Application of Fatostatin in preparation of medicine for preventing and / or treating male reproductive dysfunction

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of Fatostatin in preparation of a medicine for preventing and / or treating male reproductive dysfunction. Through an orchitis mouse model, it is proved that Fatostatin can increase the testis weight of an orchitis mouse and significantly improve abnormal sperm quality and abnormal sperm number of the orchitis mouse. According to the application, the Fatostatin is used for preventing or treating the male reproductive dysfunction for the first time, an ideal treatment effect is achieved, a new prevention and treatment choice is provided for clinical application and treatment of male sterility, especially male sterility caused by inflammation infection, and the application has extremely high application value and social benefits.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Fatostatin in preparation of a medicine for preventing and / or treating male reproductive dysfunction. BACKGROUND

[0002] Low fertility rate has become the main risk affecting the balanced development of China's population, and the health status of the childbearing population is an important guarantee for improving the fertility rate. According to statistics, the incidence of infertility of the childbearing population in China has risen from 11.9% in 2007 to 17.6% in 2020, about 33 million childbearing couples are troubled by fertility problems, about 50% of which are related to male factors. Therefore, in-depth study of the pathogenesis of male spermatogenesis and related infertility is not only of great significance to solving the problem of infertility, but also is a key measure to improve the overall reproductive health level of China and cope with the challenge of population negative growth.

[0003] The causes of male infertility are complex and diverse, among which testicular inflammation is one of the important pathological factors causing male infertility. Testicular inflammation mainly originates from systemic viral infection or local bacterial infection. Although most testicular inflammations are self-limiting, and the infection symptoms of most patients can be relieved within 2 weeks, about 50% of patients will have a certain degree of testicular atrophy, which will persist after puberty and lead to low fertility or even infertility. The cause may be that bacterial or viral infection causes immune cell infiltration, leading to an increase in testicular inflammatory factors, and then a large number of germ cells are lost and spermatogenic function is impaired. However, the pathogenesis of testicular inflammation leading to reproductive disorders has not been fully elucidated, which limits the clinical treatment. Therefore, it is imperative to further explore the potential pathological mechanism of testicular inflammatory spermatogenic disorders and develop new effective treatment methods.

[0004] Previous studies have shown that the pathogenesis of testicular inflammatory spermatogenic disorders is closely related to local inflammatory microenvironment disorder and metabolic homeostasis imbalance. Among them, cholesterol metabolism is particularly important, which not only participates in the maintenance of the body's physiological homeostasis, but also is closely related to the production of inflammatory factors. In Ch25h gene knockout mice, the accumulation of a large amount of cholesterol can inhibit the respiratory function of mitochondria, promote the release of mtDNA and the activation of inflammasomes, and then lead to the production of IL-1β inflammatory factors. Correspondingly, in the endotoxin shock model induced by bacterial lipopolysaccharides (LPS), inhibiting the activity of sterol regulatory element binding protein 2 (SREBP2) can prevent the synthesis of cholesterol, thereby effectively inhibiting inflammation. Disordered local cholesterol metabolism in the testis can cause lipid peroxidation, mitochondrial dysfunction and apoptosis of spermatogenic epithelial cells, and ultimately lead to spermatogenic disorders.

[0005] Fatostatin, a novel SREBP inhibitor, has been shown to specifically block the nuclear translocation and transcriptional activity of SREBP2, demonstrating significant efficacy in the treatment of metabolic diseases. However, whether fatostatin can improve spermatogenesis disorders associated with orchitis by regulating the SREBP2 signaling pathway remains unstudied. This study aims to investigate the protective effect of fatostatin-targeted inhibition of SREBP2 on spermatogenesis in orchitis mice and its molecular mechanism, providing experimental evidence for developing novel treatment strategies for male infertility. Summary of the Invention

[0006] The purpose of this invention is to provide new pharmaceutical uses for Fatostatin in order to solve the problems existing in the background art described above.

[0007] Another object of the present invention is to provide the use of Fatostatin in the preparation of a medicament having at least one of the following functions: 1)-4)

[0008] 1) Increase sperm concentration;

[0009] 2) It increases the number and orderly arrangement of spermatogenic cells in the testes;

[0010] 3) Reduce the degree of testicular fibrosis;

[0011] 4) Improves lipid metabolism disorders in the testes.

[0012] To achieve the above objectives, this application employs the following technical solution:

[0013] A novel pharmaceutical use for Fatostatin is its application in the preparation of drugs for the prevention and / or treatment of male reproductive dysfunction.

[0014] Furthermore, the male reproductive dysfunction may specifically be male reproductive dysfunction caused by inflammation.

[0015] Furthermore, the aforementioned male reproductive dysfunction specifically refers to testicular spermatogenesis dysfunction.

[0016] Improving lipid metabolism disorders in the testes refers to at least one of the following: 1) reducing lipid droplet deposition in the testes; 2) reducing the expression level of SREBF2 (cholesterol regulatory element binding protein 2), a key transcription factor regulating cholesterol metabolism, in the testes.

[0017] The beneficial effects of this invention are:

[0018] The application establishes a mouse model of orchitis, and systematically evaluates the effect of Fatostatin on improving the spermatogenic function related to orchitis. The research results show that Fatostatin can significantly improve the sperm concentration and survival rate of orchitis mice. Further observation of the morphological changes of testicular tissue by HE and Masson staining shows that Fatostatin can improve the morphology of testicular tissue, increase the number of various reproductive cells, and reduce the degree of fibrosis. The immunofluorescence detection results further verify that the number of sperm cells significantly increases after Fatostatin treatment. In addition, Fatostatin effectively reduces the lipid metabolism disorder level of testicular tissue in orchitis mice.

[0019] Fatostatin used in the application has a clear chemical structure and specific target, and is mainly used to improve male reproductive dysfunction caused by inflammation, which is different from traditional drugs aimed at improving sperm quality. The application solves the serious shortage of drugs for preventing or treating male reproductive dysfunction caused by inflammation in clinical practice, and for the first time finds that Fatostatin significantly improves the spermatogenic function of orchitis mice, providing a new prevention and treatment option for male reproductive dysfunction. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings are included to provide a further understanding of the application, and constitute a part of the specification, which together with the embodiments of the application are used to explain the application, and do not constitute a limitation of the application. In the drawings:

[0021] Figure 1 : Mice testis weight, testis oil red O staining results and changes in gross and testis weight ratio results.

[0022] Figure 2 : Mice testicular tissue PNA staining results.

[0023] Figure 3 : Mice sperm concentration, survival rate and sperm motility index detection results.

[0024] Figure 4 : Mice sperm motility intrinsic parameter detection results.

[0025] Figure 5 : Mice testicular tissue HE and Masson staining results.

[0026] Figure 6 : Mice peritubular myoid cells and interstitial cells SREBP2 staining results. DETAILED DESCRIPTION

[0027] The preferred embodiments of the application are described below in conjunction with the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the application, and do not limit the application.

[0028] The terms used in the present application have meanings generally understood by those of ordinary skill in the art, unless otherwise defined.

[0029] Unless otherwise specified, the specific techniques or conditions in the examples are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0030] Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test materials used in the following examples are commercially available products.

[0031] Example 1

[0032] 1. Experimental animals:

[0033] In this experiment, 8-week-old ICR male mice (SPF level) were used, which were provided by the Experimental Animal Center of the Institute of Animal Sciences, Chinese Academy of Sciences. The mice were randomly divided into three groups: control group, orchitis group and Fatostatin treatment group. Three days before the experiment, the control group of mice was injected with an equal volume of normal saline daily; the orchitis group and the Fatostatin group of mice were injected with lipopolysaccharide (LPS, 2.5 mg / kg) daily to induce orchitis model, and the injection was continued for 3 days. On the 4th to 6th day, the control group continued to be injected with normal saline, the orchitis group was injected with corn oil intraperitoneally, and the Fatostatin group was injected with Fatostatin (20 mg / kg) intraperitoneally daily for 3 consecutive days. After all the mice were treated, they were sacrificed, and the testes and epididymides were removed, the testes were weighed, and used for subsequent histological and molecular biological analysis.

[0034] The animal experiments strictly followed the management regulations of the Experimental Animal Center of the Institute of Animal Sciences, Chinese Academy of Sciences, and were reviewed by the Animal Experiment Ethics Committee.

[0035] (1) Tissue collection: After the experiment, the mice were first weighed, and the mass of the testicular tissue was measured. Fresh epididymis was immediately collected for sperm quality detection. One side of the testicular tissue was fixed in animal testicular tissue fixative, and then subjected to routine alcohol dehydration, paraffin embedding and preparation of tissue sections; the other side of the testicular tissue was half for frozen embedding section, and the other half was directly used for protein extraction or liquid nitrogen storage for subsequent experiments.

[0036] (2) Detection of the effect on the morphology of the seminiferous tubules in the testicular tissue: The paraffin sections of the testicular tissue were subjected to routine histological staining (hematoxylin-eosin staining) to observe the morphological changes of the seminiferous tubules in the testicular tissue of the mice in each group, such as the arrangement of the seminiferous tubules, the shape of the lumen, the arrangement and number of the spermatogenic cells at each level, etc.

[0037] (3) Testis lipid abnormal accumulation detection: According to the oil red O staining kit of Solerbio Company, the testis frozen section was stained, and the changes of lipid droplet deposition in the testis tissue of each group of mice were observed.

[0038] (4) Detection of the effect on testis tissue fibrosis: According to the Masson's trichrome staining kit of Sevier Company, the testis paraffin section was stained, the changes of collagen fiber distribution and content in the testis tissue of each group of mice were observed, and the degree of testis tissue fibrosis was evaluated.

[0039] (5) Determination of the effect on semen quality: Fresh epididymis, epididymal sperm was collected by diffusion method and the computer-aided semen analysis system (Beijing Suijia Software Co., Ltd.) was used to detect the number of sperm, sperm survival rate, fast forward movement and forward movement, linear velocity, curved velocity, average path velocity, linearity, head lateral displacement, and linearity of mice.

[0040] (6) Testis immunofluorescence detection of SREBP2 changes: After the paraffin section of testis tissue was routinely dewaxed, it was antigen repaired in 95℃ modified sodium citrate for 20 minutes, then endogenous peroxidase was blocked with hydrogen peroxide, and 5% fetal bovine serum and 0.3% Triton X-100 were used for blocking and membrane breaking treatment for 1 hour, SREBP2 antibody was incubated at 4℃ overnight, secondary antibody was incubated, after antibody elution, the markers of peritubular myoid cells and interstitial cells were incubated at room temperature, after secondary antibody incubation and DAPI staining, it was photographed under Zeiss LSM880 confocal microscope.

[0041] (7) Testis sperm cell immunofluorescence detection: After the paraffin section of testis tissue was routinely dewaxed, it was antigen repaired in 95℃ modified sodium citrate for 15 minutes, 5% fetal bovine serum was used for blocking for half an hour, sperm cell marker PNA was incubated for 1 hour and 30 minutes, after DAPI staining, it was photographed under Zeiss LSM880 confocal microscope.

[0042] (8) All data represent at least three independent experiments, and the data are expressed as mean ± standard deviation (SD). The difference between the two groups was statistically analyzed by the stat_compare_means() function of the ggpubr package in R language based on the t test (unpaired t test) between the two groups, and the difference was considered statistically significant when P value <0.05, * represented P<0.05, ** represented P<0.01, *** represented P<0.001, and **** represented P<0.0001.

[0043] 2. Experimental results:

[0044] 2.1 Changes in the size of mouse testis and lipid deposition results

[0045] First, to evaluate the effect of Fatostatin on testicular morphology and lipid metabolism, we compared the testicular weight, gross and testicular weight ratio of the control group (Con group), orchitis group (Orchitis group) and treatment group (Fatostatin group) mice. The results show (see Figure 1 ): Compared with the Con group, the testis / body weight ratio and testis weight of the Orchitis group mice decreased significantly, and the oil red O staining results showed that the lipid droplet deposition in the testicular tissue increased significantly. In contrast, the testis / body weight ratio and testis weight of the Fatostatin group mice increased significantly, P < 0.05, and the lipid droplet deposition in the testicular tissue decreased significantly.

[0046] 2.2 Changes in mouse sperm cells

[0047] The immunofluorescence results of mouse testicular tissue (see Figure 2 ) show that the expression of sperm cell marker (PNA) in Orchitis mice is significantly lower than that in Con mice, and the number of sperm cells in Fatostatin mice is significantly more than that in Orchitis mice, indicating that Fatostatin treatment can increase the number of sperm in orchitis mice.

[0048] 2.3 Mouse sperm concentration and sperm motility index

[0049] We detected the semen quality of the three groups of mice at the end of treatment by computer-aided semen analysis, and the results showed (see Figure 3 ): Compared with Con mice, the sperm concentration, viability and forward movement of Orchitis mice were significantly reduced, P < 0.05. After treatment with Fatostatin, the above semen parameters were significantly improved, and the sperm concentration, viability and forward movement ability were significantly higher than those of the Orchitis group, P < 0.05, suggesting that Fatostatin can effectively improve the semen quality of orchitis mice.

[0050] 2.4 Intrinsic parameters of mouse sperm motility

[0051] The intrinsic parameters of sperm motility of each group of mice were detected by computer-aided semen analysis (see Figure Four ), compared with Con mice, the intrinsic motility parameters of Orchitis mice, including forwardness (STR), whipping frequency (BCF) and straight sperm head side swing displacement (ALH), were reduced, while Fatostatin treatment can significantly improve the linear motion speed (VSL), curve motion speed (VCL), average path speed (VAP), forwardness (STR), whipping frequency (BCF) and straight sperm head side swing displacement (ALH), P < 0.05.

[0052] 2.5 HE and Masson staining results of mouse testis tissue

[0053] The morphological changes of testis tissue of each group of mice were observed after hematoxylin-eosin (HE) staining (see Figure 5 ), and the results showed that the structure of the spermatogenic tubules of the Con group mice was complete, the arrangement of the spermatogenic cells at all levels was neat, and a large number of mature sperm could be seen in the lumen; the structure of the spermatogenic tubules of the Orchitis group mice was disordered, the number of spermatogenic cells was significantly reduced, the arrangement was disordered, and almost no mature sperm was found in the lumen; while the number of spermatogenic cells at all levels in the spermatogenic tubules of the Fatostatin treatment group mice was significantly increased, the arrangement was relatively regular, and a certain amount of sperm could be seen in the lumen.

[0054] 2.6 SREBP2 staining results of mouse testis tissue

[0055] The immunofluorescence results of mouse testis tissue (see Figure 6 ) showed that compared with the Con group, the expression level of sterol regulatory element binding protein SREBP2 in the peritubular myoid cells and interstitial cells of the Orchitis mice was significantly increased, while after treatment with Fatostatin, the expression level of SREBP2 was significantly reduced, P<0.05, indicating that Fatostatin can effectively improve the lipid metabolism disorder of orchitis mice.

[0056] From the above examples, it can be seen that based on the testis of orchitis mice after treatment with Fatostatin, through computer-aided semen analysis system, testis histological staining, immunofluorescence detection, it is found that Fatostatin can significantly increase the sperm concentration and number of orchitis mice, improve the morphological structure of testis tissue, promote the spermatogenesis process, and effectively alleviate the lipid metabolism disorder caused by orchitis. Therefore, the present application proposes the application of Fatostatin in the preparation of a therapeutic drug for improving the reproductive dysfunction caused by orchitis, which has clear scientific basis and potential clinical application value.

[0057] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. The use of Fatostatin in the preparation of drugs for the prevention and / or treatment of male reproductive dysfunction.

2. The application as described in claim 1, characterized in that, The male reproductive dysfunction mentioned refers to male reproductive dysfunction caused by inflammation.

3. The application according to any one of claims 1 to 2, characterized in that: The aforementioned male reproductive dysfunction is a disorder of spermatogenesis.

4. The application according to any one of claims 1 to 3, characterized in that: The aforementioned male reproductive dysfunction refers to abnormal sperm quality or abnormal sperm count.

5. The application according to any one of claims 1 to 3, characterized in that: The male reproductive dysfunction mentioned refers to azoospermia, oligospermia, asthenospermia, and / or teratospermia.

6. The application according to any one of claims 1 to 5, characterized in that: The male reproductive dysfunction is caused by an inflammatory infection.

7. The application according to any one of claims 1 to 6, characterized in that: The term "male" refers to male mammals.

8. The application according to claim 7, characterized in that: The mammals are selected from the following: mice, rats, rabbits, cats, dogs, or primates.

9. The application according to claim 7, characterized in that: The term "male" refers to male mammals selected from humans.

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