阅读说明：本技术 达格列净及其类似物在制备防治雄性生殖功能障碍的药物中的用途 (Application of dapagliflozin and analogues thereof in preparation of medicines for preventing and treating male reproductive dysfunction ) 是由 魏蕊 金滋润 张哲� 杨进 魏天娇 洪天配 姜辉 杨宇卓 于 2021-12-13 设计创作，主要内容包括：本发明属于生物医药技术领域。具体涉及SGLT2抑制剂在制备防治雄性生殖功能障碍的药物中的用途。本发明的研究结果表明,达格列净治疗能够增加db/db小鼠的睾丸重量及睾丸/体重比,以及能够显著改善db/db小鼠精子质量异常、精子数量异常和精子运动异常。本发明首次将SGLT2抑制剂用于预防或治疗雄性生殖功能障碍,并取得较为理想的治疗效果,为临床应用治疗雄性不育,特别是由糖尿病或高血糖导致的雄性不育,提供了一种新的预防和治疗选择,具有极高的应用价值和社会效益。(The invention belongs to the technical field of biological medicines. In particular to application of an SGLT2 inhibitor in preparing a medicament for preventing and treating male reproductive dysfunction. The research result of the invention shows that the treatment of dapagliflozin can increase the testis weight and the testis/body weight ratio of a db/db mouse and can obviously improve the abnormal sperm quality, the abnormal sperm quantity and the abnormal sperm movement of the db/db mouse. The SGLT2 inhibitor is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment selection for clinical application and treatment of male sterility, particularly male sterility caused by diabetes or hyperglycemia, and has extremely high application value and social benefit.)

1. Use of Sodium-glucose cotransporter 2 (SGLT 2) inhibitors in the manufacture of a medicament for the prevention or treatment of male reproductive dysfunction.

2. Use according to claim 1, characterized in that: the SGLT2 inhibitor is selected from one or more of the following: dapagliflozin, engagliflozin, egagliflozin, ruagliflozin, or togagliflozin.

3. Use according to claim 1 or claim 2, characterized in that: the SGLT2 inhibitor is dapagliflozin.

4. Use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is a spermatogenic disorder.

5. Use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is abnormal sperm quality, abnormal sperm count, and/or abnormal sperm motility.

6. Use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is azoospermia, oligospermia, asthenospermia and/or teratospermia.

7. Use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is caused by diabetes or hyperglycemia.

8. Use according to claim 1 or claim 2, characterized in that: the male refers to a male mammal, preferably the mammal is selected from the following: mouse, rat, rabbit, cat, dog or primate, more preferably the mammal is selected from the group consisting of humans.

Technical Field

The invention belongs to the technical field of biological medicines. In particular to application of an SGLT2 inhibitor (especially dapagliflozin and analogues thereof) in preparing a medicament for preventing and treating male reproductive dysfunction.

Background

In recent years, the incidence of male infertility tends to increase year by year with the increase in environmental pollution and the postponed growth age. The most direct consequence of the increase of the incidence rate of infertility is the reduction of fertility rate, which further accelerates the progress of China to the aging society, especially the seventh national population census finds that the fertility of the domestic population is gradually reduced, which brings serious challenges to the sustainable development of national economy. The occurrence of infertility is related to a plurality of factors, the individual difference is large, the pathogenic factors are complex, and the problems are brought to the research and treatment of diseases. The onset of male infertility is related to various factors such as environment, heredity, lifestyle habits, endocrine diseases, and the like. The pathological changes mainly comprise spermatogenesis and maturation disorders, which are generally clinically manifested as oligospermia, asthenospermia, teratospermia and azoospermia, and the oligospermia caused by spermatogenesis and maturation disorder accounts for more than half of male infertility. The pathogenic mechanism of oligoasthenospermia is complex, the heterogeneity of the population is strong, and effective and targeted treatment measures are lacked. Therefore, the research on pathogenic mechanisms of the spermatogenesis dysfunction is deeply explored, and the medicine for effectively preventing or treating the diseases related to the spermatogenesis dysfunction is developed, thereby having great strategic research significance for improving the population quality and the life quality of China and supporting the construction of healthy China.

Diabetes is one of the most common metabolic diseases affecting male fertility in clinic, diabetes onset has a tendency of becoming younger in recent years, and epidemiological investigation shows that about 4.2 million diabetes patients exist in the world by 2014, so that the number of diabetes patients in the child bearing age affected by diabetes is large, and particularly the number of diabetes patients in children and teenagers is increased year by year. The male reproductive system is one of the more common serious complications of diabetes, the physiological structure and function of the testis of a diabetic patient are seriously damaged, androgen is low, the quantity, activity, abnormality rate and sperm DNA integrity of sperms are damaged to different degrees, but the pathogenesis of the abnormality of the sperms caused by the diabetes is not completely clarified. At present, the decline of male fertility caused by diabetes mainly refers to antioxidant symptomatic treatment, empirical treatment of traditional Chinese medicine and assisted reproduction technology, the treatment effect of part of patients is poor, and a targeted prevention and treatment scheme is lacked. In view of the huge number of people with diabetes worldwide, there is an urgent need to explore the molecular mechanisms of diabetic testicular spermatogenic dysfunction and male infertility and to develop drugs for effectively preventing or treating male reproductive dysfunction caused by diabetes clinically.

Sodium-glucose cotransporter 2 (SGLT 2) inhibitors are novel hypoglycemic drugs and can exert a hypoglycemic effect by inhibiting the reabsorption of glucose in the proximal tubule of the kidney. The SGLT-2 selective inhibitor serving as a new target of the hypoglycemic drug has no obvious influence on other tissues and organs due to the specific distribution of the SGLT-2 selective inhibitor in the kidney; insulin resistant diabetics can still benefit; and has the advantages of being not easy to cause hypoglycemia risk, not increasing the weight of the diabetic patients and the like. Currently, 6 SGLT2 inhibitors are on the market globally, which are: canagliflozin, Dapagliflozin, Empagliflozin, Ipagliflozin, Luseoglliflozin, and Tofogliflozin. In recent years, large-scale cardiovascular fate studies have shown that various SGLT2 inhibitors, including dapagliflozin, can significantly improve cardiovascular and renal fates in type 2 diabetic patients [ N Engl J Med, 2017, 377(7): 644-. Therefore, the medicine is the first choice for patients with type 2 diabetes mellitus and combined cardiovascular disease and diabetic nephropathy. However, the effect of such drugs on reproductive function has not been reported at present.

Disclosure of Invention

In order to solve the problem of serious shortage of drugs which can be effectively used for preventing or treating male reproductive dysfunction clinically, the inventor discovers that the SGLT2 inhibitor has a treatment effect on a male sterility animal model for the first time, and provides a new prevention and treatment selection for male reproductive dysfunction.

Specifically, the invention is realized by the following technical schemes:

the invention provides application of an SGLT2 inhibitor in preparation of a medicament for preventing and treating male reproductive dysfunction.

Preferably, the SGLT2 inhibitor is selected from one or more of the following: dapagliflozin, engagliflozin, egagliflozin, ruagliflozin, or togagliflozin.

More preferably, the SGLT2 inhibitor is dapagliflozin.

In one embodiment, the male reproductive dysfunction is a spermatogenic disorder.

In a preferred embodiment, the male reproductive dysfunction is abnormal sperm quality, abnormal sperm count, and/or abnormal sperm motility.

In another preferred embodiment, the male reproductive dysfunction is azoospermia, oligospermia, asthenospermia and/or teratospermia.

In yet another preferred embodiment, the male reproductive dysfunction is caused by diabetes or hyperglycemia.

Further, the male refers to a male mammal.

Preferably, the mammal is selected from the following: mouse, rat, rabbit, cat, dog, or primate.

More preferably, the mammal is selected from humans.

Compared with the prior art, the invention has the following beneficial effects:

the SGLT2 inhibitor (especially dapagliflozin and analogues thereof) is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment selection for clinically treating male sterility (especially male sterility caused by diabetes or hyperglycemia), and has extremely high application value and social benefit.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1: results of changes in body weight and blood glucose levels in mice.

FIG. 2: gross mouse and testicular weight ratio.

FIG. 3: and (5) mouse testicle tissue HE staining results.

FIG. 4: and (5) identifying the germ cell marker of the mouse testicular tissue.

FIG. 5: and (5) detecting the sperm density and the survival rate of the mouse.

FIG. 6: and (5) detecting the sperm movement index of the mouse.

FIG. 7: and detecting the intrinsic parameters of the mouse sperm movement.

FIG. 8: TUNEL staining of mouse testis tissue.

FIG. 9: and (3) expressing apoptosis-related protein in mouse testis tissues.

FIG. 10: and (3) expressing apoptosis-related protein in mouse testis tissues.

FIG. 11: results of oxidative stress levels in mouse testis tissue.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example (b):

1. the experimental method comprises the following steps:

classical type 2 diabetic mice: a leptin receptor double knockout mouse (db/db mouse, purchased from Jiangsu Jiejiao medicine Kangbiotech GmbH) is selected from 8-week-old mice, after the mice are adaptively fed for 1 week, the random blood sugar of the mice is detected to be more than or equal to 16.7mmol/L, and then the mice are included in the experiment. Mice were randomly divided into two groups: in the diabetic group and the intervention group, dapagliflozin (Dapa, 1 mg/kg body weight/day) and water (equal volume) were administered, respectively, and the gavage was performed once a day for 5 weeks. Littermate wild-type mice were also selected as normal controls. 9-10 of them in each group. Determination of fasting body weight, random body weight, fasting blood glucose and random blood glucose in mice before and after intervention

Typical characteristics of leptin receptor deletion model mice (db/db) include metabolic disorders such as hyperleptin syndrome, obesity, hyperglycemia, hyperinsulinemia, a decrease in prolactin in serum and an increase in prolactin in pituitary, and typical infertility [ science 1966 Sep 2;153(3740): 1127-. Humans with leptin or a mutation in its receptor also exhibit obesity and infertility [ Endocrine reviews. 2006; 27: 710-. The classical animal model db/db mice of type 2 diabetes show pathological phenotypes in male reproductive system injury very similar to those of diabetic male patients, including spermatogenic dysfunction and sexual dysfunction, and are one of the most common models for studying male sterility and sexual dysfunction [ Biochemical and biological Research Communications, 2017; 485: 686-692; diabetologia 202009, 63(9) ]. We therefore chose this typical type 2 diabetic mouse as a model for male testicular tissue damage for study.

The animal experiment strictly follows the regulations of animal experiment management of Beijing university and is approved by the Ethics Committee of animal experiment of Beijing university.

Material draw and subsequent experiments were performed the day after completion of dosing.

(1) Tissue retention: mouse weight, testis tissue weight. And (5) reserving fresh epididymis. Fixing testis tissue on one side with paraformaldehyde, dehydrating with conventional alcohol, and slicing with paraffin; and directly extracting protein from the testis tissue on the other side or freezing and storing by using liquid nitrogen.

(2) The influence on the morphology of seminiferous tubules of testis tissue is detected: and (3) carrying out paraffin sectioning on the testis tissues, and carrying out conventional histomorphology staining (hematoxylin-eosin staining method) to observe the morphological changes of seminiferous tubules of the testis tissues of each group of mice, such as the arrangement of the seminiferous tubules, the shape of the lumen, the arrangement and the number of the seminiferous cells at each level and the like. The expression of germ cell marker proteins such as DAZL (spermatogonium), SYCP3 (spermatocyte), TNP1 (spermatid), PGK2 (spermatid), SOX9 (supporting cell) and the like is detected by an immunofluorescence method.

(3) Determination of the effect on semen quality: collecting epididymis sperm by diffusion method, and detecting sperm number and sperm survival rate of mouse by computer-assisted semen analysis system (WLJY-9000, model number); rapid forward movement and forward movement; linear velocity, curvilinear velocity, average path velocity, linearity, head roll displacement, and linearity.

(4) Effects on oxidative stress and apoptosis are clear: paraffin sections of testis tissues are subjected to one-step TUNEL apoptosis detection kit (green fluorescence) to detect the apoptosis condition in the paraffin sections of the testis tissues of mice of each group. Extracting proteins from fresh or liquid nitrogen frozen testis tissues, detecting the expression of apoptosis markers (including pro-apoptotic proteins and anti-apoptotic proteins) by Western blot, and respectively detecting the total antioxidant capacity by using a total antioxidant capacity detection kit (ABTS rapid method), a total SOD activity detection kit (NBT method), a glutathione peroxidase detection kit (NADPH method), a hydrogen peroxide and lipid oxidation (MDA) detection kit and the like; SOD and glutathione peroxidase activity, hydrogen peroxide and Malondialdehyde (MDA) content, and Western blot method for detecting protein expression of 4-HNE (4-Hydroxynonenal ).

(5) Data analysis and mapping were performed on all experimental data using GraphPad Prism 7.0 statistical software. Experimental data are expressed as mean ± standard error (mean ± SEM). Comparison of differences between sets of data using analysis of variance and a Holm-Sidak post test, P <0.05 indicates that the differences are statistically significant. Represents P < 0.05; represents P < 0.01; represents P < 0.001.

2. The experimental results are as follows:

2.1 mouse weight and blood glucose level changes

First, fasting body weights and random body weights of littermate wild type mice (wild type/WT mice), leptin receptor double knockout mice (db/db mice), and db/db dapagliflozin-treated (Dapa) mice before and at the end of treatment were examined, and as a result, they were found (see fig. 1): both db/db and Dapa mice had significantly increased body weight compared to WT mice; next, blood glucose of each group of mice was measured, and as a result, it was found (see fig. 1): at the start of treatment, fasting plasma glucose and random plasma glucose were significantly elevated in db/db mice as well as in Dapa mice compared to WT mice; at the end of the treatment, the fasting plasma glucose and the random plasma glucose of db/db mice were significantly increased compared to WT mice, while Dapa treatment significantly reduced the fasting plasma glucose and the random plasma glucose of db/db mice.

2.2 weight ratio of mouse gross and testis

The body weight, testicular weight, and testicular/body weight ratio of each group of mice at the end of the treatment were examined. As a result, it was found (see fig. 2): both db/db and Dapa mice had significantly increased body weight compared to WT mice; the testis weight and testis/body weight ratio of db/db mice are significantly reduced, while the testis weight and testis/body weight ratio of db/db mice can be significantly increased by treatment with Dapa.

2.3 mouse testis tissue HE staining results

Hematoxylin-eosin (HE) staining was performed on the testis tissue of each group of mice, and as a result, it was found (see fig. 3): all levels of spermatogenic cells in spermatogenic tubules of the WT mice are arranged in order, and a large amount of mature sperms can be seen in the lumens; only a small amount of spermatogenic cells at all levels are in the spermatogenic tubules of the db/db mouse, the arrangement is disordered, and mature sperms are hardly visible in the lumen; all levels of spermatogenic cells reappear in the spermatogenic tubules of the db/db mice treated by Dapa, the arrangement is more regular, and meanwhile, a small amount of spermatozoa can also exist in the lumens.

2.4 mouse testis tissue germ cell marker identification results

The expression of various spermatogenic cell marker proteins in the testis tissues of each group of mice is detected by an immunofluorescence method. As a result, it was found (see fig. 4): similar to the HE results, the seminiferous cell markers (DAZL/SYCP 3), the sperm cell marker (TNP1), the sperm marker (PGK 2) and the supporting cell marker (sox 9) were all significantly reduced in the seminiferous tubules of db/db mice compared to WT mice; however, after Dapa treatment, these markers were all significantly increased.

2.5 mouse sperm quality results

The semen quality of each group of mice was tested by computer-assisted semen analysis and was found (see fig. 5): compared with WT mice, the sperm density of db/db mice is obviously reduced, while the sperm density of Dapa-treated db/db mice is obviously increased; similarly, a significant decrease in sperm survival was observed in db/db mice, while there was a trend toward an increase in sperm density in Dapa-treated mice.

Meanwhile, as shown in fig. 6, the rapid forward movement and forward movement of the db/db mouse sperm were significantly decreased, while the rapid forward movement and forward movement of the Dapa-treated mouse sperm were significantly increased.

As shown in fig. 7, the intrinsic movement indexes of the mouse sperms in db/db include linear movement Velocity (VSL), curvilinear movement Velocity (VCL), average path Velocity (VAP), Linearity (LIN) and straight sperm head side-sway displacement (ALH), while the Dapa treatment can improve the intrinsic movement indexes of the sperms except for Linearity (LIN), and neither the simple db/db nor the Dapa treatment has any influence on the linearity of the mouse sperms.

2.6 mouse testis tissue test results

Apoptosis was detected in testis tissue of each group of mice by TUNEL staining, and as a result, it was found (see fig. 8): apoptosis in the testis tissue of db/db mice was significantly increased, whereas apoptosis in the testis tissue of Dapa-treated db/db mice was significantly decreased.

Changes of related protein expression in two typical pathways influencing apoptosis in mouse testis tissues are detected by a Western blot method. As a result, it was found (see fig. 9): the expression of an anti-apoptotic protein Bcl2 in the testis tissues of db/db mice is obviously reduced, while the expression of an apoptotic protein BAX protein has an increasing trend; meanwhile, the expression of the anti-apoptotic protein Bcl2 in the testis tissue of Dapa-treated db/db mice tends to increase. The expression of Bcl2/BAX protein was significantly reduced in the testis tissue of db/db mice, while it was significantly up-regulated in the testis tissue of Dapa-treated db/db mice.

In another apoptotic pathway, it was found that (see fig. 10): the expression of the anti-apoptotic protein XIAP in the testis tissue of the db/db mouse is obviously reduced, the expression of the apoptotic protein caspase8 protein is increased, the expression of the caspase9 protein is obviously increased, and meanwhile, the expression of the anti-apoptotic protein XIAP in the testis tissue of the db/db mouse treated by Dapa is obviously up-regulated. The expression of XIAP/caspase3 protein is reduced in the testis tissue of db/db mouse, and is obviously up-regulated in the testis tissue of db/db mouse treated by Dapa; XIAP/caspase8 protein expression was significantly reduced in the testis tissue of db/db mice, whereas it was significantly upregulated in the testis tissue of Dapa-treated db/db mice; protein expression of XIAP/caspase9 was significantly reduced in the testis tissue of db/db mice, but was significantly up-regulated in the testis tissue of Dapa-treated db/db mice.

Finally, the change of the oxidative stress level in the testis tissue of each group of mice was detected by the kit method, and as a result, it was found (see fig. 11): the total antioxidant capacity in the testis tissues of db/db mice is obviously reduced, the activities of antioxidant stress enzyme superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are obviously reduced, and the protein expression of oxidative stress product dodecahydroxynonavaleraldehyde (4-HNE) is obviously increased; meanwhile, the total antioxidant capacity of the testis tissues of db/db mice treated by Dapa tends to be increased, the antioxidant kinase SOD activity is obviously increased, the GSH-Px activity tends to be increased, and the protein expression of the oxidative stress product 4-HNE is obviously reduced.

3. Conclusion of the experiment

The dapagliflozin treatment can increase the testis weight and the testis/body weight ratio of a db/db mouse and can remarkably improve the abnormal sperm quality, the abnormal sperm quantity and the abnormal sperm movement of the db/db mouse.

In conclusion, the SGLT2 inhibitor is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment selection for clinically treating male sterility, particularly male sterility caused by diabetes or hyperglycemia, and has extremely high application value and social benefit.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.