CN115645407A - Application of pyrroloquinoline quinone in product for improving obesity male sterility disorder - Google Patents

Abstract

The invention discloses application of pyrroloquinoline quinone in products for improving obese male infertility, and the application is application in products for preventing, improving and/or treating obese male infertility. The invention adopts high fat feed to construct an obese mouse model, researches the protective effect of PQQ on the reproductive function of obese mice, and experimental results show that the intervention of PQQ can obviously improve the testicular development condition of obese mice, reduce the scorching level of interstitial cells, increase the number of sperms, reduce the teratogenesis rate, express testosterone synthesis key enzyme and increase the testosterone content, reduce the body weight and serum lipid metabolism hormone level, reduce the fat content of abdominal cavity, improve cholesterol metabolism disorder and improve the lipid metabolism of liver.

Description

Application of pyrroloquinoline quinone in product for improving obesity male sterility disorder

Technical Field

The invention relates to the field of biological medicines, in particular to application of pyrroloquinoline quinone in a product for improving male sterility disorder caused by obesity.

Background

Obesity (obesitiy) is a common chronic endocrine-metabolic disorder caused by multiple factors. The world health organization ranks the whole body fat accumulation, lipid metabolism disorder and chronic inflammation as epidemic diseases in the 21 st century. Global survey and research forecast shows that obesity is a common metabolic abnormality disease of males with reproductive age, and by 2030, the number of overweight and obese adults can reach 19 hundred million people, which seriously affects the reproductive health and mental health of males and needs long-term prevention and treatment. Diseases such as diabetes, cardiovascular diseases, mental disorder and the like induced by obesity seriously threaten human health, wherein the induced sperm quantity reduction and the sperm quality reduction of different degrees of male of child bearing age are more important reasons for inducing male sterility, and the reproductive health of the male of child bearing age is seriously influenced. Therefore, under the large environment of increasingly declining male fertility, the molecular mechanism of the occurrence of the obese testosterone dyssynthesis is clarified, and the excavation of new drugs for preventing and treating obesity and the complications thereof is a current research hotspot.

The mechanisms by which obesity causes male reproductive dysfunction are complex and are currently unclear. Obesity destroys testosterone synthesis in mesenchymal cells, resulting in a decrease in serum testosterone levels, and is one of the most important factors in inhibiting male fertility. In addition, obesity-induced lipid metabolism abnormalities and increased levels of systemic chronic inflammation can lead to increased reactive oxygen species and fragmentation of sperm DNA, a decrease in sperm count and quality, and ultimately oligospermia in males. These findings suggest that obesity is associated with male reproductive dysfunction through a variety of mechanisms. The search for a safe drug to protect the testes from the damage of obesity complications in various ways is an important problem to be solved.

Because the clinical manifestations of obesity are highly heterogeneous and the etiology and pathogenesis of obesity have not been fully elucidated, diagnosis and treatment are affected. At present, no effective treatment means for obese male infertility exists clinically, and the main treatment is symptomatic treatment and life style improvement. Experts at home and abroad agree to advocate that obese patients need basic treatment such as lifestyle adjustment, smoking cessation, alcohol abstinence, weight reduction to a normal range and the like no matter whether the patients have fertility or not, so that symptoms of liver lipid metabolism disorder are relieved, and insulin resistance is improved. Aims to relieve symptoms caused by hyperlipidemia and prevent long-term reproductive disorder and metabolic complications. Under the current fast-paced lifestyle, the diet is regulated, the physical exercise is strengthened, the compliance is low, the symptoms of patients are difficult to be effectively improved, and especially, an effective treatment method for the fertility problem is lacked. Therefore, how to effectively treat and relieve the symptoms of infertility disorder of obese men, improve the quality of life of obese men and restore fertility thereof is a problem to be solved urgently.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an application of PQQ in preparing a medicament for treating male infertility caused by obesity; the applicant finds that after the intervention of the obese mouse with PQQ, the body weight and the abdominal fat content of the obese mouse are reduced, the serum lipid metabolism hormone level is reduced, and the primary bile acid metabolism and cholesterol metabolism level are improved; the number and the quality of sperms are increased, the apoptosis of interstitial cells is reduced, the gene expression of key enzyme for synthesizing testosterone is increased, the functions of mouse testis and epididymis are recovered, and the testosterone level is recovered; finally, the testosterone content and the fertility of the male obese mice are obviously improved.

The technical scheme adopted by the invention is as follows:

application of pyrroloquinoline quinone in products for preventing, improving and/or treating obesity male sterility disorders.

In the present invention, the product in said application is a product for increasing the diameter, and/or area of the seminiferous tubules.

In the present invention, the product in said application is a product for increasing the number of spermatozoa, and/or a product for reducing the rate of teratospermia.

In the invention, the product in the application is a product for improving the sperm motility.

In the present invention, the product in the application is a product for increasing the testicular index, and/or the epididymal index.

In the present invention, the product for use is a product for reducing body weight, and/or decreasing fat content of the abdominal cavity, and/or decreasing the diameter and area of fat cells.

In the present invention, the products in said use are products which lower TC, TG, LDL-C, raise HDL-C, and/or increase the content of bile acid metabolites.

In the invention, the product in the application is a product which can up-regulate the expression of testosterone synthesis rate-limiting enzyme genes 3 beta-HSD, stAR and P450 scc.

In the present invention, the product in said application is down-regulatedCorrelation of cell scorchingProducts expressed by genes NLRP3, caspase-1, pro-caspase-1, GSDMD and IL-1 beta.

In the invention, the products in the application comprise medicines, health-care products and foods.

In a specific cell experiment of the invention, through research of the inventor, the product is applied at a dosage of 0.001-20 μ M, and the optimal dosage is 0.1nM.

The existing research considers that interstitial cell testosterone synthesis disorder and reduction of serum testosterone level cause male endocrine disorder, sperm dysplasia, hyposexuality, erectile dysfunction and the like, and finally cause infertility. Testosterone is the most important steroid hormone for maintaining male fertility, is converted from cholesterol and is mainly responsible for normal development of testis, spermatogenesis, maintenance of secondary sexual characteristics and sexual desire generation, 95 percent of testosterone in a male body is synthesized and secreted by testicular interstitial cells, and the normal function of the interstitial cells is an important prerequisite for ensuring spermatogenesis and sperm function maturation. However, studies have shown that testosterone synthesizes a key enzyme gene in the testes of obese men and high-fat fed male mice: the expressions of steroid hormone synthesis rapid regulatory protein (StAR), cytochrome P450 cholesterol side chain lyase (P450 scc) and 3 beta-steroid dehydrogenase (3 beta-HSD) are all obviously reduced, and testosterone synthesis is inhibited. Suggesting that the change of the transport and function of the interstitial cell cholesterol caused by obesity is a key mechanism and a target point for inhibiting the synthesis of testosterone by obesity.

Abnormal lipid metabolism induces NLRP3 inflammasome-mediated cell apoptosis that may be a significant cause of low testosterone levels and fertility in obese men. Cell apoptosis is a newly discovered inflammatory programmed cell death type characterized by the formation of pores with a diameter of 10-15nm on the plasma membrane, the release of proinflammatory factors IL-1 beta, IL-18 and cell swelling death, and the classical cell apoptosis is Caspase-1/GSDMD pathway mediated by NLRP 3. Abnormal lipid metabolism in obese patients can cause a chronic, low-grade inflammatory response throughout the body, resulting in abnormal secretion of a large number of proinflammatory factors. There is increasing evidence that NLRP3 inflammasome activation is a key driver of obesity and vascular disease, that NLRP3 expression is elevated in adipose tissue of obese patients and high fat-fed mice, and that NLRP3 knockout mice are unable to develop obesity after high fat feeding. In addition, the previous research shows that the expression of NLRP3, IL-6 and TNF-alpha in the testis tissue of an obese mouse is up-regulated, the concomitant inflammatory reaction can directly inhibit the synthesis of mesenchymal testosterone, and the fertility of the mouse is reduced. And further prompting that: lipid metabolism abnormality induced interstitial cell apoptosis of obese patients is a potential target for causing reduction of testosterone level and fertility reduction of men, but related researches on inhibition of testosterone synthesis by NLRP 3-mediated interstitial cell apoptosis are rarely reported.

Pyrroloquinoline quinone (PQQ) is a newly discovered oxidoreductase coenzyme and is important for protecting the body against oxidative stress. It is a novel vitamin-like substance, is rich in o-quinone structure, can directly participate in redox reaction, and can increase the number of cell mitochondria and improve the function of the cell mitochondria.

PQQ also has various physiological functions, such as prevention of liver damage, nourishing of nerve tissue, promotion of energy metabolism, promotion of growth, anti-inflammation, enhancement of immunity, and the like. Is important for treating inflammatory diseases such as mental system diseases, cardiovascular diseases, liver diseases, cancers and the like. Previous studies show that after the PQQ is supplemented for 12 weeks, the level of LDL-C in serum can be remarkably reduced, and abnormal lipid metabolism is relieved. Research shows that the supplement of PQQ to obese female mice during pregnancy can inhibit the expression of inflammation-related factors such as liver NLRP3 and IL-6 of offspring mice, and has a lasting protective effect on hepatolipotoxicity and inflammation. In addition, PQQ can improve myocardial hypertrophy and cardiac fibrosis accompanied by diabetes by inhibiting the activation of NLRP3 inflammasome and down-regulating the expression of factors such as Caspase-1, IL-1 beta and IL-18 in a cell apoptosis signal pathway. However, no scholars have yet proposed the use of PQQ in improving fertility in obese men by inhibiting stromal cell apoptosis. Therefore, the molecular mechanism of PQQ for protecting male reproductive dysfunction caused by obesity needs to be further discussed.

The applicant finds that after PQQ is applied to a male obese mouse, the weight and the abdominal fat content of the obese mouse can be reduced, the serum lipid metabolism hormone level can be reduced, and the primary bile acid metabolism and cholesterol metabolism level can be improved; increasing the quantity and the quality of sperms, reducing the scorching of interstitial cells, increasing the expression of testosterone synthesis key enzyme genes and the testosterone content, and simultaneously recovering the structural functions of mouse testicles and epididymis; finally, the fertility of the male obese mice is obviously improved.

Drawings

FIG. 1 is a graph showing the intervention of PQQ of the present invention in the changes of body weight, abdominal fat content and hepatic fat metabolism in obese mice, wherein A: the body type of the mouse changes; b: the body weight changes in the mouse molding period (1) and the drug intervention period (2); ND, normal diet; HFD, high fat diet; c: abdominal fat weight; d: HE staining of abdominal fat; e: HE staining of liver; f, liver oil red O staining, positive cells indicated by red arrows, # p <0.05, # p <0.01;

FIG. 2 is a serum lipid profile of obese mice after intervention with PQQ of the present invention, wherein A: the serum metabolites of the OPLS-DA model analysis model group and the blank control group are distinguished in a positive ion mode (1) and a negative ion mode (2), and the serum metabolites of the PQQ drug intervention group and the model group are distinguished in a positive ion mode (3) and a negative ion mode (4) through the OPLS-DA model analysis; b: enrichment analysis of differential metabolite changes in three serum samples; c: differential metabolite metabolic pathway analysis plot as a function of log (p) (y-axis), pathway impact (x-axis) of different key metabolites between model group and blank control group (1), drug intervention group and model group (2); d: serum TC levels (1), TG levels (2), LDL-C levels (3), HDL-C levels (4) assay, # p <0.05, # p <0.01;

FIG. 3 is a diagram of the intervention of PQQ in improving testicular spermatogenesis in obese mice of the invention, wherein A: HE staining of testis of different groups; b: mouse testicular index; c: the diameter (1) and the area (2) of a mouse seminiferous tubule; d: staining epididymis HE of different groups of mice; e: mouse epididymis index; f: comparison of mouse sperm count (1), abnormal sperm rate (2), sperm motility (3) × p <0.05, × p <0.01;

FIG. 4 is a graph showing the expression change of testosterone and testosterone synthesis key enzyme genes of obese mice after the intervention of PQQ of the present invention, wherein A: detecting the level of testosterone in serum of each group of mice by a radioimmunoassay; b: qRT-PCR detected 3 β -HSD, stAR and P450scc mRNA levels in testis P < 0.05P <0.01; c: western blotting detection of 3 beta-HSD, stAR protein level (1) and Image J software in testis analysis of their statistical differences; d: immunohistochemical analysis of 3 β -HSD, stAR and P450scc expression in testis; PBST as negative control, red arrows indicate positive cells, <0.05 × p <0.01;

FIG. 5 is a graph of the change in mouse interstitial cell apoptosis following PQQ intervention of the present invention, wherein A: qPCR detection of expression of tar death gene mRNA: (1) changes in expression of NLRP3 in testis; (2) altered expression of Caspase-1 in testis; (3) altered expression of Pro-caspase-1 in testis; (4) changes in the expression of GSDMDIL-1 β in testis; (5) changes in expression of IL-1 β in testis; b: WB detects the expression of apoptosis gene proteins (NLRP 3, caspase-1, pro-Caspase-1, GSDMD, IL-1 beta); c: immunohistochemical detection of expression of apoptosis genes (NLRP 3, caspase-1, GSDMD), positive cells indicated by red arrows, <0.05, <0.01;

FIG. 6 is a graph showing the change of testosterone synthesis rate-limiting enzyme and apoptosis-related factor in the PQQ-stem-prognostic TM3 cells according to the present invention; wherein, A: the method comprises the following steps of (1) detecting toxic effect of PQQ application with different concentrations on TM3 cells by a CCK8 experiment, (2) interfering improvement effect of PQQ application with different concentrations on cell viability after 0.4mM palmitic acid treatment; b: qPCR detection of expression of tar death gene mRNA: (1) changes in expression of the TM3 cell apoptosis-related factor GSDMD; (2) altered expression of Caspase-1 in testis; c: WB detected the expression of proteins of apoptosis gene (NLRP 3, caspase-1, pro-Caspase-1, GSDMD, IL-1 beta) and testosterone synthesis rate-limiting enzyme (3 beta-HSD, stAR and P450 scc) (1), and the statistical difference (2) P <0.05 and P <0.01 was analyzed by Image J software.

Detailed Description

Example 1

1. Preparation of obese mouse model

After 4 weeks old C57BL/6J mice were adaptively fed for one week, they were randomly divided into a control group (Ctrl group) and a model group (OBE group), and the model group was fed with high fat diet fat (60.0%, protein 19.4%, carbohydrate 20.6%; calorie 5.0 kCal/g, australian Corp., beijing, K.D 12492) rich in 60% fat; the control group was fed with normal feed for 12 weeks. Mice were free to ingest water and food.

And detecting the weight of the mice after feeding for 12 weeks, and primarily judging whether the model is successfully constructed or not according to the condition that the weight of the mice in the model group is 1.2 times larger than that of the mice in the blank control group.

C57BL/6J mice with successfully constructed models are divided into OBE group (model group) and OBEPQQ group (experimental group).

After the model is successfully constructed, blood of each group of mice is collected, serum is separated, the contents of lipid metabolism hormones (TG, TC and LDL-C, HDL-C) in the serum are measured by a kit method (Nanjing Biochemical company), and the content of testosterone in the serum is measured by a radioimmunoassay (North Biochemical company). Collecting mouse material, weighing adipose tissue, fixing liver and adipose tissue, dehydrating, transparentizing, waxing and embedding to prepare paraffin section, analyzing morphological change of liver and adipose tissue by HE staining, analyzing lipid metabolism change of liver by oil red O staining, calculating diameter of fat cell, and estimating model manufacturing effect.

According to the previous related researches, the use concentration of the relevant mouse animal experimental drugs by using PQQ is 2-20mg/kg/d, however, the number of researches using PQQ as an intervention drug is small, and although the researches on the mechanism related to non-alcoholic fatty liver induced by using PQQ to intervene high-fat feed have been carried out in the present year, the literature does not report the mechanism of improving the male infertility caused by obesity by the PQQ and the model forming rate of the obese mouse model is low, the feeding is difficult, and the PQQ use dose with the best intervention effect cannot be set by a plurality of experimental intervention groups, so that the PQQ dose applied in the research is 10mg/kg/d which is relatively balanced.

2. Detection of the Effect of PQQ intervention on testis development in OBE group mice

The experimental group dosing regimen was: PQQ (Simma Bio Inc., cat # 72909-34-3) was dissolved in physiological saline and continuously administered by gavage for 8 weeks. After the treatment is finished, the weight of the mice is measured, and the treatment effect of the medicine is judged. The materials are taken after the treatment is finished.

Collecting blood of each group of mice, separating serum, determining the content of lipid metabolism hormone (TG, TC, LDL-C, HDL-C) in serum by a kit method, and determining the content of testosterone in serum by a radioimmunoassay. Collecting mouse material, weighing adipose tissue, fixing liver and adipose tissue, dehydrating, transparentizing, waxing and embedding to prepare paraffin section, analyzing morphological change of liver and adipose tissue by HE staining, analyzing lipid metabolism change of liver by oil red O staining, and calculating diameter of fat cell. Mouse testis and epididymis tissue are fixed, paraffin sections are prepared through dehydration, transparence, wax penetration and embedding, the morphological change of tissues is analyzed through HE staining, semen in epididymis is collected, semen quality analysis is carried out, and the drug treatment effect is presumed.

3. Detecting the influence of PQQ intervention on testosterone synthesis and mesenchymal cell apoptosis of OBE mice

Collecting mouse testis slices of each group, carrying out heat restoration by sodium citrate buffer solution antigen, then carrying out 1% Triton X-100-PBST permeable membrane and 5% BSA sealing, respectively dripping primary antibodies StAR, P450scc and 3 beta-HSD on a glass slide, and incubating overnight in a refrigerator at 4 ℃. PBST washing 3 times (5 min/time) and then with biotin-labeled streptavidin were incubated for 1h; PBST is washed for 3 times (5 min/time), and DAB staining solution is mixed evenly and then dripped into the slices. Developing color at room temperature, finishing counterstaining with hematoxylin, analyzing the expression of StAR, P450scc and 3 beta-HSD in testis by optical microscope imaging, observing the expression of testosterone synthesis key enzyme gene in interstitial cells, and determining the yellow or brown area as positive. Meanwhile, RNA and protein are extracted from the right testis, and expression change conditions of StAR, P450scc and 3 beta-HSD in the testis are detected by a QRT-PCR and Western blotting method.

Collecting mouse testis slices of each group, performing heat restoration by sodium citrate buffer solution antigen, blocking by 1% Triton X-100-PBST permeable membrane and 5% BSA, respectively dripping primary anti-NLRP 3, caspase-1 and GSDMD onto a glass slide, and incubating overnight in a refrigerator at 4 ℃. After PBST washing, incubating with streptavidin labeled with biotin for 1h; PBST is washed for 3 times (5 min/time), and DAB staining solution is mixed evenly and then dripped into the slices. And (3) developing at room temperature, finishing counterstaining by hematoxylin, analyzing the expression of NLRP3, caspase-1 and GSDMD in testis by imaging of an optical microscope, observing the expression condition of apoptosis key genes in interstitial cells, and determining that a yellow or tan area under the microscope is positive. Meanwhile, RNA and protein are extracted from the right testis, and the expression change conditions of NLRP3, caspase-1 and GSDMD in the testis are detected by a QRT-PCR and Western blotting method.

4. TM3 mesenchymal cell line in vitro culture, NLRP3 inhibitor and PQQ intervention

For the experimental purposes of this study, a model of cells for high-fat intervention was constructed by culturing a TM3 mesenchymal cell line (Ctrl group) in vitro, adding 0.4mM palmitic acid (Sigma Aldrich trade Co., ltd., cat # P0500, PA group), and after determining the concentration of palmitic acid, adding MCC950 nM (Shandong Cisco Biotechnology Co., ltd., cat # SJ-MX0058A, PA + MCC950 group) and PQQ (PA + PQQ group), which are specific inhibitors of NLRP3, to the medium. The CCK8 experiment detects the toxicity of the palmitic acid to TM3 cells and the improvement effect of PQQ on the palmitic acid, and the optimal intervention concentration of PQQ is screened. Experimental data show that varying concentrations of PQQ have no toxic effect on stromal cell treatment, whereas 0.1nM (i.e., 10) was used ^-1 uM) PQQ StemThe recovery of TM3 cell viability after 0.4mM palmitic acid treatment was best in advance and statistically significant, and therefore was the optimal intervention concentration for PQQ, which was chosen for intervention in later experiments.

5. Detection of the effects of PQQ intervention on Testosterone Synthesis and apoptosis in palmitic acid-treated TM3 cells

After the culture is finished, collecting each group of cells to analyze the function of interstitial cells, the cell apoptosis condition and the expression change of testosterone synthesis rate-limiting enzyme genes.

The method comprises the following specific steps of:

1. sampling blood and tissue from an orbit: treatment was started after the expiration of the dosing period and fasting was performed for 12 hours before treatment. Fixing the mouse on the animal table with the left hand, slightly pressing the head downwards to make the eyeball protrude outwards, holding a capillary tube (with an inner diameter of 1.0-1.5mm, and a sharp point, breaking the tip), inserting the capillary tube into the bottom of the eyeball from the edge of the eyeball to a depth of about 4.0-5.0 mm, rotating the capillary tube to cut the venous plexus, and allowing blood to flow into the capillary tube. Or removing eyeball with ophthalmological forceps, and collecting dropped blood with test tube. After blood sampling, the testis, epididymis, fat and liver are found in the lower abdomen of the mouse, the capsule and fat are carefully separated, the tissues are taken out and weighed, one testis is preserved in liquid nitrogen at-80 ℃, and the other tissue is fixed by 4% paraformaldehyde.

2. Extracting the semen of the mouse: the mice were weighed and recorded, and after neck-broken death, epididymal tail and vas deferens were immediately removed, surface blood vessels and fat were removed, placed in a petri dish containing 1mL of 37 ℃ physiological saline, sperm were flushed out with a syringe needle and transferred to a 1.5mL EP tube, and placed in a carbon dioxide incubator (37 ℃,5% CO) ₂ ) Incubate for 20min to allow it to be energized.

3. Extracting testicle metabolites: taking out a sample at minus 80 ℃, grinding the sample by liquid nitrogen, weighing 60 mg of the sample, adding 0.5mL of methanol acetonitrile aqueous solution (2.

4. Lipid metabolism hormone assay: the blood lipid level is measured by adopting an enzyme method, the serum of each treatment group of mice is taken out from a refrigerator at the temperature of-20 ℃, is placed at room temperature for dissolution, then the used reagent is prepared according to the kit specification, and finally the result is measured in an enzyme-labeling instrument with the wavelength of 510nm according to the kit specification. According to the kit specification, obtaining and recording the OD value of each well after the measurement is finished, firstly calculating the difference value between the sample A2 value and the sample A1 value as A, the difference value between the blank A2 value and the blank A1 value as B, then calculating the difference value between the calibration A2 value and the calibration A1 value as C, and the difference value between the blank A2 value and the blank A1 value as D, and then multiplying the difference value of ((A-B) - (C-D)) by the concentration of the calibrator to calculate the contents of TC, TG, HDL and LDL.

5. And (3) counting sperms: the prepared sperm suspension was pipetted at 10uL and dropped into a counting cell of a cell counting plate, and the number of sperm in 5 squares (denoted by a) in the central large cell was counted under a microscope by the erythrocyte counting method (x 400), and the number of sperm in the mouse was calculated by the formula a x 50000= the number of sperm (10%/mL).

6. And (3) calculating the teratospermia rate: sucking 10uL of sperm suspension by a pipette, dripping the sperm suspension on a clean glass slide, uniformly pushing the slide, naturally drying in the air, fixing for 5min by methanol, naturally drying in the air again, staining for 10min by 2% eosin solution, slightly washing by distilled water, and drying at room temperature. Under a microscope, the sperms in the parts with clear background and less overlap are found under a low power microscope, then the high power microscope is changed to sequentially check the morphology of the sperms, 200 sperms are counted in each mouse, and the overlapped and tailless sperms are not counted. Observing teratospermia including hairless, banana-shaped, amorphous, double-headed, fat-headed, tail-folded and double-tail, etc. and counting to calculate out the teratogenesis rate.

7. And (3) sperm motility detection: taking 10uL of sperm suspension, dripping into a cell counting plate, counting the number of sperms at each activity level under a microscope (continuously counting 200 sperms per mouse), and calculating the sperm motility according to the following formula, wherein the sperm motility (%) = (I + II + III)/(I + II + III + IV) multiplied by 100%. The activity of the sperm is classified into 4 grades according to the laboratory test manual of the interaction between human semen and sperm of the world health organization and cervical mucus. Respectively as follows: the movement is good, and the rapid and active linear motion is realized; II: the sperm can move, but the moving direction is not clear and the sperm can move in a rapid or dull straight line or non-straight line; III: poor movement, in-situ rotation or rotation, and poor forward movement capability; IV: sperm were immotile.

8. Serum metabonomics analysis: (1) The collection and pretreatment of the sample, for the blood sample, firstly the blood collection amount should not be less than 500 μ l/case, and secondly the respiratory anesthesia (isoflurane) is used in the collection process to avoid the interference to the mass spectrum peak line. If plasma is needed, a heparin sodium anticoagulation tube is used for collecting plasma, a common tube is used for collecting serum, the collected serum is kept stand for 30 minutes at room temperature after collection, and the collected serum is stored for use after centrifugation. And (2) performing statistical analysis on the data, namely performing principal component analysis. Principal component analysis can reveal the internal structure of the data, and thus better interpret data variables. The data was formatted using SIMCA or QI software and then subjected to automated modeling analysis. A principal component analysis score scatter plot may be obtained for all samples. The second is orthogonal partial least squares-discriminant analysis. The standard generally used is the standard generally accepted by the academia, namely student T test (R0.05), and the projection importance of the variable of the first principal component of the orthogonal partial least squares-discriminant analysis model is greater than 1. And finally, performing pathway analysis in a database of Kyoto gene and genome encyclopedia metabolic pathways.

The statistical method comprises the following steps: all data were replicated three more times. Quantitative data are expressed by mean (M) + -Standard Deviation (SD), SPSS 20 software is used for carrying out statistical analysis on the data, one-way ANOVA method is adopted for comparison among a plurality of groups, t-test method is adopted for comparison in pairs, and P is less than or equal to 0.05, so that the difference has statistical significance.

The raw materials and reagents used in the application of the pyrroloquinoline quinone in the products for male infertility caused by obesity provided by the invention can be purchased from the market.

As shown in figure 1, PQQ intervenes in weight loss in obese mice; the fat content in the abdominal cavity is reduced, and the lipid metabolism disorder of the liver is improved; as shown in the figure, fat mice are enlarged in body type, the fat content in abdominal cavity is obviously increased, the HE staining of slices of the fat mice shows that the diameter and the area of fat cells are obviously increased, and meanwhile, the morphological structure of liver is disordered, the cell nucleus is deeply stained with solid shrinkage, and the oil red O staining shows that the content of fat droplets in liver is increased. The morphological changes described above can be significantly improved by intervention with PQQ.

As shown in figure 2, serum lipid metabolism was improved in obese mice following PQQ intervention; * P <0.01, the difference is statistically significant; the results show that: PQQ can increase the content of primary bile acid metabolites; reducing the content of serum triglyceride, cholesterol and low-density lipoprotein, and increasing the content of high-density lipoprotein; and has statistical significance. As shown in panel a, samples from different groups were clustered on both sides of the graph, indicating that there was a difference between the groups and a small error within the group. The graph B is a heat map of serum-differential metabolite concentration analysis, which shows that metabolites such as taurine and arachidonic acid involved in the regulation of serum cholesterol level are different between groups, and in the serum of obese mice, the content of taurine, which is one of primary cholesterol metabolism products, is reduced (blue, the shade of color represents the degree of reduction of the amount of the metabolite), the content of blank and PQQ-treated groups is increased (red, the shade of color represents the degree of increase of the amount of the metabolite), and the reduction of cholesterol decomposition may be one of the causes of the increase of serum cholesterol level of obese patients. Simultaneously, carrying out channel enrichment analysis on all serum differential metabolites, and enriching all the differential metabolites to obtain the following 11 metabolic channels as shown in a C diagram, wherein arginine and proline metabolic channels; the glycine, serine and threonine metabolic pathways phenylalanine, tyrosine; a phenylalanine metabolic pathway; biosynthesis of phenylalanine and tryptophan; metabolic pathways for taurine and hypotaurine; a primary bile acid biosynthetic purine pathway; and the arachidonic acid metabolic pathway has difference among all groups, which indicates that the bile acid metabolism is one of the ways of improving the fat metabolism of obese mice by PQQ. The serum lipid metabolism indexes TC, TG, LDL-C and HDL-C are detected to verify whether PQQ can regulate the serum lipid metabolism of the obese mice. The results show that: PQQ can increase the content of primary bile acid metabolites; reducing the content of serum triglyceride, cholesterol and low-density lipoprotein, and increasing the content of high-density lipoprotein; and has statistical significance.

As shown in figure 3, PQQ intervention improved testicular spermatogenic function in obese mice; * P <0.01, the difference is statistically significant; the results show that: PQQ can improve the testis and epididymis structural disorder of obese mice and improve organ index; can also improve the semen quality, increase the total number of sperms and the sperm motility, and reduce the teratospermia rate, and figure 3 shows the research for evaluating PQQ to improve the semen quality of the food-borne obese mice for the first time.

As shown in fig. 4, the expression of testosterone and testosterone synthesis key enzyme genes of obese mice after PQQ intervention changes; the results show that: compared with a control group, the expressions of testosterone and testosterone synthesis key enzyme genes (3 beta-HSD, stAR and P450 scc) in the testis of the obese mouse are both reduced at the protein level and the mRNA level, and the PQQ stem prognosis is up-regulated; after the intervention of PQQ, the number of cells of the testosterone synthesis key enzyme gene positively expressed by interstitial cells is increased.

As shown in figure 5, changes in mouse stromal cell apoptosis following PQQ intervention; the results show that: after PQQ intervention, the expression of testis focal death key factors (NLRP 3, caspase-1, pro-caspase-1, GSDMD and IL-1 beta) of obese mice is obviously reduced, and the difference has statistical significance; as shown in the figure, under the condition of high-fat feed feeding, the cell apoptosis factor is positively expressed in the mesoplasmic cell in the seminiferous tubule (the positive cell is brown), and the PQQ is improved after intervention. Since mesenchymal cells are responsible for the synthesis of testosterone, we speculate that PQQ may improve testosterone synthesis in obese mice by improving mesenchymal apoptosis.

As shown in fig. 6, PQQ stem prognosis TM3 cells changes in testosterone synthesis rate-limiting enzymes and apoptosis-related factors; the results show that: after PQQ intervention, the expression of critical factors (NLRP 3, caspase-1, pro-caspase-1, GSDMD and IL-1 beta) of mouse interstitial cell line TM3 cells under simulated high-fat environment by palmitic acid intervention is remarkably reduced, the expression of testosterone synthesis rate-limiting enzyme genes (3 beta-HSD, stAR and P450 scc) is improved, and the difference has statistical significance.

In recent years, although research has proved that NLRP3 can be used as a target for improving diseases by aiming at inflammation, the testosterone synthesis disorder mechanism of obese men is unknown, and no related research discloses that PQQ can reduce interstitial cell apoptosis and improve the action mechanism of testosterone synthesis disorder by inhibiting the expression of NLRP3 apoptosis related factors. Therefore, the research simultaneously applies a cell model to verify the action of PQQ on interstitial cells under a high-fat environment and the mechanism of the PQQ. Based on a palmitic acid treated cell model, a specific inhibitor PCC950 of NLRP3 is added, and the activation of Caspase-1/GSDMD classical cell apoptosis pathway mediated by NLRP3 in a high-fat environment is firstly proved to trigger mesenchymal cell apoptosis, so that the synthesis of testosterone of male mice is reduced. While inhibition of NLRP3 factor can reduce the level of apoptosis in cells and thereby improve testosterone synthesis. In addition, the molecular mechanism of PQQ is similar to that of MCC950 treatment, namely the expression of cell apoptosis factors such as NLRP3 and the like which are increased in expression under high lipid environment is reduced, and the expression of a testosterone synthesis rate-limiting enzyme gene of mesenchymal cells is improved. Meanwhile, in the measurement of the previous cell experiment by using PQQ (mostly 0.001-20 mu M), the intervention dosage of PQQ for improving the activity and the scorching of mesenchymal cells in a high-fat environment and having the strongest effect of promoting the synthesis of testosterone is found to be 0.1nM.

Table 1 shows that PQQ intervention remarkably improves the body weight and the abdominal fat weight of obese mice. ( Ctrl is blank control group; and (3) OBE: a model group; OBEPQQ: PQQ drug intervention group. Each set of specific intervention was as described in example one )

Table 2 shows that PQQ intervention remarkably improves serum lipid metabolism hormone content of obese mice.

Table 3 shows that PQQ intervention remarkably improves the testicular epididymis index, the content of testosterone in serum and the semen quality of obese mice.

Table 4 represents all raw data used for the calculation of Table 1 data.

Table 5 represents all raw data used for Table 2 data calculation.

Table 6 indicates all raw data used for Table 3 data calculation.

Table 1.Effects of PQQ treatments on the body weight and fat mice in obese mice.

*P<0.05,**P＜0.01vs.Ctrl； ^# P<0.05, ^## P＜0.01vs.OBE

Table 2.Effects of PQQ treatments on the serum lipid levels in obese mice.

*P<0.05,**P＜0.01vs.Ctrl； ^# P<0.05, ^## P＜0.01vs.OBE

Table 3.Effects of PQQ treatments on the structure of testis and epididymis,testosterone and sperm parameters in obese mice.

*P<0.05,**P＜0.01vs.Ctrl； ^# P<0.05, ^## P＜0.01vs.OBE

Table 4.The row data of table 1.

Table 5.The row data of table 2.

Table 6.The row data of table 3.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. Application of pyrroloquinoline quinone in products for improving obese male infertility disorders, which is characterized in that the application is application in products for preventing, improving and/or treating obese male infertility disorders.

2. Use according to claim 1, wherein the product is a product for increasing seminiferous tubule diameter, and/or area.

3. Use according to claim 1, wherein the product is a product that increases sperm count, and/or a product that decreases teratospermia rate.

4. Use according to claim 1, wherein the product is a product for enhancing sperm motility.

5. Use according to claim 1, wherein the product is a product that increases the testicular index, and/or a product that increases the epididymal index.

6. Use according to claim 1, wherein the product is a weight-loss product, and/or a product that reduces the fat content of the abdominal cavity, and/or a product that reduces the diameter and area of fat cells.

7. Use according to claim 1, wherein the product is a product for lowering TC, TG, LDL-C, raising HDL-C, and/or a product for increasing the content of bile acid metabolites.

8. The use according to claim 1, wherein the product is a product that upregulates the expression of the testosterone synthesis rate-limiting enzyme gene 3 β -HSD, stAR, P450 scc.

9. The use according to claim 1, wherein the product is a product that down-regulates expression of the apoptosis-related genes NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-1 β.

10. The use according to claim 1, wherein the product comprises a pharmaceutical, nutraceutical, food product.

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