoracic irradiation.
a: lung flora abundance-histogram; b: intestinal flora abundance-histogram; c: lung flora abundance-heat diagram; d: intestinal flora abundance-heat diagram.
(C: Normal control group; RT: thoracic irradiation group only; PC + RT: thoracic irradiation group one month after PC pre-administration; RT + PC: PC adminis
tration group after thoracic irradiation).
lung cells and further increase the permeability of pulmonary vascular can be reduced. Studies have shown that the progression of pulmonary
endothelial cells and alveolar epithelium, leading to pulmonary edema fibrosis is related to the ongoing inflammation, mainly related to
and thrombosis. These processes eventually cause pulmonary fibrosis. increased interleukins and tumor necrosis factor [17,18].
The release of pro-inflammatory cytokines is related to various signaling In this study, PC intervention significantly reduced the levels of TNF-
pathways, such as the activation of NF-κB. If the expression of inflam α, LPS and IL-6, in the lung tissue, intestine, and blood of a mouse model
matory cytokines can be reduced, the progression of pulmonary fibrosis with lung injury induced by chest irradiation. This indicates that the
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anti-pulmonary fibrosis effect of PC may be related to the reduction of PC intervention significantly reduced the relative abundance of these
inflammation and lipopolysaccharide levels. It has been shown that in strains and reduced the inflammatory damage of the bacteria to the
allergic inflammation, PC can significantly inhibit the release of TNF-α, tissue.
IL-6, IL-1β, and neutrophil infiltration. PC can reduce the increase of Secondly, a large number of studies show that many bacteria are
TNF-α, IL-1β, and IL-6 caused by LPS-induced microglia [19]. In addi involved in the production of short-chain fatty acids (SCFAs), such as
tion, PC can also have an important protective effect on inflammatory Faecalibacterium, Ruminiclostridium, Muribaculum, Anaerotruncus, Rom
bowel diseases and macrophage activation [20]. These results indicate boutsia, Turicibacter, and Clostridiales [34–38]. They are beneficial bac
that PC can reduce the symptoms of various diseases by inhibiting the teria, so they can ferment and produce SCFA in the intestine. SCFAs can
inflammatory response, including pulmonary fibrosis induced by chest reduce the level of intestinal inflammation, thereby protecting the in
irradiation. These results indicate that PC can inhibit related diseases by testinal barrier [39]. SCFAs can inhibit histone deacetylase (HDAC) and
inhibiting inflammation. The anti-pulmonary fibrosis effect of PC may G protein coupled receptor (GPCR), while GPCRs are involved in lung
also be related to the reduction of inflammatory factors and LPS levels. diseases [40]. Compared with the irradiated group, the therapeutic
In allergic inflammation, PC can significantly inhibit the release of in administration of PC significantly increased the relative abundance of
flammatory factors and the infiltration of neutrophils [19,20]. These Faecalibacterium, Lachnoclostridium, Ruminiclostridium, and Clostridiales,
results indicate that PC can reduce lung tissue damage caused by chest increased the synthesis of SCFAs, and enhanced its inhibitory effect on
irradiation by inhibiting inflammation. HDACs and GPCRs.
By analyzing the correlation between the flora and the physiological In addition, many microorganisms are probiotics, such as Lactoba
indicators of pulmonary fibrosis, we determined the importance of PC in cillus, Lactococcus, and Bifidobacteriu [41]. Probiotics can improve the
regulating the flora. First, a large number of studies have shown that a immunity of human mucosa by increasing immunoglobulins, and play
variety of flora is involved in inflammation [21–23]. For example, Hel an important role in human health [42]. Probiotics can regulate the
icobacter can induce various cytokines and chemokines, such as TNF-α balance of intestinal flora in the body by inhibiting the increase of
and IL-6 [24]. Helicobacter can produce ammonia and other harmful pathogenic bacteria, which can help treat diseases [43]. For example,
substances, causing damage to the intestinal wall. Some substances such Lactobacillus interacts with many pathogens by secreting salivary hor
as phospholipase and cytotoxin produced by Helicobacter can destroy the mone B (an antibiotic) [44]. Other studies have found that the
tight junctions of epithelial cells and cause apoptosis [25]. Helicobacter fermentation of lactic acid bacteria can not only enhance the physio
can cause inflammatory bowel disease in immuno-deficient mice [26], logical activity of intestinal peristalsis, but also improve immunity and
and is associated with a variety of enteritis diseases [27]. Alistairs were avoid hyperglycemia [45,46]. Bifidobacterium is also a beneficial bac
shown capable of inducing inflammation, and their abundance was teria in the intestinal tract, and it has immuno-modulation and hypo
positively correlated with blood levels of IL-6 and LPS [28,29]. Other glycemic effects [47]. In this study, the relative abundance of
bacteria related to inflammation are Parabacteroides and Bacteroides. Lactobacillus, Lactococcus, and Bifidobacterium increased significantly
Studies have shown that they were related to chronic inflammatory after PC intervention compared with the irradiated group alone,
bowel disease and are more abundant in chronic inflammatory bowel improving the immunity and balance ability of the flora.
disease [30,31]. Moreover, Intestinimonas could increase in abundance Dysfunctional flora promotes the production of lipopolysaccharide
in animal models of inflammation [14]. Mucispirillum showed high (LPS). By binding to Toll-like receptor 4 (TLR4), LPS induces chronic
abundance in the intestinal tract with oxidative stress and inflammation, low-grade inflammation [48]. LPS could not only directly damage tis
which was also a sign of serious indigestion [32,33]. After thoracic sues, but also cause systemic inflammatory cascades through blood
irradiation, Helicobacter, Alisipes, Parabacteroides, Bacteroides, Intestini circulation, leading to the release of inflammatory cytokines [49]. In this
monas, and Mucispirillum in the intestine proliferated in large quantities. study, LPS levels were increased in lung tissue, blood, and feces after
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