Application of cordyceps sobolifera polysaccharide in preparation of medicine for intervening in male dyszoospermia and reproductive injury

Abstract

The invention discloses an application of cordyceps sobolifera polysaccharide in preparation of a medicine for intervening in male dyszoospermia and reproductive injury, the molecular weight of the cordyceps sobolifera polysaccharide is 2.92 * 10 < 4 > Da, the cordyceps sobolifera polysaccharide is composed of galactose, mannose and glucose according to the molar ratio of 68.125: 43.875: 12, a novel cordyceps sobolifera homogeneous polysaccharide component CCP-1 is separated and purified, and the cordyceps sobolifera polysaccharide can be used for preparing a medicine for intervening in male dyszoospermia and reproductive injury. The influence on reproductive injury of a cyclophosphamide-induced immunocompromised male mouse and a potential mechanism thereof are discussed through an HPT axis; an apoptosis inducer is used for inducing mouse spermatogont-GC-1spg cells to construct an apoptosis model to discuss the influence on cell apoptosis; the understanding of the cordyceps sobolifera polysaccharide CCP-1 as a potential health supplement for intervening male dyszoospermia and reproductive injury is improved.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to an application of Cordyceps sinensis polysaccharide in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage. Background Art

[0002] Spermatogenesis disorder is a common clinical disease in recent years and the main cause of male infertility. Due to the increasing impact of environmental pollution and life pressure, the number of male spermatogenesis disorders is increasing, and the incidence rate is rising year by year. Cyclophosphamide can cause oxidative stress damage in the body, resulting in a decrease in sperm count and vitality, an increase in the deformity rate, and spermatogenesis dysfunction. The hypothalamus-pituitary-testis (HPT) axis plays a vital role in regulating male hormone production and spermatogenesis, which is essential for the male reproductive function of vertebrates. Disorders of the HPT axis can lead to impaired spermatogenesis. The hypothalamus secretes gonadotropin-releasing hormone (GnRH), which is released into the pituitary portal vein system to stimulate the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone (FSH) in the anterior pituitary, thereby stimulating the synthesis of testosterone (T) by interstitial cells in the testis and promoting sperm production in the male gonads. At present, the therapeutic drugs for the treatment of cyclophosphamide-induced spermatogenesis dysfunction are still limited to traditional Chinese medicine formulas.

[0003] Cordyceps cicadae, also known as golden cicada flower, is one of the traditional precious Chinese medicinal materials in my country and is now listed in the new resource food sequence. Modern medical research shows that Cordyceps cicadae contains a large amount of functional ingredients such as cordycepic acid, cordyceps polysaccharides, adenosine, alkaloids and ergosterol, which can improve renal function, relieve chronic renal failure, calm and hypnotize, and resist radiation. At present, there are not many studies on Cordyceps cicadae polysaccharides, and there is no research on the effect of polysaccharides of uniform components on male spermatogenesis disorders.

[0004] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of how to use Cordyceps sinensis polysaccharide to intervene in male spermatogenesis disorder, and provide an application of Cordyceps sinensis polysaccharide in the preparation of drugs for intervention in male spermatogenesis disorder and reproductive damage.

[0006] In order to achieve the above-mentioned object, the present invention discloses an application of Cordyceps sinensis polysaccharide in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, wherein the molecular weight of the Cordyceps sinensis polysaccharide is 2.92×10 4 Da, composed of galactose, mannose and glucose with a molar ratio of 68.125:43.875:12.

[0007] The method for purifying Cordyceps sinensis polysaccharide comprises the following steps:

[0008] S1, dissolving the crude polysaccharide of Cordyceps sinensis in pure water and filtering it through a microporous membrane filter, and then loading the filtrate onto a DEAE agarose FF anion exchange chromatography column for purification;

[0009] S2, eluting the chromatographic column with ultrapure water and sodium chloride solutions of different concentrations, collecting the components, tracking with the phenol-sulfuric acid method, and detecting with an ELISA reader at 490 nm;

[0010] S3, the largest fraction of the eluate was collected, dialyzed, and then freeze-dried at -60 °C using Superdex TM The purified product was further purified by 75% chromatography, and the eluate was collected and combined, and then freeze-dried to obtain the purified Cordyceps sinensis polysaccharide.

[0011] In step S1, the method for preparing crude polysaccharide of Cordyceps sinensis comprises the following steps:

[0012] S11, crushing the fruiting bodies of Cordyceps sinensis and sieving to obtain Cordyceps sinensis fruiting body powder;

[0013] S12, adding distilled water at a solid-liquid ratio of 1:6-10 for ultrasonic extraction, then placing in a water bath at 80-90° C. for 1.5-2.5 h, filtering and collecting the supernatant multiple times and combining them;

[0014] S13, subjecting the supernatant to ultra-high pressure treatment at a pressure of 400-450 MPa, a ultra-high pressure treatment time of 10-15 min, and maintaining the temperature at 20° C.;

[0015] S14, concentrating the treated supernatant and adding ethanol, collecting the precipitate by centrifugation, and adding water to dissolve the precipitate to obtain a crude polysaccharide aqueous solution;

[0016] S15, adding the same volume of Sevag reagent to the crude polysaccharide aqueous solution, magnetically stirring and then centrifuging to collect the supernatant; repeating until the protein is completely removed, then concentrating the polysaccharide solution to remove small molecules, dialyzing with flowing water using a dialysis bag, concentrating by rotary evaporation to a thick liquid, and vacuum freeze-drying to obtain crude polysaccharides from Cordyceps sinensis fruiting bodies, and the volume ratio of chloroform and n-butanol in the crude polysaccharide aqueous solution and the Sevag reagent polysaccharide aqueous solution is 5:4:1.

[0017] In the step S11, the fruiting bodies of Cordyceps militaris are crushed and then passed through a 60-mesh sieve.

[0018] In step S15, the molecular weight cut-off of the dialysis bag is 3500 Da.

[0019] In the step S1, the pore size of the microporous membrane filter is 0.45 μm.

[0020] In step S2, the concentration of the sodium chloride solution is 0.2-1 mol / L, and the flow rate is 1.0 mL / min.

[0021] In step S2, the concentration of the sodium chloride solution is 1 mol / L, and the flow rate is 1.0 mL / min.

[0022] In step S3, Superdex is used TM When further purified by chromatography at 75 °C, the flow rate was 0.5 mL / min.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention separates and purifies a new uniform polysaccharide component CCP-1 from Cordyceps sinensis, and explores its effect on reproductive damage induced by cyclophosphamide in immunocompromised male mice and its potential mechanism through the HPT axis, thereby improving the understanding of Cordyceps sinensis polysaccharide CCP-1 as a potential health supplement for intervening in male spermatogenesis disorders and reproductive damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The elution curves of four CCP components (CCP-W, CCP-0.2M, CCP-0.5M and CCP-1.0M) through ultrapure water, 0.2mol / L sodium chloride solution, 0.5mol / L sodium chloride solution and 1mol / L sodium chloride solution;

[0025] Figure 2 (A) HPGPC spectrum of CCP-1; (B) standard monosaccharide mixture; (C) monosaccharide composition of CCP-1 (*-solvent peak, 6-galactose, 7-glucose and 10-mannose analysis; (D) FT-IR spectrum of CCP-1;

[0026] Figure 3 Effects of CCP-1 on the organ parameters of testis (A) and epididymis (B) in CTX-induced mice. Data are expressed as mean±SD (n=8), *p<0.05, **p<0.01 vs. control group. # p<0.05, ## p<0.01, vs CTX model group;

[0027] Figure 4 The effect of CCP-1 on testicular antioxidant parameters including SOD activity (A), GSH-Px level (B), MDA content (C) and serum FSH (D), LH (E) and T (F) levels. Data are expressed as mean ± SD (n = 8). *p < 0.05, **p < 0.01 vs. control group, # p<0.05, ## p<0.01, vs CTX model group;

[0028] Figure 5 The effect of CCP-1 on the reproductive system of male mice, including: (A) sperm micrograph on the left (400×) (red arrows point to sperm without head and short tail), sperm staining micrograph on the right (400×). Sperm count (B), sperm deformity rate (C) and sperm mortality rate (D), data are expressed as mean ± SD (n = 8). *p < 0.05, **p < 0.01 vs. control group. # p<0.05, ## p<0.01, vs CTX model group;

[0029] Figure 6 In the figure, (A) is a microscopic picture of HE tissue staining of mouse testis (scale 100 μm), (B) is a microscopic picture of mouse kidney (scale 100 μm);

[0030] Fig. 7A The cells in the seminiferous tubules in the testis are evaluated for the cell populations of spermatogonia (red arrows), primary spermatocytes (green arrows), secondary spermatocytes (yellow arrows), and spermatids (white arrows) (scale bar 50 μm).

[0031] Figure 7B To count the number of germ cells in the seminiferous tubules of testicular tissue;

[0032] Figure 7C is the diameter of interstitial cells (scale bar 50 μm);

[0033] Fig.7D is the nuclear diameter of interstitial cells (scale bar 50 μm);

[0034] Fig. 7E The diameter and volume of interstitial cells in each group, the nuclear diameter and volume of interstitial cells were measured by statistical methods. The data are expressed as mean ± SD (n = 8), *p < 0.05, **p < 0.01 vs. control group, # p<0.05, ## p<0.01, vs CTX model group;

[0035] Fig. 8A This is a microscope photo of mouse hypothalamus HE histological staining;

[0036] Figure 8B Microscope photo of mouse hypothalamus stained with Neil;

[0037] Figure 8C The number of Nissl bodies in the hypothalamus was determined by statistical methods;

[0038] Fig.8D The expression of GnRH in the hypothalamus was detected by immunohistochemical staining (original magnification, 400×);

[0039] Fig. 8E Statistical analysis of immunohistochemical staining, data are expressed as mean ± SD (n = 8), *p < 0.05, **p < 0.01 vs. control group, # p<0.05, ## p<0.01, vs CTX model group;

[0040] Fig. 9 In the figure, (A) is the expression of SCF, C-Kit, Bcl-2, Bax and Caspase-3 in testis detected by immunohistochemical staining (original magnification, 400×; scale bar 100 μm), (B) is the statistical analysis of immunohistochemical staining, (C) is the results of protein immunoblotting of SCF, C-kit, Bcl-2, Bax and Caspase-3, and the quantitative analysis of SCF, C-kit, Bcl-2, Bax and Caspase-3 proteins, and the data are expressed as mean ± SD (n = 8). *p < 0.05, **p < 0.01 vs. control group. # p<0.05, ## p<0.01, vs CTX model group;

[0041] Fig.10 In the figure, (A) is the survival rate of GC-1spg cells after treatment with different concentrations of CCP-1 (0, 25, 50, 100, 200, 400, 800 μg / mL). Five samples were analyzed in each group, and the results are expressed as mean ± standard deviation; (B) is the survival rate of GC-1spg cells after treatment with apoptosis inducer TS at different dilution ratios (1:500, 1:1000 and 1:1500) for 0, 4, 8 and 12 hours. Five samples were analyzed in each group, and the results are expressed as mean ± standard deviation; (C) is the GC-1spg cells induced with apoptosis inducer TS diluted 1:1000 for 4 hours, and then treated with 100 μg / mL CCP-1 to observe the apoptosis of the cells and perform statistical analysis. Five samples were analyzed in each group, and the results are expressed as mean ± standard deviation;

[0042] Fig.11A To detect the fluorescence intensity of SCF in GC-1spg cells by immunofluorescence and to perform statistical analysis of the results;

[0043] Fig. 11B To detect the fluorescence intensity of C-Kit in GC-1spg cells by immunofluorescence and perform statistical analysis of the results, 5 samples were analyzed in each group, and the results are expressed as mean ± SD, *p < 0.05, **p < 0.01 vs control group. # p<0.05, ## p<0.01, vs CTX model group;

[0044] Fig. 11C To detect the fluorescence intensity of Bcl-2 in GC-1spg cells by immunofluorescence and perform statistical analysis of the results, 5 samples were analyzed in each group, and the results are expressed as mean ± SD, *p < 0.05, **p < 0.01 vs control group. # p<0.05, ## p<0.01, vs CTX model group;

[0045] Fig.11D To detect the fluorescence intensity of Bax in GC-1spg cells by immunofluorescence and perform statistical analysis of the results, 5 samples were analyzed in each group, and the results are expressed as mean ± SD, *p < 0.05, **p < 0.01 vs control group. # p<0.05, ## p<0.01, vs CTX model group;

[0046] Fig.11E To detect the fluorescence intensity of Cleaved-Caspase3 in GC-1spg cells by immunofluorescence method and perform statistical analysis, 5 samples were analyzed in each group, and the results are expressed as mean ± SD, *p < 0.05, **p < 0.01 vs control group. # p<0.05, ## p<0.01, vs CTX model group;

[0047] Fig.12 (A) HPGPC spectrum of CCP-2; (B) standard monosaccharide mixture; (C) monosaccharide composition of CCP-2 (*-solvent peak, 6-galactose, 7-glucose and 10-mannose analysis; (D) FT-IR spectrum of CCP-2;

[0048] Fig.13 The effect of CCP-1 on the organ parameters of testis (A) and epididymis (B) in CTX-induced mice. Data are expressed as mean ± SD (n = 8), *p < 0.05, **p < 0.01 vs. control group;

[0049] Fig.14 The effect of CCP-2 on serum FSH (A), LH (B) and T (C) levels. Data are expressed as mean ± SD (n = 8), *p < 0.05, **p < 0.01 vs. control group. DETAILED DESCRIPTION

[0050] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0051] Each experiment in this application was repeated 3-4 times. SPSS22.0 software was used for statistical analysis, and all data results were expressed as mean ± standard deviation. The difference was considered significant when the P value was less than 0.05.

[0052] 1. Isolation, purification and characterization of Cordyceps sinensis polysaccharide CCP-1

[0053] First, prepare the crude polysaccharide of Cordyceps sinensis, and the specific process is as follows:

[0054] (1) crushing the fruiting bodies of Cordyceps sinensis and passing through a 60-mesh sieve to obtain a powder of the fruiting bodies of Cordyceps sinensis;

[0055] (2) Distilled water was added at a ratio of 1:10 and ultrasonic extraction was performed for 20 min. Then, the mixture was placed in a water bath at 80°C for 2 h, filtered through four layers of gauze, and the supernatant was collected. This process was repeated three times and the supernatants were combined.

[0056] (3) subjecting the supernatant to ultrahigh pressure treatment at a pressure of 400 MPa, an ultrahigh pressure treatment time of 12 min, and a temperature of 20° C.;

[0057] (4) The treated supernatant was concentrated to 150 ml by rotary evaporation, and then 600 ml of anhydrous ethanol was added, and the mixture was allowed to stand at 4°C for 12 h, and the precipitate was collected by centrifugation at 5000 rpm / min, and water was added to dissolve the precipitate to obtain a crude polysaccharide aqueous solution;

[0058] (5) adding approximately the same volume of Sevag reagent as in step (4), the volume ratio of the polysaccharide aqueous solution to the chloroform and n-butanol in the Sevag reagent polysaccharide aqueous solution being 5:4:1, magnetic stirring for 2 hours, and centrifuging to collect the supernatant; repeating until the protein is completely removed, and then concentrating the polysaccharide solution to remove small molecules, dialyzing with flowing water using a dialysis bag, rotary evaporation concentration to a thick liquid, and vacuum freeze drying to obtain the Cordyceps sinensis fruiting body crude polysaccharide;

[0059] After the crude polysaccharide of Cordyceps sinensis is dissolved in pure water, it is filtered through a microporous membrane filter (pore size of 0.45μm), and then the filtrate is loaded onto a DEAE agarose FF anion exchange chromatography column for purification. Ultrapure water and sodium chloride solutions of different concentrations (0.2-1mol / L, flow rate of 1.0mL / min) were used to elute the chromatographic column and collect each component, which was tracked by the phenol-sulfuric acid method and detected by an enzyme marker at 490nm. The eluent components obtained were named: CCP-W, CCP-0.2, CCP-0.5 and CCP-1.0. The eluent components with the largest proportion were collected, dialyzed, and then freeze-dried at -60°C. Superdex was used. TM75 chromatography was further purified (flow rate of 0.5 mL / min), and the eluate was collected and combined, and the purified polysaccharide component was obtained after freeze-drying, which was named CCP-1. The purity and molecular weight of CCP-1 were determined on a Shimadzu LC-10A instrument equipped with a BRT105-104-10-102 column (7.8×300 mm) and a differential detector (Shimadzu RI-502). The sample solution (5 mg / mL) was centrifuged at 12000 rpm for 10 min, and then the supernatant was collected and loaded into the HPGPC system. Dextran standards of different molecular weights were used as calibration standards. The monosaccharide composition of CCP-1 was detected by high-performance ion chromatography (HPIC) (ICS5000) equipped with an electrochemical detector and a Dionex CarbopacTMPA20 column (3×150 mm). 5 mg of CCP-1 was hydrolyzed with trifluoroacetic acid (2 mol / L, 10 mL) at 120°C for 3 hours. Subsequently, 100 μL of the acid hydrolysis solution was evaporated and dissolved in 900 μL. After centrifugation at 12000 rpm for 5 min, the supernatant was collected and loaded into the HPIC system for elution at a flow rate of 0.3 mL / min, a temperature of 30 °C, and an injection volume of 10 μL. Standards used trehalose (Fuc), rhamnose (Rha), arabinose (Ara), glucosamine hydrochloride (GlcN), galactose (Gal), glucose (Glc), N-acetyl D-glucosamine (GlcNAc), xylose (Xly), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (GalA), glucuronic acid (GlcA), galactosamine hydrochloride (GulA), uric acid, and mannuronic acid (ManA). The chemical structure of CCP-1 was characterized by Fourier transform infrared (FT-IR) spectroscopy. 5 mg of CCP-1 was ground with 200 mg of dry potassium bromide powder and then pressed into 1 mm pellets for infrared measurement, which was recorded in the frequency range of 4000-400 cm-1 using an infrared spectrometer (Nicolet 6700).

[0060] Figure 1 The components of the four CCPs obtained (CCP-W, CCP-0.2M, CCP-0.5M and CCP-1.0M) are shown. CCP-W, as the main CCP component, was purified by Superdex TM 75 column for further purification and freeze-dried to obtain polysaccharide CCP-1. Figure 2 As shown in A, the HPGPC chromatogram shows that CCP-1 exhibits a single and symmetrical peak, indicating that it is uniformly divided into polysaccharides. According to the dextran standard curve equation (logMw = -0.2006x + 12.618, R 2 =0.9996) and its average molecular weight is about 2.92×10 4 Da. like Figure 2 As shown in B and C, CCP-1 is mainly composed of galactose, mannose and glucose with a molar ratio of 68.125:43.875:12. The FT-IR spectrum of CCP-1 is shown in Figure 2 D, at 3388cm -1 The absorption peak at 2927cm is caused by the stretching vibration of OH in the sugar residue, which is the characteristic peak of sugar. -1 The weaker absorption peak at 1652 cm -1 The absorption peak at 1413 cm represents the stretching vibration of the C=O bond. -1 The absorption peak at 1216 cm -1 and 1049cm -1 The absorption peak at is due to the angular vibration of OH.

[0061] 2. Construction of immunocompromised mouse model

[0062] 48 SPF healthy male BALB / c mice (6-8 weeks old, weighing 18±2g) were purchased from Anhui Medical University. After one week of adaptive feeding, 40 mice were randomly selected and intraperitoneally injected with cyclophosphamide (80mg / kg, dissolved in 0.9% saline) once a day for 3 consecutive days, and the other 8 mice were intraperitoneally injected with the same dose of saline (0.9%) as the control group (Con). Subsequently, these 40 mice were randomly divided into 5 groups: CTX model group, positive control group, CCP-1 low-dose group, CCP-1 medium-dose group, and CCP-1 high-dose group. CTX model group: continued to be gavaged with saline (0.9%) once a day for a total of 12 times. Positive control group: continued to be gavaged with levamisole hydrochloride (LMH, 40mg / kg·d) once a day for a total of 12 times. CCP-1 low-dose group: continued to be gavaged with CCP-1 solution (50mg / kg·d) once a day for a total of 12 times. CCP-1 medium-dose group: continued oral administration of CCP-1 solution (100 mg / kg·d), once a day, for a total of 12 times. CCP-1 high-dose group: continued oral administration of CCP-1 solution (200 mg / kg·d), once a day, for a total of 12 times. Control group: continued oral administration of normal saline (0.9%), once a day, for a total of 12 times. After the experiment, all mice were fasted for one day and then killed under chloral hydrate anesthesia (10%, 0.35 mL / 100 g).

[0063] 3. Testis / epididymis index measurement

[0064] Before killing the mice, blood samples were collected from the eye sockets, centrifuged at 2000 rpm for 20 min, and serum was obtained and stored at -80°C for biochemical analysis. The testicles of the mice were collected and weighed, and the organ indexes of the testicles and epididymis were calculated.

[0065] Testis / epididymis index = (testis / epididymis mass (mg)) / body mass (g) × 100%

[0066] like Figure 3 As shown in the figure, compared with the control group, the organ indexes of the testis and epididymis in the CTX model group were significantly decreased, indicating that CTX induced a decrease in the relative weight of the testis and epididymis. However, compared with the CTX model group, the organ indexes of the testis and epididymis increased after CCP-1 treatment. The above results show that CCP-1 intervention reversed the atrophy of reproductive organs such as the testis and epididymis induced by CTX.

[0067] 4. Analysis of testicular and serum biochemical indicators

[0068] 0.2 g of testicular tissue was homogenized in each group and 10% homogenate was generated using pre-frozen physiological saline. The levels of antioxidant parameters, including superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-pX) level, and malondialdehyde (MDA) content, were determined using commercial test kits. The levels of steroids such as FSH, LH, and T in serum samples were determined using ELISA kits.

[0069] like Figure 4 As shown in the results, compared with the control group, the levels of SOD and GSH-Px in the testicular tissue of mice in the CTX model group were decreased, while the content of MDA was increased. However, after CCP-1 intervention, the levels of SOD and GSH-Px increased in a dose-dependent manner, and the content of MDA in the testicular tissue decreased, almost reaching a level similar to that of LMH. The results indicate that CCP-1 can improve the antioxidant activity of testicular tissue. Compared with the control group, the levels of serum hormones such as FSH, LH, and T in mice in the CTX model group were significantly decreased. Compared with the CTX group, LMH and CCP-1 intervention significantly increased the levels of these three serum hormones, and in a concentration-dependent manner in the CCP-1 treatment group.

[0070] 5. Sperm quality evaluation

[0071] One testis was fixed with 4% paraformaldehyde, and the other testis was stored at -80°C to prepare tissue homogenate for biochemical parameter determination. The bilateral epididymis of the mouse was placed in 200 μL human tubal fluid (HTF) in vitro fertilization medium and cut into small pieces with micro scissors. Then, the epididymal tissue was gently squeezed with eye forceps to release sperm into the culture medium. After removing the left epididymal tissue, the released sperm was incubated in an incubator at 37°C for 30 minutes to prepare sperm suspension, and then the sperm quality level was evaluated.

[0072] Sperm count: Dilute the sperm suspension 20 times and mix thoroughly. Then, add 10 μL of the suspension into a hemocytometer and count using the hemocytometer.

[0073] Sperm deformity rate: 10 μL of sperm suspension was placed on a glass slide and stained with 2% eosin solution. One hour later, the sperm morphology was observed under a microscope at ×400 magnification and the sperm deformity rate was calculated.

[0074] Sperm mortality: 500 μl of sperm suspension was centrifuged (1000 rpm, 5 min) to a sperm concentration of 1-6×10 6 ml -1 Subsequently, the sperm were suspended in 100 μL 1× binding buffer containing 6 μL Annexin V-FITC and 5 μL PI staining solution and incubated in the dark at room temperature for 10 min. Then 400 μL 1× binding buffer was added and sperm mortality was analyzed and evaluated using a FACScan flow cytometer within 1 hour.

[0075] like Figure 5 As shown in A, the lower picture shows the microscopic appearance of sperm morphology, and the upper picture shows the sperm morphology stained with eosin. Figure 5 In BC, the sperm count of mice in the CTX model group was reduced, and abnormal sperm including headless sperm and short-tailed sperm increased. However, LMH treatment reversed these phenomena. Abnormal sperm were almost not seen in the HCCP-1 group, and the sperm count was significantly increased. Sperm mortality was assessed by flow cytometry ( Figure 5 D) It was found that the sperm mortality rate in the control group was 1.30%, while that in the CTX model group increased to 32.2%. After CCP-1 intervention, the sperm mortality rate was reduced to 9.43%, which was comparable to that in the LMH group. These results indicate that CCP-1 intervention can effectively improve CTX-induced sperm deformity and increase sperm survival, thereby protecting male mice from reproductive damage.

[0076] VI. Histopathological Analysis

[0077] The hypothalamus, kidneys and right testis were fixed with 10% neutral buffer formalin, then dehydrated and embedded in paraffin. The tissue was then cut into 5 μm thick cross sections and stained with hematoxylin-eosin (HE). Paraffin-embedded hypothalamic coronal sections were processed into 5 μm thick tissue sections and Nissl stained. The histopathological changes of the hypothalamus, kidneys and right testis were observed under an electron microscope.

[0078] VII. Analysis of spermatogenesis kinetics

[0079] Eight HE-stained sections were randomly selected from each group for testicular spermatogenesis dynamics analysis. The seminiferous tubules were observed under a microscope, and the number of spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids in each tubule was determined.

[0080] 8. Measuring interstitial cell volume

[0081] HE-stained testicular tissue sections were randomly selected, and the cell diameter and nuclear diameter were measured using the Image J system, and then the volumes of interstitial cells and nuclei were calculated.

[0082] like Figure 6 As shown in A, the structure of the seminiferous tubules of the control group mice was intact, the sperm cells were neatly arranged in layers, and there were many spermatids and mature sperm in the middle cavity. The basement membrane of the seminiferous tubules of the testicular mice in the CTX model group was ruptured, accompanied by vacuolation of the spermatogenic epithelium, a decrease in the number of spermatogenic cells, disordered arrangement, and cavities in the tubular lumen, indicating obvious testicular damage. LMH intervention improved the integrity of the basement membrane of the seminiferous tubules, but did not significantly increase the number of spermatids. However, CCP-1 alleviated the damage to the seminiferous tubules by increasing the number and orderly arrangement of spermatogenic cells and reducing the cavities in the tubular lumen. The improvement effect of high-dose CCP-1 was particularly obvious. Figure 6 As shown in B, the renal sections of the control group showed intact basement membrane, intact tubular morphology, and normal distribution of mesangial cells. In contrast, the renal tubular basement membrane of mice in the CTX model group was widened and thickened, the renal corpuscles were swollen and deformed, and the mesangial cells were proliferative and cytoplasmic vacuolated. After treatment with LMH and CCP-1, the morphological changes of the kidneys were significantly improved. In particular, supplementation with a high dose of CCP-1 significantly improved the swelling and deformation of the kidneys of male mice and alleviated kidney damage.

[0083] like Fig. 7A and Figure 7B As shown, spermatogonia, spermatocytes, and spermatids are all present in the seminiferous tubules of normal mice. Spermatogonia (indicated by the red arrow) are oval in shape near the basement membrane. Primary spermatocytes (indicated by the green arrow) are located near the luminal side of spermatogonia, are larger and appear dark purple. Secondary spermatocytes (indicated by the yellow arrow) are located on the distal luminal side of spermatogonia and are smaller than primary spermatocytes. Spermatids (indicated by the white arrow) are shaped like tadpoles and are located in the middle of the lumen. In the CTX model group, these cells were in a disordered state, their number was significantly reduced, and a cavity was formed in the middle of the lumen. LMH treatment increased the number of cell populations in the seminiferous tubules to a certain extent, but caused the cells to be loosely arranged and there were holes at the junction of the seminiferous tubules and the basement membrane. In the CCP-1 intervention group, a large number of neatly arranged sperm were found in the center of the lumen, among which the effect of HCCP-1 was the most significant. In addition, from Figure 7C-Figure 7E The measurement results in the study showed that, according to statistical studies, the diameter and volume of the interstitial cells and nuclei of the testicular seminiferous tubules in the CTX model group were significantly reduced compared with the control group. However, after LMH or CCP-1 intervention treatment, the diameter and volume were observed to increase significantly, and HCCP-1 had the most significant improvement effect.

[0084] IX. Immunohistochemical Analysis

[0085] Immunohistochemistry was used to detect the expression of SCF, C-Kit, Bcl-2, Bax and Caspase-3 in testicular tissue, and the expression of GnRH in the hypothalamus. The fixed testicular and hypothalamic tissues were embedded and sliced, then removed with xylene, hydrated with graded ethanol (95%, 85%, 75%) for 10 minutes, and then rinsed with distilled water. The sections were treated with 3% hydrogen peroxide solution for 25 minutes at room temperature to block endogenous peroxidase activity, and then treated with peroxidase blocker for another 10 minutes and steamed in citrate buffer for 3 minutes. The sections were then incubated with goat serum for 5 minutes and incubated with primary antibodies overnight at 4°C. After washing with PBS for 1 hour, they were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 1 hour. After washing twice in PBS, the sections were incubated with 3,3'-diaminobenzidine (DAB) for 8 minutes for color development, rinsed with distilled water to stop the color development, counterstained with hematoxylin, and dehydrated and mounted. Finally, images were captured using an IX73 Olympus microscope equipped with a DP80 camera.

[0086] like Fig. 8A As shown in the figure, in the control group, the size and morphology of the hypothalamic neurons were normal, and the nuclear structure was clear and complete. CTX induction caused obvious vacuolation of the neurons, accompanied by cytoplasmic swelling and nuclear condensation to a certain extent, as shown by the red arrows. However, after treatment with LMH and CCP-1, the number of vacuolated cells decreased significantly, the number of normal cells increased significantly, and the degree of nuclear condensation decreased significantly. As shown in Figures 8B and Figure 8C As shown, Nissl staining shows blue-purple Nissl bodies, which represent neurons or nerve cells in the hypothalamus. Compared with normal mice, the number of neurons in the lateral hypothalamic (LH) and arcuate nucleus (Arc) of CTX-induced mice was significantly reduced, while LMH and CCP-1 treatment reversed this trend. Notably, CCP-1 intervention led to a dose-dependent increase in the number of neuronal cells. These results indicate that CCP-1 has a neuroprotective effect on CTX-induced hypothalamic damage in male mice.

[0087] like Fig.8D and Fig. 8E As shown in the figure, GnRH is mainly located in the arcuate nucleus and is widely distributed. Compared with the control group, the expression of GnRH in the arcuate nucleus secreted by the hypothalamus of mice in the CTX model group was significantly reduced. However, the intervention of CCP-1 significantly increased the expression of GnRH in a dose-dependent manner, among which HCCP-1 had the most significant effect. Compared with the CTX model group, the secretion of FSH and LH in the anterior pituitary and the secretion of testicular T in the CCP-1 intervention group further supported this finding. The results show that CCP-1 protects the spermatogenesis of male mice from CTX-induced damage by regulating the expression of GnRH.

[0088] 10. Immunoblotting

[0089] The expression of SCF, C-Kit, Bcl-2, Bax, and Caspase-3 in testicular tissue was detected by immunoblotting. Testicular tissue was extracted using 800 μL of radioimmunoprecipitation (RIPA) lysis buffer containing 1 mmol / L phenylmethylsulfonyl fluoride, and then the protein content was assessed using a bicinchoninic acid (BCA) detection kit. 50 μg of protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed three times with three buffered saline containing 0.1% Tween-20 (TBST) and then detected with the corresponding secondary antibodies at room temperature for 40 min. Protein bands were visualized using enhanced chemiluminescence detection reagents and subsequently scanned by autoradiography. The optical density of the relevant bands was determined using Labworks gel imaging and software (Labworks LLC, Lehi, USA).

[0090] like Fig. 9 A- Fig. 9 As shown in B, compared with the control group, the expression of reproductive regulatory factors such as SCF and C-Kit decreased in the CTX-induced group, and the intervention of CCP-1 gradually increased their expression in a dose-dependent manner. Compared with the control group, the expression of Bax and cleaved-caspase-3 increased after CTX treatment, while the expression of the anti-apoptotic protein Bcl-2 decreased. However, after CCP-1 intervention, the expression of Bax and cleaved-caspase-3 decreased significantly, while the level of Bcl-2 increased. In addition, as Fig. 9 As shown in C, western blot analysis was consistent with the above immunohistochemistry results. The results showed that CCP-1 played a protective role in the reproductive system of male mice by activating reproductive regulatory pathways such as the SCF / C-Kit pathway and inhibiting apoptotic signaling pathways such as the Bax / Caspase-3 pathway.

[0091] XI. Establishment of GC-1spg cell apoptosis model

[0092] Mouse spermatogonia—GC-1spg cells were selected and placed in high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in a CO2 incubator at 37°C, 5% carbon dioxide, and 95% air. After the culture was completed, GC-1spg cells (1×10 4Cells / well) were seeded in 96-well plates at 37°C overnight and cultured in serum-free fresh medium with different concentrations of CCP-1 (0, 25, 50, 100, 200, 400, 800 μg / mL) for 24 hours. After that, the culture medium was removed, 20 μL MTT (5 mg / mL, 20 μL) was added to each well, incubated for 4 hours, and 100 μL of dimethyl sulfoxide (DMSO) solution was added after removing the supernatant. The cells were shaken in the dark for 10 minutes, and the absorbance at 570 nm was measured by a spectrophotometer. The absorbance value was positively correlated with cell viability, so as to select the optimal CCP-1 treatment concentration. Subsequently, GC-1spg cells were treated with apoptosis-inducing agents (TS, TNF-α+SM-164). The optimal concentration and induction time of apoptosis inducers were determined by the above-mentioned MTT method.

[0093] The MTT method was used to detect the effect of CCP-1 on the viability of GC-1spg cells. Fig.10 As shown in A, when the concentration of CCP-1 was lower than 400 μg / mL, the cell viability of the CCP-1-treated group was higher than that of the control group. When the concentration of CCP-1 was between 0 and 100 μg / mL, the viability of GC-1spg cells increased, but when the concentration of CCP-1 exceeded 100 μg / mL, the cell viability gradually decreased. It can be seen that when the concentration of CCP-1 was 100 μg / mL, the cell viability of GC-1spg cells reached its maximum value. Therefore, the final concentration of CCP-1 in subsequent experiments was 100 μg / mL, and the culture time was 24 hours. The MTT method was used to detect the viability of GC-1spg cells induced by the apoptosis reagent TS. The changes in cell viability after treatment with apoptosis inducers at different dilution rates (0 hours, 4 hours, 8 hours and 12 hours) were observed. As shown Fig.10 As shown in B, when the dilution rate of apoptosis inducer TS was 1:1000, after inducing cells for 4 hours, cell viability dropped to 33%, indicating that the cell apoptosis rate reached its peak at this time. Therefore, in subsequent experiments, apoptosis inducer TS with a dilution rate of 1:1000 was selected to treat cells for 4 hours.

[0094] 12. Effect of CCP-1 on apoptosis of GC-1spg cells

[0095] The apoptosis model was established by treating GC-1spg cells with the optimal concentration of apoptosis inducers, and then incubating GC-1spg cells with the optimal concentration of CCP-1 for 24 hours. Finally, the AO / PI double staining method was used to visually evaluate the improvement effect of CCP-1 on GC-1spg cell apoptosis. 5The cells were placed in a confocal culture dish and cultured for 12 hours. The cells were first cultured in a serum-free medium containing an apoptosis inducer for 4 hours, and the supernatant was discarded. The cells were then cultured in a serum-free medium containing CCP-1 for 24 hours, and the supernatant was discarded. 5 μL of acridine orange (AO) dye and 10 μL of propidium iodide (PI) dye were added, and double-stained in the dark for 20 minutes. The cells were observed using a confocal microscope (LSCM) at an excitation wavelength of 488 and an emission wavelength of 630 at a magnification of 100 times.

[0096] To further investigate whether CCP-1 can inhibit apoptosis of GC-1spg cells, GC-1spg cells were treated with apoptosis inducer TS at a dilution ratio of 1:1000 for 4 hours, and then incubated with 100 μg / mL CCP-1 for 24 hours. Fig.10 As shown in C, after treatment with apoptosis inducers, the number of round GC-1spg cells with green fluorescence (representing living cells) decreased significantly, while the number of GC-1spg cells with red fluorescence (representing apoptotic cells) increased significantly. MTT quantitative experiments further showed that the cell survival rate decreased to 25% and the cell apoptosis rate increased to 75%. However, after CCP-1 treated the cells, the survival rate of GC-1spg cells increased to 95% and the cell apoptosis rate decreased to 5%. The results show that CCP-1 can effectively alleviate the apoptosis of GC-1spg cells induced by the apoptosis inducer TS.

[0097] 13. Effects of CCP-1 on the expression of apoptosis and reproductive regulatory factors

[0098] Immunofluorescence was used to detect the expression of SCF, C-Kit, Bcl-2, Bax and Cleaved-caspase-3 in GC-1spg cells. The method was as follows: GC-1spg cells (1×10 5 After culturing 12 hours of cells / well in a 6-well cell slide, the culture medium was removed, the cells were washed twice with phosphate buffer, and then fixed with paraformaldehyde (4%, 500μL) for 2 hours, and the cells were blocked with 3% BSA at room temperature for 30 minutes. Then 500μL of a mixed solution of 0.3% TritonX-100 and 1% serum blocking solution was added and incubated for 30 minutes. The primary antibody prepared in a certain proportion was added to the cell well plate and incubated overnight at 4°C. After shaking and washing on a decolorizing shaker for 3 times, the secondary antibody was added to cover the cells and incubated at room temperature for 50 minutes. After shaking and washing on a shaker for 3 times, DAPI staining solution was added and incubated at room temperature for 30 minutes away from light. After washing 3 times, the slide was sealed with an anti-fluorescence quenching sealing agent, and the image was observed and collected under a fluorescence microscope.

[0099] After successfully establishing the GC-1spg cell apoptosis model, the effect of CCP-1 on the expression of related proteins was further studied. As shown in Figure 11, compared with the control group, after treatment with apoptosis inducers, the fluorescence intensity of Bax and Cleaved-caspase-3 increased, while the fluorescence intensity of SCF, C-Kit and Bcl-2 decreased. However, after treatment with CCP-1, the fluorescence intensity of Bax and Cleaved-caspase-3 was significantly reduced, while the fluorescence intensity of SCF, C-kit and Bcl-2 was enhanced. These results indicate that CCP-1 can inhibit the apoptosis of GC-1spg cells by downregulating the expression of pro-apoptotic proteins such as Bax and Cleaved-caspase-3 and upregulating the expression of reproductive regulatory factors such as Bcl-2, SCF and C-Kit.

[0100] 14. Characterization and performance of CCP-2

[0101] 1. Characterization of CCP-2

[0102] Figure 1 The four CCP components obtained are shown (CCP-W, CCP-0.2M, CCP-0.5M and CCP-1.0M). The CCP-0.2M component was taken and the TM 75 column for further purification and freeze-dried to obtain polysaccharide CCP-2. Fig.12 As shown in A, the HPGPC chromatogram shows that CCP-2 exhibits a single and symmetrical peak, indicating that it is uniformly divided into polysaccharides. According to the standard curve equation of dextran (logMw = -0.1805x + 11.673, R 2 =0.9997) and its average molecular weight is about 2.12×10 4 Da. like Fig.12 As shown in B and C, CCP-2 is mainly composed of galactose, mannose and glucose with a molar ratio of 23.52:15:1. The FT-IR spectrum of CCP-2 is shown in Fig.12 D, at 3416cm -1 The absorption peak at 2934cm is caused by the stretching vibration of OH in the sugar residue, which is the characteristic peak of sugar. -1 The weaker absorption peak at 1652 cm is attributed to the CH stretching vibration of the methine (-CH2-) in sugar. -1 The absorption peak at 1055 cm represents the stretching vibration of the C=O bond. -1 The absorption is characteristic of sugar ring.

[0103] 2. Effects of CCP-2 on organ indexes of testis and epididymis

[0104] As above, CTX was used to induce male mice to establish a reproductive injury mouse model, and then CCP-2 was used for intervention. Fig.13 As shown in the figure, compared with the control group, the organ indexes of the testis and epididymis in the CTX model group were significantly decreased, indicating that CTX induced a decrease in the relative weight of the testis and epididymis. However, compared with the CTX model group, the organ indexes of the testis and epididymis did not change significantly after CCP-2 treatment. The above results show that CCP-2 has no improvement effect on CTX-induced atrophy of mouse reproductive organs such as the testis and epididymis.

[0105] 3. Effects of CCP-2 on serum hormone levels

[0106] exist Fig.14 As shown in the figure, compared with the control group, the levels of serum hormones such as FSH, LH and T in the CTX model group mice were significantly reduced. Compared with the CTX group, there was no significant change in serum hormone levels after CCP-2 intervention, indicating that CCP-2 could not improve the decrease of male hormones in mice caused by CTX.

[0107] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A use of Cordyceps sinensis polysaccharide in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: The molecular weight of the Cordyceps sinensis polysaccharide is 2.92×10 4 Da, composed of galactose, mannose and glucose with a molar ratio of 68.125:43.875:

12.

2. The use of a Cordyceps sinensis polysaccharide as claimed in claim 1 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: The method for purifying Cordyceps sinensis polysaccharide comprises the following steps: S1, dissolving the crude polysaccharide of Cordyceps sinensis in pure water and filtering it through a microporous membrane filter, and then loading the filtrate onto a DEAE agarose FF anion exchange chromatography column for purification; S2, eluting the chromatographic column with ultrapure water and sodium chloride solutions of different concentrations, collecting the components, tracking with the phenol-sulfuric acid method, and detecting with an ELISA reader at 490 nm; S3, the largest fraction of the eluate was collected, dialyzed, and then freeze-dried at -60 °C using Superdex TM The purified product was further purified by 75% chromatography, and the eluate was collected and combined, and then freeze-dried to obtain the purified Cordyceps sinensis polysaccharide.

3. The use of a Cordyceps sinensis polysaccharide as claimed in claim 2 in the preparation of a drug for intervening in male spermatogenesis disorders and reproductive damage, characterized in that: In step S1, the method for preparing crude polysaccharide of Cordyceps sinensis comprises the following steps: S11, crushing the fruiting bodies of Cordyceps sinensis and sieving to obtain Cordyceps sinensis fruiting body powder; S12, adding distilled water at a solid-liquid ratio of 1:6-10 for ultrasonic extraction, then placing in a water bath at 80-90° C. for 1.5-2.5 h, filtering and collecting the supernatant multiple times and combining them; S13, subjecting the supernatant to ultra-high pressure treatment at a pressure of 400-450 MPa, a ultra-high pressure treatment time of 10-15 min, and maintaining the temperature at 20° C.; S14, concentrating the treated supernatant and adding ethanol, collecting the precipitate by centrifugation, and adding water to dissolve the precipitate to obtain a crude polysaccharide aqueous solution; S15, adding the same volume of Sevag reagent to the crude polysaccharide aqueous solution, magnetically stirring and then centrifuging to collect the supernatant; repeating until the protein is completely removed, then concentrating the polysaccharide solution to remove small molecules, dialyzing with flowing water using a dialysis bag, concentrating by rotary evaporation to a thick liquid, and vacuum freeze-drying to obtain crude polysaccharides from Cordyceps sinensis fruiting bodies, and the volume ratio of chloroform and n-butanol in the crude polysaccharide aqueous solution and the Sevag reagent polysaccharide aqueous solution is 5:4:

1.

4. The use of a Cordyceps sinensis polysaccharide as claimed in claim 3 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In the step S11, the fruiting bodies of Cordyceps militaris are crushed and then passed through a 60-mesh sieve.

5. The use of a Cordyceps sinensis polysaccharide as claimed in claim 3 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In step S15, the molecular weight cut-off of the dialysis bag is 3500 Da.

6. The use of a Cordyceps sinensis polysaccharide according to claim 2 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In the step S1, the pore size of the microporous membrane filter is 0.45 μm.

7. The use of a Cordyceps sinensis polysaccharide according to claim 2 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In step S2, the concentration of the sodium chloride solution is 0.2-1 mol / L, and the flow rate is 1.0 mL / min.

8. The use of a Cordyceps sinensis polysaccharide as claimed in claim 2 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In step S2, the concentration of the sodium chloride solution is 1 mol / L, and the flow rate is 1.0 mL / min.

9. The use of a Cordyceps sinensis polysaccharide as claimed in claim 2 in the preparation of a drug for intervening in male spermatogenesis disorder and reproductive damage, characterized in that: In step S3, Superdex is used TM When further purified by chromatography at 75 °C, the flow rate was 0.5 mL / min.

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