Application of tremella aurantialba polysaccharide in preparation of medicine for treating male immunological dyszoospermia

Abstract

The invention discloses application of tremella aurantialba polysaccharide in preparation of a medicine for treating male immunological dyszoospermia, and belongs to the field of biological medicine. The invention provides a new application of tremella aurantialba polysaccharide, and experiments prove that the tremella aurantialba polysaccharide can improve dyszoospermia induced by cyclophosphamide, significantly increase the number of sperms of mice, reduce the abnormal rate of sperms and promote secretion of testosterone. According to the invention, cyclophosphamide is adopted to construct a male mouse dyszoospermia model, tremella aurantialba polysaccharides extracted in different manners are subjected to intragastric administration, the action mechanism of NAPs for improving mouse dyszoospermia is researched, the mechanism of NAPs for treating spermatogenesis is disclosed, and a basis is provided for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Tremella fuciformis polysaccharide in the preparation of a medicine for treating male immune spermatogenesis disorder. Background Art

[0002] Among male infertility, spermatogenesis disorders are a major cause. The spermatogenesis process involves spermatogonia mitosis, spermatocyte meiosis, and spermatid deformation into tadpole-shaped sperm. Defects in any of these stages can lead to male infertility. Clinically, spermatogenesis disorders primarily manifest as azoospermia, oligospermia, teratozoospermia, and / or asthenozoospermia, with azoospermia being the most severe. In recent years, the incidence of male spermatogenesis disorders has shown a significant upward trend, becoming a significant issue in the global reproductive health field. Male infertility accounts for 40%-50% of infertility in couples of reproductive age. Globally, approximately 10%-15% of couples of reproductive age experience infertility, with male factors accounting for nearly half of these cases. Asthenozoospermia (sperm progressive motility rate <32%) accounts for 60%-70% of male infertility cases, making it the most common type of spermatogenesis disorder.

[0003] Cyclophosphamide (CTX) has a modulatory effect on the immune system, inhibiting the proliferation and function of immune cells, including T and B cells, thereby suppressing the body's immune response. At the cellular level, upon entry into the body, its active metabolites directly attack the spermatogenic epithelial cells within the testicles, interfering with the normal division and differentiation of spermatogonia. This leads to a blockage of key steps in spermatogenesis, significantly reducing sperm count and even causing azoospermia. Furthermore, it damages sperm genetic material, significantly increasing sperm abnormalities and impairing fertilization. From an endocrine perspective, cyclophosphamide can interfere with the normal function of the hypothalamic-pituitary-gonadal axis, disrupting the secretion of gonadotropin-releasing hormone (GnRH) and leading to an imbalance in the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary. This ultimately reduces the ability of Leydig cells to synthesize and secrete testosterone. Reduced testosterone levels not only affect male secondary sexual characteristics but also alter the microenvironment for sperm maturation, further impairing reproductive function. Furthermore, cyclophosphamide can directly impact semen quality, reducing sperm motility and forward motility, making it difficult for sperm to swim smoothly within the female reproductive tract and fertilize the egg. Long-term or high-dose use of the drug can also cause testicular atrophy, permanently damaging the structure and function of the seminiferous tubules and resulting in irreversible damage to reproductive function. Furthermore, the combined effects of low testosterone levels and psychological stress can cause some men to experience sexual dysfunction, including erectile dysfunction and decreased libido. Overall, cyclophosphamide's impact on male reproductive function is multifaceted and long-term, posing a serious threat to patients' fertility and reproductive health.

[0004] Tremella fuciformis polysaccharide is the main active ingredient of Tremella fuciformis, and has antioxidant, anti-inflammatory, lipid-lowering, and anti-thrombotic effects. Existing technology reports that Tremella fuciformis polysaccharides obtained from different origins and methods can enhance the phagocytic function of mouse peritoneal macrophages and the production of mouse antibodies. Tremella fuciformis polysaccharide has an immune-protective effect on cyclophosphamide-induced immunosuppression in mice, can promote mouse diet, and effectively increase the number of white blood cells and lymphocytes in the mouse blood. However, there are no reports on the effect of Tremella fuciformis polysaccharide on alleviating cyclophosphamide-induced spermatogenesis disorders in male mice. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of Tremella fuciformis polysaccharide in preparing a medicine for treating male immune spermatogenesis disorder.

[0006] The present invention first provides an application of Tremella fuciformis polysaccharide in preparing a medicine for treating male infertility.

[0007] In the above application, the infertility is oligospermia, azoospermia or spermatogenic dysfunction caused by immunosuppression.

[0008] Specifically, the infertility is oligospermia, azoospermia or spermatogenic dysfunction caused by cyclophosphamide.

[0009] The present invention further provides an application of Tremella fuciformis polysaccharide in the preparation of any of the following medicines: (1) Drugs that protect testicular and / or epididymal tissues; (2) Drugs that improve sperm quality; (3) Drugs to increase sperm count; (4) Drugs that reduce sperm deformity rates; (5) Drugs that increase testosterone levels; (6) Drugs that improve testicular function.

[0010] In the above applications, the diseased subject is a human or a male mouse.

[0011] In the above application, the Tremella fuciformis polysaccharide is water-insoluble Tremella fuciformis polysaccharide.

[0012] Specifically, the method for extracting Tremella fuciformis polysaccharide comprises the following steps: (1) adjusting the water content and pH value of the deep eutectic solvent to obtain DES1; the pH value of the DES1 is 11-13; The deep eutectic solvent is composed of n-octanoic acid and sodium octanoate; the molar ratio of n-octanoic acid to sodium octanoate is 2-3:1; (2) mixing Tremella fuciformis and the DES1, heating and extracting the mixture to obtain a precipitate 1 and a supernatant; adding anhydrous ethanol to the supernatant to obtain a precipitate that is a water-soluble polysaccharide; (3) adjusting the water content and pH value of the deep eutectic solvent to obtain DES2; the pH value of DES2 is 1-3; (4) The precipitate 1 obtained in step (2) and the DES2 are mixed, heated for extraction, and the supernatant is obtained; anhydrous ethanol is added to the supernatant, and the resulting precipitate is a water-insoluble polysaccharide, namely, the Tremella fuciformis polysaccharide.

[0013] In the above application, the molar ratio of n-octanoic acid to sodium octanoate is 2:1.

[0014] In the above application, in step (1), the volume percentage water content of the DES1 is 82%-98%; preferably 84%-88%; In step (3), the volume percentage water content of the DES2 is 10%-90%, preferably 20%-40%.

[0015] In the above application, in step (2), the material-liquid ratio of the edible fungus and DES1 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h; preferably 1.75-2.25 h; In step (4), the material-liquid ratio of the precipitate 1 and DES2 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h, preferably 1.75-2.25 h.

[0016] In the above application, in steps (2) and (4), the volume ratio of the supernatant to anhydrous ethanol is 1:4-5.

[0017] In the above application, step (2) further comprises the steps of re-dissolving the precipitate obtained after adding anhydrous ethanol with water and freeze-drying; Step (4) also includes a step of freeze-drying the precipitate obtained after adding anhydrous ethanol.

[0018] The present invention has the following advantages: (1) The yield of NAP-DES2 is high, the process is time-saving and the operation is simple.

[0019] (2) NAP-DES2 significantly improved cyclophosphamide-induced spermatogenesis disorders, significantly increased the sperm count of mice, reduced the sperm deformity rate, and promoted the secretion of testosterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1is the scavenging ability of NAPs on hydroxyl radicals.

[0021] Figure 2 Scanning electron microscope images of NAPs.

[0022] Figure 3 The effects of NAPs on the testicular index and epididymal index of mice.

[0023] Figure 4 Effects of NAPs on sperm density in mice.

[0024] Figure 5 The effect of NAPs on mouse sperm morphology (red arrows indicate abnormal sperm).

[0025] Figure 6 Effects of NAPs on serum testosterone levels in mice.

[0026] Figure 7 These are testicular pathological sections (100×, 200×).

[0027] Figure 8 The effects of NAPs on the expression of related genes in mouse testicular tissue. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0030] The quantitative tests in the following examples were performed in triplicate unless otherwise specified, and the results were averaged.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1: Extraction and physicochemical property determination of Tremella fuciformis polysaccharide 1. Preparation of NAP-DES1 Tremella fuciformis polysaccharide and NAP-DES2 Tremella fuciformis polysaccharide Octanoic acid, sodium octanoate according to n(C8H 16 O2):n(C8H 15 NaO2) = 2:1 and then stirred continuously at 80 ° C until a transparent and stable liquid is formed, which is named deep eutectic solvent (DES); the obtained DES is stored in a dry and cool place for future use.

[0033] An appropriate amount of Tremella fuciformis (NA) was dried and pulverized, passed through a 100-mesh sieve, and the powder was collected for later use. The pH of DES was adjusted to 12 using a NaOH solution, and distilled water was added to prepare DES1 with a water content of 84% (volume percentage) for later use. 1 g of NA was added to DES1 at a solid-liquid ratio of 1:47.5 (g:mL). The mixture was heated in a 100°C water bath for 2.25 hours, followed by centrifugation at 8500 rpm for 10 minutes. The supernatant was collected and designated as Precipitate 1. Anhydrous ethanol was added to the supernatant at a V (supernatant) to V (anhydrous ethanol) ratio of 1:4 (volume ratio), allowed to stand for 12 hours, and then centrifuged. The precipitate was reconstituted with distilled water and freeze-dried to obtain water-soluble Tremella fuciformis polysaccharide (NAP-S). After alcohol precipitation, the supernatant was rotary evaporated to collect DES, which was then adjusted to pH 2 with 3 mol / L HCl solution. Distilled water was then added to prepare DES2 with a water content of 40%. DES2 was then added at a solid-liquid ratio of 1:42.5 (g:mL). Extraction was performed in a 100°C waterbath for 1.75 h, followed by centrifugation at 8500 rpm for 10 min. The supernatant was then added with anhydrous ethanol at a volume ratio of V (supernatant): V (anhydrous ethanol) of 1:4. The supernatant was allowed to stand for 12 h before centrifugation. The precipitate was then freeze-dried to obtain water-insoluble Tremella fuciformis polysaccharide (NAP-I). The purities of NAP-S and NAP-I polysaccharides extracted using this method were 72.86% and 64.38%, respectively.

[0034] 2. Water-extracted Tremella fuciformis polysaccharide (NAP-W) An appropriate amount of Tremella fuciformis (NA) was dried and pulverized, passed through a 100-mesh sieve, and the powder was collected for later use. 50 g of Tremella fuciformis powder was added to distilled water at a material-liquid ratio of 1:40 (g:mL), mixed thoroughly, and heated in a 100°C waterbath for 1.5 h. The mixture was then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was retained, and the precipitate was subjected to a second extraction using the same method. The two supernatants were combined and concentrated to 1 / 5 of the original volume by rotary evaporation, and the spun liquid was collected. 109.5 g of zinc acetate was added to 500 mL of distilled water to prepare a zinc acetate solution. 53 g of potassium ferrocyanide was added to 500 mL of distilled water to prepare a potassium ferrocyanide solution. The protein was removed at a ratio of V (zinc acetate solution): V (potassium ferrocyanide solution): V (spin solution) = 1:1:10. After 30 minutes, the solution was centrifuged at 10,000 r / min for 10 minutes, and the supernatant was retained. The supernatant was dialyzed using a 3500 Da dialysis bag until the conductivity was lower than 20 μS / cm (taking 48 h-72 h). Anhydrous ethanol was added at a volume ratio of V (dialysate): V (anhydrous ethanol) = 1:4, and the solution was precipitated at 4°C for 24 hours. After that, the precipitate was collected after centrifugation at 10,000 r / min for 10 minutes, reconstituted with distilled water, and freeze-dried to obtain water-extracted Tremella fuciformis polysaccharide (NAP-W).

[0035] 3. Acid-extracted Tremella fuciformis polysaccharide (NAP-A) An appropriate amount of Tremella fuciformis (NA) was dried and pulverized, passed through a 100-mesh sieve, and the powder was collected for later use. 50 g of NA powder was added to 0.1 mol / L citric acid at a material-liquid ratio of 1:40 (g:mL), mixed thoroughly, and heated in an 80°C water bath for 3 h. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was retained, and the precipitate was subjected to a second extraction using the same method. The two supernatants were combined, and the pH was adjusted to neutral with 0.1 mol / L NaOH solution. The supernatant was then concentrated to 1 / 5 of its original volume by rotary evaporation, and the spun liquid was collected. 109.5 g of zinc acetate was added to 500 mL of distilled water to prepare a zinc acetate solution. 53 g of potassium ferrocyanide was added to 500 mL of distilled water to prepare a potassium ferrocyanide solution. The protein was removed at a ratio of V (zinc acetate solution): V (potassium ferrocyanide solution): V (spin solution) = 1:1:10. After 30 min, the solution was centrifuged at 10,000 r / min for 10 min, and the supernatant was retained. The supernatant was dialyzed with a 3500 Da dialysis bag until the conductivity was lower than 20 μS / cm (taking 48 h-72 h). Anhydrous ethanol was added at a ratio of V (dialysate): V (anhydrous ethanol) = 1:4 (volume ratio), and the solution was allowed to stand at 4°C for 24 h. After that, the precipitate was collected after centrifugation at 10,000 r / min for 10 min, reconstituted with distilled water, and freeze-dried to obtain acid-extracted Tremella fuciformis polysaccharide (NAP-A). 4. Alkali Extraction of Tremella Polysaccharide (NAP-AL) An appropriate amount of Tremella fuciformis (NA) was dried and pulverized, passed through a 100-mesh sieve, and the powder was collected for later use. 50 g of NA powder was added to a 0.1 mol / L NaOH solution at a material-liquid ratio of 1:40 (g:mL), mixed and stirred, and then heated and leached in a 100°C waterbath for 1.5 h. The mixture was then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was retained, and the precipitate was subjected to a second leaching using the same method. The two supernatants were combined, the pH adjusted to neutral with 0.1 mol / L HCl solution, and the supernatant was concentrated to 1 / 5 of its original volume by rotary evaporation. The spun liquid was collected. 109.5 g of zinc acetate was added to 500 mL of distilled water to prepare a zinc acetate solution. 53 g of potassium ferrocyanide was added to 500 mL of distilled water to prepare a potassium ferrocyanide solution. The protein was removed at a ratio of V (zinc acetate solution): V (potassium ferrocyanide solution): V (spin solution) = 1:1:10. After 30 min, the solution was centrifuged at 10,000 r / min for 10 min, and the supernatant was retained. The supernatant was dialyzed using a 3500 Da dialysis bag until the conductivity was lower than 20 μS / cm (taking 48 h-72 h). Anhydrous ethanol was added at a volume ratio of V (dialysate): V (anhydrous ethanol) = 1:4, and the solution was precipitated at 4°C for 24 h. After that, the precipitate was collected after centrifugation at 10,000 r / min for 10 min, reconstituted with distilled water, and freeze-dried to obtain alkali-extracted Tremella fuciformis polysaccharide (NAP-AL).

[0036] 5. Comparison of Tremella fuciformis polysaccharides obtained by different extraction methods 5.1 Physical and chemical properties As shown in Table 1, NAP-W is composed of galactose, glucose, xylose, mannose, and glucuronic acid in a molar ratio of 0.020:0.074:0.241:0.614:0.051. NAP-AL polysaccharide is primarily composed of galactose, glucose, xylose, mannose, glucuronic acid, and glucosamine hydrochloride in the following molar ratios: 0.018:0.113:0.240:0.579:0.047:0.004. NAP-A polysaccharide, in addition to the monosaccharide composition of NAP-AL, also contains fucose and arabinose, with the molar ratios of these monosaccharides being 0.003, 0.002, 0.001, 0.018, 0.101, 0.252, 0.564, and 0.059, respectively. NAP-S is composed of galactose, glucose, xylose, mannose, glucuronic acid, glucosamine hydrochloride, arabinose, galactosamine hydrochloride, and rhamnose, with molar ratios of 0.005, 0.295, 0.116, 0.515, 0.056, 0.003, 0.002, 0.001, and 0.007. NAP-I polysaccharide, based on NAP-S, adds fucose, with the molar ratios of the monosaccharides being 0.025, 0.166, 0.235, 0.496, 0.063, 0.002, 0.005, 0.002, 0.002, and 0.004, respectively. This indicates that the extraction method has a major impact on the monosaccharide composition and ratio, with NAP-S and NAP-I having significantly higher monosaccharide compositions compared to the first three polysaccharides. In addition, the yields of NAP-S and NAP-I were 46.6% and 29.3%, respectively, which were significantly increased compared with the yields of the first three polysaccharides.

[0037] Table 1 Chemical composition of NAP extracted by different methods

[0038] 5.2 Hydroxyl Radical Scavenging Ability of NAPs Hydroxyl radicals are extremely aggressive free radicals that can easily cause oxidative damage to DNA and cell membranes.

[0039] The five Tremella fuciformis polysaccharides were tested for scavenging hydroxyl radicals. The specific method is as follows: Sample Preparation: Polysaccharides obtained by different extraction methods were prepared into aqueous solutions with the following concentration gradients (0.25, 0.5, 1, 2, and 4 mg / mL). Water-insoluble polysaccharides were prepared using 0.05 M NaCl solution. The procedure for the hydroxyl radical scavenging ability assay kit (see Table 2) was then strictly followed. Vortex mix thoroughly and incubate in a 37°C water bath for 60 min. Centrifuge at 10,000 rpm for 10 min at room temperature. 200 μL of the supernatant was collected in 1 mL micro-cuvettes and the absorbance at 536 nm was measured. These were designated as A blank, A control, and A assay, respectively.

[0040] Table 2 Operation steps for hydroxyl radical scavenging ability

[0041] The hydroxyl radical scavenging rate was calculated according to the formula: Hydroxyl free radical scavenging rate D% = (A determination - A control) ÷ (A blank - A control) × 100% like Figure 1 The five NAPs showed hydroxyl radical scavenging activity in the range of 0.25-4 mg / mL. At 4 mg / mL, the scavenging abilities of NAP-W, NAP-AL, NAP-A, NAP-S, and NAP-I were 20.34±0.24%, 17.16±0.11%, 20.86±0.23%, 12.84±0.12%, and 52.5±0.85%, respectively. NAP-I had the highest scavenging rate for •OH, while NAP-S had the lowest. P <0.05). The three methods of extracting Tremella fuciformis polysaccharides showed different antioxidant capacities. All NAPs had antioxidant capacity and could be used as sources of active ingredients.

[0042] 5.32.3 Scanning Electron Microscopy Scanning electron microscopy (SEM) is an effective method for observing the surface characteristics of materials. The surface morphology of Tremella fuciformis polysaccharides obtained by different extraction methods was studied using SEM ( Figure 2 ).Depend on Figure 2 As can be seen, the surface morphologies of NAPs obtained from different extraction media vary in shape and size. NAP-W exhibits a loose, flexible, and smooth flaky structure; NAP-AL exhibits a dense, uneven, and rough network structure, likely due to the stronger interactions between polysaccharide chains under alkaline conditions. NAP-A and NAP-I exhibit relatively dense and smooth flaky structures. NAP-S exhibits a rough, dispersed, fibrous structure.

[0043] Example 2 1. Materials and Methods 1.1 Materials Experimental animals: 6-week-old Balb / C male mice weighing 18-22 g were purchased from Suzhou Sibeifu Biotechnology Co., Ltd. Throughout the experiment, all laboratory animal testing standards and regulations of Shanxi Agricultural University were strictly adhered to. Bedding, animal feed, and drinking water all met SPF animal care standards.

[0044] Test materials: 5 types of Tremella fuciformis polysaccharides extracted from Example 1.

[0045] 1.2 Experimental Methods 1.2.1 Animal grouping and drug administration One hundred and twenty six-week-old male BALB / c mice, weighing 18-22 g, were randomly divided into eight groups, 15 in each: normal group (NC), model group (CY), water-extracted group (NAP-W), acid-extracted group (NAP-A), alkaline-extracted group (NAP-AL), soluble group (NAP-S), insoluble group (NAP-I), and positive group (LH). Mice were housed at 25 ± 2°C with a 12-h light cycle and fed a maintenance diet. After one week of adaptive feeding, multiple low-dose cyclophosphamide injections were administered to model the disease and to treat the disease. Mice in all other groups, except the normal group, received a daily intraperitoneal injection of 80 mg / kg cyclophosphamide on days 8, 9, and 10. From day 11 to day 20, they were gavaged once daily with various polysaccharides according to Table 3. On days 21 and 22, mice were again given a daily intraperitoneal injection of 80 mg / kg cyclophosphamide to assess their resistance to the cyclophosphamide-induced immune spermatogenesis model. Mice were fasted for 24 hours prior to the experiment. Specific groupings are shown in Table 3.

[0046] Table 3 Modeling information table

[0047] 1.2.2 Sample collection and processing After a 24-hour fast, mice were sacrificed by cervical dislocation and blood was collected from their eyeballs (i.e., on the 23rd day of the experiment). Blood was collected in 1.5 mL centrifuge tubes and centrifuged to obtain serum, which was then frozen at −80°C for subsequent analysis. The testicles and epididymis, covered by the fat layer under the lower abdomen of the mice, were removed and the fat removed. The testicles and epididymis were weighed, and the left testicle was fixed with 4% paraformaldehyde solution. The right testicle and epididymis were frozen in liquid nitrogen and stored at −80°C. The left epididymis was then removed for sperm analysis.

[0048] 1.2.3 Testicular and epididymal index determination Mice were sacrificed by cervical dislocation. The testicles and epididymis were removed, fat removed, rinsed in saline, and the water removed with absorbent paper. The testicular index and epididymal index were calculated using the following formula:

[0049] 1.2.4 Determination of sperm count and morphology The left epididymis of a mouse was placed in a culture dish. 2-3 mL of normal saline was injected into the epididymis using a syringe. The epididymis was then minced to release the sperm. The liquid was aspirated into a centrifuge tube and incubated at 37°C for 20 minutes. Impurities were filtered to obtain a sperm suspension. 5 μL of the sperm suspension was diluted 5-fold with 0.9% normal saline. A 5-μL droplet was placed into the counting chamber of a cell counting chamber and allowed to stand for 10 minutes to allow the sperm to fully motility and settle before counting. Sperm were counted under a microscope (×100) for observation. The sperm were counted by counting the number of sperm within the four middle grids (counting sperm near the edges was based on counting up, not down, and counting left, not right). This yielded the sperm count A, which was then calculated using the formula.

[0050]

[0051] Place a drop of sperm suspension on a glass slide, create a smear of appropriate thickness, and place the slide over an alcohol burner to dry. Using a rubber-tipped dropper, place 4 drops of hematoxylin stain on the slide. Gently blow with an ear bulb to evenly distribute the stain. Let it sit for 2 minutes. Place 5 drops of eosin stain on the slide, covering the hematoxylin stain and evenly distributing it. Let it sit for 2 minutes. Rinse the slide with a gentle stream of clean water and air-dry the stained side. Locate the sperm under a low-power microscope (×100) and observe under a high-power microscope (×400). Examine at least 200 intact sperm per group, count the number of sperm with abnormalities, and calculate the sperm abnormality rate. Spermatozoa without heads or tails, or those that overlap, are not counted.

[0052]

[0053] 1.2.5 Histopathological observation Remove the testicles fixed with 4% paraformaldehyde, trim the blocks, and then wash the tissue with water. Then, dehydrate the tissue in a gradient ethanol solution (30%, 50%, 70%, 80%, 95%, 100%, v / v). After dehydration, soak the tissue in xylene to make it transparent. After transparency, embed the tissue in molten paraffin to make a wax block for subsequent sectioning. Cut the wax block into 6-7 micron thick slices, attach them to a glass slide, and spread them in distilled water at 37°C. Then, bake the slices in a 40°C oven and place them in an oven. Stain and seal the slides according to the instructions for the HE staining solution, and photograph the pathological sections of the testicular tissue under an optical microscope.

[0054] 1.2.6 Detection of testosterone levels in mouse serum Whole blood samples were collected from the mouse orbital cavity and placed at 4°C overnight. The samples were centrifuged at 1000 × g for 20 minutes. The supernatant was diluted 10-fold with sample diluent and mixed thoroughly. Serum testosterone levels were determined using the Mouse High-Sensitivity Testosterone ELISA Kit (see instructions for the kit).

[0055] 1.2.7 RT-qPCR analysis of related mRNA expression in testicular tissue (1) Extraction of total RNA from mouse testicular tissue and synthesis of cDNA Total RNA was extracted from mouse testicular tissue according to the instructions of the Xinjing Animal Tissue Total RNA Extraction Kit. The extracted total RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit. The reverse transcription system is shown in Table 4.

[0056] Table 4 Reverse transcription system

[0057] Reverse transcription conditions: 37°C for 15 min; 85°C for 15 s. The cDNA template was obtained by reverse transcription. After determining the concentration of the cDNA, the sample was stored at -80°C.

[0058] (2) RT-qPCR All primers used in the experiment were synthesized by Beijing Qingke Biotechnology Co., Ltd. GAPDH was selected as the internal reference gene for control. Detailed information on the primers is shown in Table 5.

[0059] Table 5 Primer sequences for each gene

[0060] RT-qPCR reaction. The specific reaction system is shown in Table 6.

[0061] Table 6 RT-qPCR reaction system

[0062] Reaction conditions: Step 1: 95°C, 30 s; Step 2: 95°C, 5 s, 60°C, 30 s (Step 2 cycled 40 times); Step 3: Melt Curve.

[0063] Quantitative analysis: using 2 -△△Ct The relative expression levels of related genes were calculated.

[0064] 2. Conclusion 2.1 Effects of NAPs on testicular and epididymal indices like Figure 3As shown in Figures A and B, the testicular and epididymal indices of mice in the CY group were significantly lower than those in the NC group. After NAPs intervention, the testicular and epididymal indices of mice in the experimental group showed no significant downward trend, but were higher than those in the CY group. NAP-W and NAP-I significantly alleviated the testicular index of mice. There was no significant difference in the effect of the experimental groups on the epididymal index, with NAP-S and NAP-I showing the best protective effects. These results suggest that polysaccharides can effectively protect testicular and epididymal tissues and mitigate cyclophosphamide-induced organ damage.

[0065] 2.2 Effects of NAPs on sperm count Depend on Figure 4 As shown in the results, compared with the normal group, the sperm count of the model group mice decreased significantly, indicating that cyclophosphamide caused a decrease in sperm count in mice and seriously damaged sperm quality. The sperm count of mice improved after NAPs intervention, with NAP-I having the most significant effect.

[0066] 2.3 Effect of NAPs on sperm deformity rate Depend on Figure 5 As shown in the figure, compared with the normal group, the sperm deformity rate of mice in the model group was significantly increased, indicating that cyclophosphamide caused a significant increase in the sperm deformity rate in mice. After NAPs intervention, the sperm morphology of mice improved, with NAP-I having the most significant effect.

[0067] 2.4 Testosterone Content Figure 6 Testosterone levels in the model group were significantly lower than in the normal group. After NAP treatment, testosterone levels remained significantly lower than in the normal group, but recovered significantly to levels higher than in the model group. These results suggest that NAP-S and NAP-I significantly antagonize the inhibitory effect of cyclophosphamide on serum testosterone levels, indicating that NAPs can improve androgen secretion in mice, indirectly reflecting their protective effect against testicular damage caused by cyclophosphamide.

[0068] 2.5 Pathological changes of the testicles Figure 7 Under light microscopy, it was clearly observed that spermatogenic cells within the seminiferous tubules of the normal group were arranged in tight and regular layers, with clear spermatid nucleoli and numerous spermatozoa within the tubule lumen. After cyclophosphamide treatment, the testicular tissue structure of the model group mice was damaged, with a significant decrease in the number and layers of spermatogenic cells at all levels, and a decrease in spermatids and spermatogenesis within the tubule lumen. Intervention with NAPs significantly alleviated testicular pathological damage, with spermatogenic cells arranged in tight and regular layers and an increase in the number of spermatogenic cells at all levels. There were no significant differences between the treatment groups. These results indicate that NAPs can protect mouse testicular tissue from cyclophosphamide damage and have a certain effect on promoting spermatogenesis in testicular tissue.

[0069] 2.6 Effects of NAPs on the expression of related genes in mouse testicular tissue RT-qPCR technology was used to further quantify the mRNA expression of proliferation and differentiation (Pcna, C-kit, WT1, Bax, Bcl-2r, Cata4, Nr5al, Star, Arrdc5). C-kit, WT1, Pcna were used as genes that promote cell proliferation ( Figure 8 AC in the five polysaccharides), the mRNA expression levels of Pcna, C-kit, and WT1 were significantly increased after intervention. Figure 8 D and Figure 8 The experimental data of E in Figure 1 show that compared with the normal group, the model group had a significant increase in Bax mRNA expression and a significant decrease in Bcl-2r mRNA expression. This result indicates that cyclophosphamide promotes apoptosis in testicular tissue and leads to spermatogenic cell apoptosis. After administration of NAPs, Bax mRNA expression in each experimental group decreased significantly, while Bcl-2r mRNA expression increased significantly. In contrast, the polysaccharides extracted from NAP-S and NAP-I were more effective in regulating cell proliferation and apoptosis. Figure 8 As shown in the F, the lack of Arrdc5 gene will lead to reduced sperm production, slower motility, and distorted shape. Compared with the model group, the expression of Arrdc5 mRNA in the polysaccharide group increased significantly, which is consistent with the previous changes in sperm quality. NAP-A and NAP-I can effectively promote the expression of Arrdc5 mRNA. Figure 8 As shown in the GI, the expression level of testosterone synthesis genes in the model group was the lowest. After administration of NAPs, the expression levels of Cata4, Nr5al, and Star mRNA were increased. Among them, NAP-S had a better effect on Cata4 mRNA expression, NAP-W and NAP-S had a better effect on Nr5al mRNA expression, and NAP-AL and NAP-S had a better effect on Star mRNA expression.

Claims

1. Use of Tremella fuciformis polysaccharide in the preparation of a drug for treating male infertility.

2. The use according to claim 1, characterized in that: The infertility is oligospermia, azoospermia or spermatogenic dysfunction caused by low immunity.

3. The use according to claim 1, characterized in that: The infertility is oligospermia, azoospermia or spermatogenic dysfunction caused by cyclophosphamide.

4. Use of Tremella fuciformis polysaccharide in the preparation of any of the following medicines: (1) Drugs that protect testicular and / or epididymal tissues; (2) Drugs that improve sperm quality; (3) Drugs to increase sperm count; (4) Drugs that reduce sperm deformity rates; (5) Drugs that increase testosterone levels; (6) Drugs that improve testicular function.

5. The use according to any one of claims 1 to 4, characterized in that: The Tremella fuciformis polysaccharide is water-insoluble Tremella fuciformis polysaccharide.

6. The use according to any one of claims 1 to 5, characterized in that: The extraction method of Tremella fuciformis polysaccharide comprises the following steps: (1) adjusting the water content and pH value of the deep eutectic solvent to obtain DES1; the pH value of the DES1 is 11-13; The deep eutectic solvent is composed of n-octanoic acid and sodium octanoate; the molar ratio of n-octanoic acid to sodium octanoate is 2-3:1; (2) mixing Tremella fuciformis and the DES1, heating and extracting the mixture to obtain a precipitate 1 and a supernatant; adding anhydrous ethanol to the supernatant to obtain a precipitate that is a water-soluble polysaccharide; (3) adjusting the water content and pH value of the deep eutectic solvent to obtain DES2; the pH value of DES2 is 1-3; (4) The precipitate 1 obtained in step (2) and the DES2 are mixed, heated for extraction, and the supernatant is obtained; anhydrous ethanol is added to the supernatant, and the resulting precipitate is a water-insoluble polysaccharide, namely, the Tremella fuciformis polysaccharide.

7. The use according to claim 6, characterized in that: The molar ratio of n-octanoic acid to sodium octanoate is 2:

1.

8. The use according to claim 6 or 7, characterized in that: In step (1), the volume percentage water content of DES1 is 82%-98%, preferably 84%-88%; In step (3), the volume percentage water content of the DES2 is 10%-90%, preferably 20%-40%.

9. The use according to any one of claims 6 to 8, characterized in that: In step (2), the material-liquid ratio of the edible fungus and DES1 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h; preferably 1.75-2.25 h; In step (4), the material-liquid ratio of the precipitate 1 and DES2 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h, preferably 1.75-2.25 h.

10. The use according to any one of claims 6 to 9, characterized in that: In steps (2) and (4), the volume ratio of the supernatant to anhydrous ethanol is 1:4-5.

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