Application of mangiferin in preparation of protective agent for testicular sertoli cells and preparation of medicine for treating male infertility diseases

Abstract

The invention discloses application of mangiferin in preparation of a protective agent for testicular sertoli cells and preparation of a medicine for treating male infertility diseases, and relates to the technical field of medicine application. Wherein the mangiferin can play a role in protecting the sertoli cells, and the protection role is reflected in ways of improving the cell viability of the sertoli cells, inhibiting the apoptosis of the sertoli cells, inhibiting the oxidative stress of the sertoli cells, inhibiting the inflammatory response of the sertoli cells and the like; meanwhile, mangiferin can be used for treating male infertility diseases in ways of relieving weight loss, repairing testis, improving endocrine system functions, inhibiting oxidative stress of testis tissues, inhibiting inflammatory response of the testis tissues and the like. Wherein the mangiferin can inhibit the inflammatory reaction mainly by inhibiting the activation of NLRP3 inflammasome to inhibit the NLRP3 pathway, so that the inflammation and even pyroptosis of testicular sertoli cells are remarkably reduced to improve spermatogenesis, and the male infertility disease is well treated.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical production, and particularly relates to the application of mangiferin in the preparation of a protective agent for Sertoli cells and in the preparation of a drug for treating male infertility diseases. Background Art

[0002] With the aggravation of problems such as work burden, employment pressure, genetic factors, psychological stress, diet, and environmental pollution, the incidence of male infertility continues to rise. WHO data shows that about 20% of childbearing couples are troubled by infertility, among which 40 - 60% are caused by male factors, and this proportion shows an increasing trend year by year. Male infertility caused by semen abnormalities accounts for 70 - 80%, and oligospermia accounts for about 18%. More notably, the number of human sperm decreases at a rate of 2.1% per year, indicating that the spermatogenic function of the testis is declining.

[0003] Existing treatment methods, including drugs, surgery, and assisted reproductive technologies, still have obvious deficiencies. Among them, the effect of drug treatment varies greatly among individuals, and the side effects may affect the quality of life, lacking an effective solution for the cause; although surgical intervention has a certain effect, it has high risks and a long recovery period, bringing challenges to the physical and mental health of patients; assisted reproductive technologies such as in vitro fertilization can help some patients, but the cost is high, and the success rate is affected by various factors.

[0004] In summary, the current treatment methods for oligospermia have limited effects. Therefore, it is urgent to carry out research on the pathogenesis and intervention measures of oligospermia in order to provide more effective treatment plans for male infertility patients and improve the fertility rate and quality of life. Summary of the Invention

[0005] The main object of the present invention is to propose the application of mangiferin in the preparation of a protective agent for Sertoli cells and in the preparation of a drug for treating male infertility diseases, aiming to provide an effective method for treating male infertility diseases.

[0006] To achieve the above object, the present invention proposes an application of mangiferin in the preparation of a protective agent for Sertoli cells.

[0007] In one embodiment, the concentration of mangiferin in the protective agent is 25 - 400 μg / mL.

[0008] In one embodiment, the protective agent exerts a protective effect through at least one of the following ways: increasing the cell viability of Sertoli cells, inhibiting the apoptosis of Sertoli cells, inhibiting the oxidative stress of Sertoli cells, and inhibiting the inflammatory response of Sertoli cells.

[0009] In one embodiment, the inhibition of the oxidative stress of Sertoli cells includes increasing the content of superoxide dismutase and decreasing the content of malondialdehyde; and / or,

[0010] The inhibition of the inflammatory response of Sertoli cells includes inhibiting the expression of TNF-α and IL-1β, and / or inhibiting the expression of NLRP3, ASC, Caspase-1, and GSDMD genes.

[0011] The present invention also provides an application of mangiferin in the preparation of a drug for treating male infertility diseases.

[0012] In one embodiment, the male infertility diseases include oligospermia, asthenospermia, azoospermia, or teratozoospermia.

[0013] In one embodiment, the oral dosage of mangiferin in the drug is 15 - 60 mg / kg.

[0014] In one embodiment, the drug exerts its effect through at least one of the following pathways: alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting oxidative stress in testicular tissue, and inhibiting the inflammatory response in testicular tissue.

[0015] In one embodiment, the repair of the testis includes repairing testicular function and / or repairing the testicular tissue structure:

[0016] Among them, the repair of testicular function includes enhancing sperm motility, increasing sperm concentration, and reducing the sperm malformation rate;

[0017] The repair of the testicular tissue structure includes improving the epithelial thickness of seminiferous tubules, improving the diameter of seminiferous tubules, and improving the cross-sectional area of seminiferous tubules.

[0018] In one embodiment, the improvement of the function of the endocrine system includes inhibiting the expression of luteinizing hormone and follicle-stimulating hormone and enhancing the expression of testosterone; and / or,

[0019] The inhibition of oxidative stress in testicular tissue includes increasing the content of superoxide dismutase, increasing the content of glutathione peroxidase, and reducing the content of malondialdehyde; and / or,

[0020] The inhibition of the inflammatory response in testicular tissue includes inhibiting the expression of TNF-α and IL-1β, and / or inhibiting the expression of NLRP3, ASC, Caspase-1, and GSDMD genes.

[0021] In the technical solution of the present invention, mangiferin can play a protective role in Sertoli cells, and this protective role is reflected in improving the cell viability of Sertoli cells, inhibiting the apoptosis of Sertoli cells, inhibiting the oxidative stress of Sertoli cells, inhibiting the inflammatory response of Sertoli cells and other ways; at the same time, mangiferin can treat male infertility diseases through ways such as alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting the oxidative stress of testicular tissue, and inhibiting the inflammatory response of testicular tissue. Among them, mangiferin inhibits the inflammatory response may mainly inhibit the NLRP3 pathway by inhibiting the activation of NLRP3 inflammasome, thereby significantly reducing the inflammation and even pyroptosis of Sertoli cells to improve spermatogenesis, and thus better treat male infertility diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the structures shown in these drawings.

[0023] Figure 1 Among them, (A) is the result diagram of the effect of mangiferin on the body weight of rats in Example 1 of the present invention; Figure 1 Among them, (B) is the result diagram of the effect of mangiferin on the testicular index of rats in Example 1 of the present invention;

[0024] Figure 2 Among them, (A) is the result diagram of the effect of mangiferin on the sperm concentration of rats in Example 1 of the present invention; Figure 2 Among them, (B) is the result diagram of the effect of mangiferin on the sperm motility of rats in Example 1 of the present invention; Figure 2 Among them, (C) is the result diagram of the effect of mangiferin on the sperm malformation rate of rats in Example 1 of the present invention; Figure 2 Among them, (D) is the result diagram of the effect of mangiferin on the sperm state of rats in Example 1 of the present invention;

[0025] Figure 3 Among them, (A) is the result diagram of the effect of mangiferin on the diameter of seminiferous tubules of rats in Example 1 of the present invention; Figure 3 Among them, (B) is the result diagram of the effect of mangiferin on the epithelial thickness of seminiferous tubules of rats in Example 1 of the present invention; Figure 3 Among them, (C) is the result diagram of the effect of mangiferin on the cross-sectional area of seminiferous tubules of rats in Example 1 of the present invention; Figure 3 Among them, (D) is the result diagram of the effect of mangiferin on the pathological morphology of testicular tissue of rats in Example 1 of the present invention;

[0026] Figure 4Figure (A) shows the results of the effect of mangiferin on testosterone levels in rat serum in Example 1 of the present invention; Figure 4 Figure (B) shows the results of the effect of mangiferin on luteinizing hormone levels in rat serum in Example 1 of the present invention; Figure 4 Figure (C) shows the results of the effect of mangiferin on follicle-stimulating hormone levels in rat serum in Example 1 of the present invention;

[0027] Figure 5 Figure (A) shows the results of the effect of mangiferin on the content of superoxide dismutase in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (B) shows the results of the effect of mangiferin on the content of malondialdehyde in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (C) shows the results of the effect of mangiferin on the content of glutathione peroxidase in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (D) shows the results of the effect of mangiferin on TNF-α levels in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (E) shows the results of the effect of mangiferin on IL-1β levels in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (F) shows the results of the effect of mangiferin on IL-6 levels in rat testicular tissue in Example 1 of the present invention; Figure 5 Figure (G) shows the results of the effect of mangiferin on IL-10 levels in rat testicular tissue in Example 1 of the present invention;

[0028] Figure 6 Figure (A) shows the results of the effect of mangiferin on the mRNA level of NLRP3 in rat testicular tissue in Example 1 of the present invention; Figure 6 Figure (B) shows the results of the effect of mangiferin on the mRNA level of ASC in rat testicular tissue in Example 1 of the present invention; Figure 6 Figure (C) shows the results of the effect of mangiferin on the mRNA level of Caspase-1 in rat testicular tissue in Example 1 of the present invention; Figure 6 Figure (D) shows the results of the effect of mangiferin on the mRNA level of GSDMD in rat testicular tissue in Example 1 of the present invention;

[0029] Figure 7 Figure (A) shows the results of the effect of mangiferin on the cell viability of Sertoli cells in the testis of oligospermia rats in Example 2 of the present invention; Figure 7 Figure (B) shows the results of the effect of mangiferin on the apoptosis level of Sertoli cells in the testis of oligospermia rats in Example 2 of the present invention; Figure 7 Figure (C) shows the results of Hoechst 33342 staining of apoptosis of Sertoli cells in the testis of oligospermia rats by mangiferin in Example 2;

[0030] Figure 8(A) shows the effect of mangiferin on the content of superoxide dismutase in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (B) shows the effect of mangiferin on the content of malondialdehyde in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (C) shows the effect of mangiferin on the content of glutathione peroxidase in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (D) shows the effect of mangiferin on the level of TNF-α in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (E) shows the effect of mangiferin on the level of IL-1β in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (F) shows the effect of mangiferin on the level of IL-6 in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 8 (G) shows the effect of mangiferin on the level of IL-10 in testicular Sertoli cells of oligospermia in Example 2 of the present invention;

[0031] Figure 9 (A) shows the effect of mangiferin on the protein expression bands of NLRP3, Caspase-1, and GSDMD in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 9 (B) shows the effect of mangiferin on the gray scale ratio of NLRP3, Caspase-1, and GSDMD proteins in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 9 (C) shows the effect of mangiferin on the protein bands of NLRP3, Caspase-1, and GSDMD in normal testicular Sertoli cells in Example 2 of the present invention; [[ID=2,1]] Figure 9 (D) shows the effect of mangiferin on the gray scale value ratio of NLRP3, Caspase-1, and GSDMD proteins in normal testicular Sertoli cells in Example 2 of the present invention; Figure 9 (E) shows the effect of mangiferin on the amplification fold of NLRP3 mRNA in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 9 (F) shows the effect of mangiferin on the amplification fold of ASC mRNA in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 9 (G) shows the effect of mangiferin on the amplification fold of Caspase-1 mRNA in testicular Sertoli cells of oligospermia in Example 2 of the present invention; Figure 9 (H) shows the effect of mangiferin on the amplification fold of GSDMD mRNA in testicular Sertoli cells of oligospermia.

[0032] The realization, functional features, and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed implementation mode

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchases. In addition, the meaning of "and / or" appearing throughout the text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or a solution that satisfies both A and B simultaneously. In addition, the technical solutions between the embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] With the aggravation of problems such as work burden, employment pressure, genetic factors, psychological stress, diet, and environmental pollution, the incidence of male infertility has been continuously rising. WHO data shows that about 20% of childbearing-age couples are troubled by infertility, among which 40-60% are caused by male factors, and this proportion is increasing year by year. Male infertility caused by abnormal semen accounts for 70-80%, and oligospermia accounts for about 18%. More notably, the number of human sperm is decreasing at a rate of 2.1% per year, indicating that the spermatogenic function of the testis is declining. Existing treatment methods, including drugs, surgery, and assisted reproductive technologies, still have obvious deficiencies. Among them, the effect of drug treatment varies greatly due to individual differences, and the side effects may affect the quality of life, lacking an effective solution for the cause; although surgical intervention has a certain effect, it has a high risk and a long recovery period, posing challenges to the physical and mental health of patients; assisted reproductive technologies such as in vitro fertilization can help some patients, but the cost is high, and the success rate is affected by various factors. In summary, the current treatment methods for oligospermia have limited effects. Therefore, it is urgent to carry out research on the pathogenesis and intervention measures of oligospermia in order to provide more effective treatment solutions for male infertility patients and improve the fertility rate and quality of life.

[0035] In view of this, the present invention provides an application of mangiferin in the preparation of a protective agent for Sertoli cells.

[0036] In the technical solution of the present invention, mangiferin can play a protective role in Sertoli cells, and this protective role is reflected in improving the cell viability of Sertoli cells, inhibiting the apoptosis of Sertoli cells, inhibiting the oxidative stress of Sertoli cells, inhibiting the inflammatory response of Sertoli cells and other ways; at the same time, mangiferin can treat male infertility diseases through ways such as alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting the oxidative stress of testicular tissue, and inhibiting the inflammatory response of testicular tissue. Among them, mangiferin inhibits the inflammatory response may mainly inhibit the NLRP3 pathway by inhibiting the activation of the NLRP3 inflammasome, thereby significantly reducing the inflammation and even pyroptosis of Sertoli cells to improve spermatogenesis, and thus better treat male infertility diseases.

[0037] In some embodiments, the concentration of mangiferin in the protective agent is 25-400 μg / mL. Preferably, the concentration of mangiferin in the protective agent is 200 μg / mL. At this concentration, the cell viability of Sertoli cells with oligospermia treated with benzimidazole is optimally improved.

[0038] In some embodiments, the protective agent plays a protective role through at least one of the ways of improving the cell viability of Sertoli cells, inhibiting the apoptosis of Sertoli cells, inhibiting the oxidative stress of Sertoli cells, and inhibiting the inflammatory response of Sertoli cells. Among them, in some embodiments, the inhibition of the oxidative stress of Sertoli cells includes increasing the content of superoxide dismutase and decreasing the content of malondialdehyde. In some embodiments, the inhibition of the inflammatory response of Sertoli cells includes inhibiting the expression of TNF-α and IL-1β, and / or inhibiting the expression of NLRP3, ASC, Caspase-1, GSDMD genes.

[0039] The present invention also provides an application of mangiferin in the preparation of a drug for treating male infertility diseases. In some embodiments, the male infertility diseases include oligospermia, asthenospermia, azoospermia or teratospermia. Among them, oligospermia refers to the sperm count in the semen of men being lower than the normal range, affecting the fertility of men, and its core pathological feature is the overall decline in sperm quality.

[0040] In some embodiments, the oral dosage of mangiferin in the drug is 15-60 mg / kg, and its converted oral dosage for humans is 1.5-6 mg / kg. The oral dosage of mangiferin can be 15 mg / kg, 30 mg / kg or 60 mg / kg, and its oral dosage within the above range can ensure better therapeutic effects on male infertility diseases such as oligospermia. Preferably, the oral dosage of mangiferin is 30 mg / kg (converted to an oral dosage for humans of 3 mg / kg). At this oral dosage, the drug has the best therapeutic effects and plays a role by alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting oxidative stress in testicular tissue, and inhibiting the inflammatory response in testicular tissue.

[0041] In some embodiments, the drug plays a role through at least one of the following pathways: alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting oxidative stress in testicular tissue, and inhibiting the inflammatory response in testicular tissue. Among them, in some embodiments, the repair of the testis includes repairing testicular function and / or testicular tissue structure: among them, the repair of testicular function includes enhancing sperm motility, increasing sperm concentration, and reducing the sperm deformity rate; the repair of testicular tissue structure includes improving the epithelial thickness of seminiferous tubules, improving the diameter of seminiferous tubules, and improving the cross-sectional area of seminiferous tubules. In some embodiments, the improvement of the endocrine system function includes inhibiting the expression of luteinizing hormone and follicle-stimulating hormone and enhancing the expression of testosterone. In some embodiments, the inhibition of oxidative stress in testicular tissue includes increasing the content of superoxide dismutase, increasing the content of glutathione peroxidase, and reducing the content of malondialdehyde. In some embodiments, the inhibition of the inflammatory response in testicular tissue includes inhibiting the expression of TNF-α and IL-1β, and / or inhibiting the expression of NLRP3, ASC, Caspase-1, and GSDMD genes.

[0042] It should be noted that pyroptosis is a programmed cell death mode mediated by inflammasomes, which amplifies tissue damage by releasing pro-inflammatory factors and reactive oxygen species (ROS). The NLRP3 inflammasome is the core molecular switch connecting oxidative stress and pyroptosis. In the oligospermia model, toxic substances such as busulfan induce K +Outflow, ROS accumulation, and lysosome rupture trigger the assembly of NLRP3 inflammasome. Activated NLRP3 recruits Caspase-1 through ASC, which cleaves pro-IL-1β and pro-IL-18 into active forms, and simultaneously cleaves GSDMD to form GSDMD-N pores, driving pyroptosis. In addition, NLRP3 activation can also promote ROS production through a positive feedback loop, exacerbating oxidative stress. Therefore, in the present invention, it is found that mangiferin has an inhibitory effect on the expression of NLRP3, ASC, Caspase-1, and GSDMD genes (including proteins and mRNAs) in the NLRP3 activation pathway. Therefore, mangiferin can inhibit the inflammatory response and even pyroptosis of testicular tissue by inhibiting the NLRP3 activation pathway, thereby protecting testicular tissue and treating male infertility diseases.

[0043] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0044] Example 1 Establishment of a busulfan-induced oligospermia rat model and mangiferin intervention

[0045] Model establishment: Sixty 8-week-old male SD rats were randomly divided into 6 groups, with 10 rats in each group. A busulfan solution was prepared using normal saline containing 4% dimethyl sulfoxide as a solvent. Five groups of rats were intraperitoneally injected at a dose of 3 mg / kg once a day for 5 consecutive days to obtain 5 groups of oligospermia model rats. Another group of rats was injected with an equal amount of the above solvent every day, and this group was designated as the blank control group or the normal group.

[0046] Mangiferin solutions at different concentrations (15, 30, 60 mg / kg) and vitamin E (VE) solution (100 mg / kg, a positive control drug) were prepared using 0.2% sodium carboxymethyl cellulose (CMC-Na) as a solvent. Four groups of oligospermia model rats were intragastrically administered once a day for 30 consecutive days to obtain mangiferin intervention groups at different concentrations (15, 30, 60 mg / kg) and a VE intervention group (100 mg / kg). Another group of oligospermia model rats without drug treatment was the model group. It should be noted that during the above four groups of drug intervention treatments, the blank control group and the model group were intragastrically administered with an equal dose of solvent (0.2% sodium carboxymethyl cellulose) every day.

[0047] The following tests were performed on the rats in the 6 groups of treatments: the blank control group (Ctrl), the model group (BU(M)), the 15 mg / kg mangiferin intervention group (M+MG-15), the 30 mg / kg mangiferin intervention group (M+MG-30), the 60 mg / kg mangiferin intervention group (M+MG-60), and the VE intervention group (M+VE).

[0048] (1) Body weight observation and calculation of testicular index

[0049] From the first day of model establishment, the body weight was recorded every 3 days. The coat color, mental state, etc. of the rats were observed daily. 24 hours after the last gavage of the rats, they were weighed, anesthetized with 1% sodium amobarbital (50 mg / kg) to take blood, and then sacrificed. The testes were dissected and the testicular index was calculated. Testicular index (mg / g) = testicular mass (mg) / body weight (g). The results are as Figure 1 shown in Table 1. In Table 1, the differences in body weight data between groups were compared (horizontal comparison between different treatment groups). *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively.

[0050] Table 1 Body weight record table

[0051]

[0052]

[0053] As Figure 1 can be seen, the body weights of rats in all groups showed a trend of first increasing and then stabilizing. However, from the 9th day, the body weight growth in the model group slowed down significantly and was significantly lower than that of the normal group. It should be noted that although the early body weight changes in the mangiferin intervention group were similar to those in the model group, with the prolongation of the dosing time, the downward trend of its body weight was effectively alleviated and was significantly better than the model group after 30 days, while the positive control VE group had limited effect on improving body weight ( Figure 1 in A and Table 1). In terms of testicular index, the model group showed obvious testicular atrophy characteristics, and its testicular index was significantly lower than that of the normal group, while this index increased significantly after treatment with mangiferin ( Figure 1 in B), indicating its repair effect on testicular tissue. These results together indicate that mangiferin can effectively improve the systemic metabolic state and testicular function of oligospermia rats.

[0054] (2) Detection of sperm count, sperm motility, and sperm deformity rate

[0055] 10 μL of the sperm sample obtained from the epididymis was dropped onto a cell counting plate, photographed under a 200× microscope, and a 30-s video was recorded for analysis. Referring to the fifth edition of the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen, the sperm concentration and total sperm motility of rat semen were recorded. 10 μL of the sperm sample was stained by the Diff-quick staining method and photographed under a microscope ( Figure 2 in D). Referring to the fifth edition of the World Health Organization Laboratory Manual for the Examination and Processing of Human Semen to determine the sperm deformity criteria, the sperm deformity rate was statistically analyzed. The results are as Figure 2 shown.

[0056] AsFigure 2 It can be seen that the sperm motility in the model group (busulfan group) was significantly reduced ( Figure 2 B in the figure), the malformation rate was significantly increased ( Figure 2 C in the figure), and at the same time, the sperm concentration showed a downward trend ( Figure 2 A in the figure). These changes are consistent with the typical characteristics of oligospermia. It is worth noting that all the mangiferin intervention groups at different doses could significantly increase the sperm concentration and motility, and at the same time effectively reduce the sperm malformation rate. In terms of the dose-effect relationship, the improvement effects of the three dose groups on sperm concentration and motility were similar, but in terms of reducing the malformation rate, the effects of the 30 mg / kg and 60 mg / kg dose groups were significantly better than those of the 15 mg / kg group. In contrast, the positive control VE intervention group only showed a significant effect on reducing the sperm malformation rate. These data fully demonstrate that mangiferin has reproductive protection effects in multiple aspects. It can not only effectively increase the sperm quantity and quality, but also significantly improve the sperm morphology. Its comprehensive curative effect is better than that of the traditional therapeutic drug VE, providing a new potential option for the treatment of oligospermia.

[0057] (3) Histopathological morphological observation of testis tissue

[0058] One side of the testis of the rats was fixed with 4% paraformaldehyde, and then the specimens were subjected to a series of operations including alcohol dehydration, paraffin embedding, sectioning, dewaxing with xylene, hematoxylin staining, alcohol differentiation, rinsing, dehydration with absolute ethanol, eosin staining, clearing with xylene, neutral resin fixation, and microscopic imaging. The results are shown as Figure 3 shown in (D) in the figure. And the key morphological indexes such as the thickness, diameter, and area of the seminiferous tubule epithelium were detected. The results are shown as Figure 3 shown in (B), (A), and (C) in the figure respectively.

[0059] The integrity of the testicular tissue structure is crucial for spermatogenesis, and the morphological changes of the seminiferous tubules can directly reflect the degree of testicular damage in oligospermia rats. It can be seen from Figure 3 that the model group was significantly lower than the normal group in these three parameters, indicating obvious structural damage to its testicular tissue. After intervention with mangiferin, all the dose groups could improve these indexes to varying degrees. Among them, the 30 mg / kg dose group was the most prominent, and the recovery effects of this group in terms of the thickness, diameter, and area of the seminiferous tubule epithelium were better than those of the other two dose groups. These morphological improvements confirm that mangiferin can effectively repair the damaged testicular tissue structure and reconstruct the microenvironment conducive to spermatogenesis, providing a histological basis for explaining the mechanism of its improvement of sperm quality. In particular, the significant therapeutic effect shown by the 30 mg / kg dose suggests that this dose may be the optimal choice for mangiferin to repair testicular tissue.

[0060] (4) Detection of sex hormones in rat serum

[0061] The blood obtained after anesthetizing the rats was allowed to stand for 2 h, and then centrifuged at 3500 rpm for 20 min. The upper serum was taken to detect the levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum. The measurement method was carried out strictly according to the method provided in the kit instructions. The results are shown respectively as Figure 4 shown in (A), (B), and (C) in

[0062] The maintenance of testicular function depends on the precise endocrine regulation system of the hypothalamic-pituitary-gonadal axis, and its dysfunction often leads to disorders of serum reproductive hormone levels. As Figure 4 can be seen, compared with the normal group, the level of T in the serum of the model group rats decreased significantly, and the levels of LH and FSH increased significantly. This characteristic change reflects the dysfunction of the endocrine system in OA rats. After VE intervention, the levels of LH and FSH decreased significantly, but it had a negative effect on the level of T. In contrast, the mangiferin treatment group showed an obvious dose-dependent regulatory effect, and the 30 mg / kg dose group had the most prominent effect. It could significantly reduce the elevated levels of LH and FSH induced by busulfan and restore them to near the normal range. Although the effect on the level of T was relatively limited, the level of T also had an upward trend. These results confirmed the regulatory effect of mangiferin on the endocrine system, and the 30 mg / kg dose showed the best hormone regulation effect.

[0063] (5) Detection of oxidative stress-related indicators

[0064] The testicular tissue was homogenized, and the supernatant was taken after centrifugation to detect the protein concentration by the BCA method. Then, the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were detected according to the steps described in the biochemical kit instructions. The results are shown as Figure 5 shown in (A), (B), and (C) in

[0065] As Figure 5 shown in (A), (B), and (C), the testicular tissue of busulfan-induced oligospermia rats showed abnormal changes in oxidative stress markers (decrease in SOD and GSH-Px, increase in MDA), and treatment with 30 mg / kg mangiferin could significantly improve or partially improve these pathological indicators.

[0066] (6) Detection of inflammatory factor levels

[0067] The testicular tissue was homogenized, and the supernatant was taken after centrifugation to detect the protein concentration by the BCA method. Then, the levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) were detected according to the steps described in the Elisa kit instructions. The results are shown as Figure 5as shown in (D), (E), (F), and (G) in

[0068] As can be seen from Figure 5 (D)-(G) in , the testicular tissues of busulfan-induced oligospermia rats showed abnormal changes in inflammatory factors (TNF-α, IL-1β), and treatment with 30 mg / kg mangiferin could significantly improve or partially improve these pathological indexes.

[0069] (7) Detection of the expression levels of NLRP3, ASC, Caspase-1, and GSDMD by RT-qPCR

[0070] The testicular tissues were homogenized with Trizol lysis buffer. After adding chloroform, it was divided into organic and aqueous phases, and the upper aqueous phase RNA was transferred to a RNase-free centrifuge tube. An equal volume of isopropanol was added for RNA precipitation. 1 mL of 75% ethanol was added to each 1 mL of sample to wash the RNA precipitate. After centrifugation, the ethanol solution was aspirated, and the total RNA was obtained after drying at room temperature. The RNA was dissolved with RNase-free water, and the concentration and purity of RNA were determined by ultraviolet absorption method. Template cDNA was synthesized according to the instructions of the cDNA synthesis kit. Finally, cDNA was used for RT-qPCR, and the reaction conditions were pre-denaturation at 95 °C for 10 min, 45 cycles at 95 °C for 15 s, and annealing at 60 °C for 1 min. Quantitative analysis was based on the expression of the GAPDH gene as an internal reference, and the results were expressed as Ct values. The 2 -ΔΔCt method was used to calculate the relative expression level of the target gene. The results are as Figure 6 shown in (A), (B), (C), and (D) in

[0071] As can be seen from Figure 6 (A) to (D) in , mangiferin downregulated the mRNA levels of NLRP3, ASC, Caspase-1, and GSDMD in the testicular tissues of busulfan-induced oligospermia rats, and had an inhibitory effect on the mRNA levels of the NLRP3-ACS-Caspase-1-GSDMD signaling pathway.

[0072] Example 2 Establishment of a oligospermia cell model and mangiferin intervention

[0073] Sertoli cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured at 37°C with 5% CO2 in a saturated humidity environment, and the medium was changed every two days. The experiment was divided into a control group (Ctrl), a model group (DRB(M)), and different concentration mangiferin intervention groups (M+MG-400, M+MG-200, M+MG-100, M+MG-50, M+MG-25). The model group was the benzimidazole group, and an in vitro oligospermia cell model was established. The cells were cultured in a benzimidazole (1000 μg / mL) solution in an incubator at 37°C with 5% CO2 for 24 h, and then changed to normal medium and continued to be cultured for 24 h. After the intervention groups were cultured in a benzimidazole (1000 μg / mL) solution for 24 h, mangiferin solutions with final concentrations of 400, 200, 100, 50, and 25 μg / mL were added to each well, and the cells were continued to be cultured for 24 h. The control group was added with normal medium to Sertoli cells and cultured in an incubator at 37°C with 5% CO2 for 24 h.

[0074] The following detections were performed on the above 7 groups of cells:

[0075] (1) Cell viability detection

[0076] Cells (1000 cells / well) were seeded in 96-well plates and grown for 24 h. After removing the medium, a benzimidazole-induced oligospermia model was established, and then the benzimidazole was removed. Different concentrations of drugs were added to each well, with 6 replicates per well, and the cells were incubated in an incubator for 24 h. After removing the medium, 10% CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 1 h. The OD value was measured at 450 nm using a microplate reader, and the experiment was repeated 3 times. The results are shown in Figure 7 as shown in (A).

[0077] As Figure 7 shown in (A), benzimidazole treatment significantly reduced cell viability, successfully simulating the pathological state of oligospermia. It is worth noting that cell viability was significantly improved after mangiferin intervention, and the concentration of 200 μg / mL showed the best protective effect (this concentration was used in subsequent mechanism studies, that is, the Figure 7 , Figure 8 , Figure 9 M+MG group in).

[0078] (2) Cell apoptosis detection

[0079] First, soak the clean coverslips in 75% alcohol and place them in the laminar flow hood overnight. Before plating the Sertoli cells, take out the coverslips, rinse them 3 times with DMEM medium to wash away the alcohol, and place them in a 6-well plate. After establishing the oligospermia model with benzimidazole, remove the benzimidazole, add 200 μg / mL of mangiferin to each well, rinse the cells in the 6-well plate with phosphate buffer solution (PBS), add 0.5 mL of paraformaldehyde solution to each well, and fix for 15 min at 37 °C. Then add 0.5 mL of Hoechst33342 solution to each well and stain for 25 min at room temperature. After discarding the staining solution, rinse the cells 3 times with PBS, shake for 3 min each time, and drop 10 μL of anti-fluorescence quencher on the glass slide, then cover with a coverslip. Finally, observe the staining results under blue excitation light using an inverted fluorescence microscope, and the results are as shown in Figure 7 shown in (C); and statistically analyze the level of apoptosis, and the results are as shown in Figure 7 shown in (B).

[0080] As can be seen from Figure 7 shown in (B) and (C), the Hoechst staining results further confirm that mangiferin can significantly inhibit benzimidazole-induced apoptosis and provide effective protection for Sertoli cells.

[0081] (3) Detection of oxidative stress-related indicators

[0082] Lyse the Sertoli cells obtained by culture, centrifuge to obtain the supernatant, and detect the protein concentration by the BCA method. Then, detect the levels of SOD, MDA, and GSH-Px according to the steps described in the biochemical kit instructions. The results are as shown in Figure 8 shown in (A), (B), and (C).

[0083] As can be seen from Figure 8 shown in (A), (B), and (C), in the in vitro model, it was observed that benzimidazole-induced Sertoli cells showed typical oxidative stress characteristics, manifested as a significant decrease in SOD activity and a significant increase in MDA content. After mangiferin intervention, these changes could be partially reversed, but the effect on the level of GSH-Px was limited.

[0084] (4) Detection of inflammatory factor levels

[0085] Lyse the Sertoli cells obtained by culture, centrifuge to obtain the supernatant, and detect the protein concentration by the BCA method. Then, detect the levels of TNF-α, IL-1β, IL-6, and IL-10 according to the steps described in the Elisa kit instructions. The results are as shown in [[ID=;29]] Figure 8 shown in (D), (E), (F), and (G).

[0086] As can be seen from Figure 8As can be seen from Figures (D)-(G), benzimidazole induced a significant increase in the levels of pro-inflammatory factors TNF-α and IL-1β, while mangiferin treatment could effectively inhibit the expression of these inflammatory factors, with relatively less effect on IL-6 and IL-10.

[0087] The in vivo experimental results and in vitro experimental results mutually confirmed these findings, that is: the testicular tissues of rats with busulfan-induced oligospermia also showed abnormal changes in oxidative stress markers (decrease in SOD and GSH-Px, increase in MDA) and inflammatory factors (TNF-α, IL-1β), and treatment with 30 mg / kg mangiferin could significantly improve or partially improve these pathological indices ( Figure 5 ). These results indicate that mangiferin effectively protects testicular cells from damage and thus plays a role in treating oligospermia through a dual mechanism of action of reducing oxidative damage and inhibiting inflammatory responses.

[0088] (5) Detection of the protein expression levels of NLRP3, Caspase-1, and GSDMD by Western blot

[0089] After centrifuging the cell lysate obtained from the culture to take the supernatant (there were 4 groups of cell lysates: control group, model group, M+MG group (or M+MG-200 group), MG group (a group of testicular Sertoli cells cultured routinely without modeling and intervened with 200 μg / mL mangiferin, that is, the control group intervened with 200 μg / mL mangiferin)), the protein concentration was measured by the BCA method. Then, after electrophoresis at a constant voltage of 80-120 V on a 10% SDS-PAGE gel with a sample loading of 10 μL / 50 μg protein, it was wet-transferred to a PVDF membrane, and then soaked in skim milk powder for 2 h and shaken evenly on a shaker at room temperature. The membrane was washed with TBST, incubated with the primary antibody rabbit anti-rat (NLRP3, Caspase-1, GSDMD) at a dilution of 1:1000, and shaken evenly on a shaker in a 4°C refrigerator overnight. After rewarming at room temperature for 30 min to wash off the primary antibody, the secondary antibody was added at a dilution of 1:4000 and incubated for 2 h at room temperature. The secondary antibody was washed off with TBST, ECL reagent was added, images were collected by AI600Images, and the protein expression gray values were recorded after exposure on the machine. The electrophoresis diagrams are as shown in Figure 9 Figures (A) and Figure 9 Figures (C); the three proteins were quantified based on β-actin, and the results are as shown in Figure 9 Figures (B) and Figure 9 Figures (D). It should be noted that in the above experimental design, in order to clarify the effect of mangiferin alone on the expression of NLRP3 inflammasome activation-related proteins in testicular Sertoli cells cultured routinely, testicular Sertoli cells cultured routinely without modeling were intervened with 200 μg / mL mangiferin as the single mangiferin intervention group.

[0090] From Figure 9It can be seen that the expression levels of NLRP3, Caspase-1, and GSDMD in the model group were all higher than those in the control group, and the expression levels decreased after mangiferin administration. Of particular note is that even in conventionally cultured cells without modeling, mangiferin treatment can downregulate the protein expression of NLRP3, Caspase-1, and GSDMD to a certain extent. These findings suggest that mangiferin, as a natural extract, can not only play a protective role under pathological conditions but may also be used as a daily dietary supplement for reproductive health maintenance.

[0091] (6) Detection of the expression levels of NLRP3, ASC, Caspase-1, and GSDMD by RT-qPCR

[0092] The cells were divided into three groups for treatment according to the aforementioned experimental requirements. After culturing for 24 h, the culture medium was removed, and Trizol lysate and chloroform were added. The upper aqueous layer of RNA was transferred to a RNase-free centrifuge tube. An equal volume of isopropanol was added and mixed to precipitate RNA. 1 mL of 75% ethanol was added to each 1 mL of the sample to wash the RNA precipitate. After centrifugation, the ethanol solution was aspirated, and the total RNA was obtained after drying at room temperature. The RNA was dissolved in RNase-free water, and the concentration and purity of RNA were determined by ultraviolet absorption method. According to the instructions of the cDNA synthesis kit, sample RNA and reverse transcriptase were added to synthesize the template cDNA. Finally, cDNA was taken for RT-qPCR, and the reaction conditions were pre-denaturation at 95 °C for 10 min, 95 °C for 15 s for 45 cycles, and annealing at 60 °C for 1 min. Quantitative analysis was based on the expression of the β-actin gene as an internal reference, and the results were expressed as Ct values. The 2 -ΔΔCt method was used to calculate the relative expression level of the target gene. The results are as Figure 9 shown in (E), (F), (G), and (H) therein.

[0093] As Figure 9 shown in (E) to (H) therein, the results showed that the change trends of the relative expression levels of NLRP3, ASC, Caspase-1, and GSDMD genes were consistent with the protein levels. The mRNA expression in the model group was significantly higher than that in the normal group and decreased significantly after mangiferin intervention.

[0094] It is worth noting that the in vivo experimental results showed the same trend as the in vitro study, that is, mangiferin downregulated the mRNA levels of NLRP3, ASC, Caspase-1, and GSDMD in the testicular tissues of busulfan-induced oligospermia rats. These results together suggest that mangiferin may treat oligospermia synergistically by inhibiting the activation of NLRP3 inflammasome and its mediated pyroptosis pathway, thereby through antioxidant stress and anti-inflammatory effects.

[0095] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the patent protection scope of the present invention.

Claims

1. Use of mangiferin in the preparation of a protective agent for Sertoli cells.

2. The application according to claim 1, characterized in that The concentration of mangiferin in the protective agent is 25 - 400 μg / mL.

3. The application according to claim 1, characterized in that, The protective agent exerts a protective effect through at least one of the following pathways: increasing the cell viability of Sertoli cells, inhibiting the apoptosis of Sertoli cells, inhibiting the oxidative stress of Sertoli cells, and inhibiting the inflammatory response of Sertoli cells.

4. The application according to claim 3, characterized in that, The inhibition of the oxidative stress of Sertoli cells includes increasing the content of superoxide dismutase and decreasing the content of malondialdehyde; and / or, The inhibition of the inflammatory response of Sertoli cells includes inhibiting the expression of TNF-α and IL-1β, and / or, inhibiting the expression of NLRP3, ASC, Caspase-1, and GSDMD genes.

5. Use of mangiferin in the preparation of a drug for treating male infertility diseases.

6. The application according to claim 5, characterized in that, The male infertility diseases include oligospermia, asthenospermia, azoospermia, or teratospermia.

7. The application according to claim 5, characterized in that, The oral dosage of mangiferin in the drug is 15 - 60 mg / kg.

8. The application according to claim 5, wherein The drug exerts its effect through at least one of the following pathways: alleviating weight loss, repairing the testis, improving the function of the endocrine system, inhibiting the oxidative stress of testicular tissue, and inhibiting the inflammatory response of testicular tissue.

9. The application according to claim 8, characterized in that The repair of the testis includes repairing testicular function and / or repairing the testicular tissue structure: Among them, the repair of testicular function includes enhancing sperm motility, increasing sperm concentration, and reducing the sperm deformity rate; The repair of the testicular tissue structure includes improving the epithelial thickness of seminiferous tubules, improving the diameter of seminiferous tubules, and improving the cross-sectional area of seminiferous tubules.

10. The application according to claim 8, wherein The improvement of the function of the endocrine system includes inhibiting the expression of luteinizing hormone and follicle-stimulating hormone, and enhancing the expression of testosterone; and / or, The inhibition of the oxidative stress of testicular tissue includes increasing the content of superoxide dismutase, increasing the content of glutathione peroxidase, and decreasing the content of malondialdehyde; and / or, The inhibition of the inflammatory response of testicular tissue includes inhibiting the expression of TNF-α and IL-1β, and / or, inhibiting the expression of NLRP3, ASC, Caspase-1, and GSDMD genes.

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