Use of Humanin polypeptide and its analogs in delaying physiological ovarian dysfunction

Abstract

The invention discloses the use of Humanin polypeptide and its analogs in preparing a drug for treating physiological ovarian dysfunction. The invention adopts naturally aged mice as an animal model and adopts HNG administration to evaluate the effect of Humanin polypeptide in treating physiological ovarian dysfunction. The experimental results show that intraperitoneal injection of Humanin polypeptide analog HNG can significantly delay the loss of ovarian follicles in naturally aged mice, increase the levels of anti-Mullerian hormone and estradiol in rats and inhibit the oxidative stress and cell apoptosis of local ovarian cells. Exogenous supplementation of HNG can significantly improve the oocyte quality of naturally aged mice, increase their pregnancy rate and litter size, and also significantly improve the health parameters such as blood sugar and blood lipids of naturally aged mice. The invention opens up a new way of drug research and development for exploring the protection of ovarian health and maintenance of fertility of elderly women in childbearing age based on Humanin polypeptide, showing significant application potential and broad development prospects.

Description

Technical Field

[0001] The present invention relates to a new application of polypeptide Humanin, namely application of Humanin in preparing a medicine for improving physiological ovarian dysfunction. Background Art

[0002] Diminished Ovarian Reserve (DOR) is characterized by a decrease in the number of remaining oocytes in the ovaries. There are many causes, including age-related ovarian dysfunction, neuroendocrine, environmental, genetic and congenital factors, and some unknown factors. Unfortunately, there is no effective program to prevent and alleviate the decline of ovarian reserve. Therefore, the occurrence of fertility problems caused by ovarian dysfunction is a major issue in the field of reproductive medicine.

[0003] Reproductive decline is considered an inevitable natural phenomenon in women's lifetime, which will have an impact on women's life choices. Women's fertility is negatively correlated with age. From the age of 35 to menopause, the ovarian reserve decreases significantly. However, due to changes in the socioeconomic environment, modern women continue to postpone their childbearing age. Fertility begins to decline significantly after about 37 years old, and it is difficult to get pregnant after 45 years old. More and more older women are turning to assisted reproductive technology to obtain their own oocytes. However, their assisted reproductive outcomes are limited by factors such as poor response to stimulation regimens, limited number and quality of retrieved oocytes, and impaired fertilization potential and development ability. Many older women are limited to oocyte donation because the possibility of live birth with their own oocytes is greatly reduced in women over 40 years old. In addition, menopause caused by women's ovarian dysfunction is a driving factor for age-related diseases. Therefore, how to effectively delay physiological ovarian dysfunction is of great clinical significance.

[0004] Humanin peptide is a 24-amino acid peptide derived from mitochondria, which is reported to be expressed in various tissues, such as brain, retinal pigment epithelial cells, blood vessels and testis, and exhibits multiple properties, such as antioxidant, anti-inflammatory, anti-apoptotic and anti-aging. Researchers have found that replacing the serine at the 14th position of ginsenoside with glycine forms a new derivative called S14G-Humanin (HNG), which has a biological activity 1000 times that of Humanin. Our previous study investigated the expression of the Humanin peptide gene in various types of cells in the ovary and endometrium and found that supplementation with Humanin peptide or HNG significantly improved insulin resistance, reduced oxidative stress and apoptosis in granulosa cells of rat models of polycystic ovary syndrome. Another study of ours found that in patients with intrauterine adhesions and rats with reduced expression levels of Humanin peptide in the endometrium, supplementation with HNG reduced endometrial fibrosis and improved fertility in rats.

[0005] It is worth noting that Humanin has been reported to reduce the functional decline of other organs, including improving age-related macular degeneration, significantly improving the cognitive ability of Alzheimer's mice, and increasing the healthy life span of mice. There are currently no reports on the role of Humanin peptide in the treatment of physiological ovarian dysfunction. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a new use of Humanin polypeptide, that is, using Humanin polypeptide and its analogs in the preparation of drugs for treating physiological ovarian dysfunction.

[0007] The present invention applies Humanin polypeptide and its analogs to drugs for treating physiological ovarian dysfunction. The ingredients (or active ingredients) of the drugs are Humanin polypeptide and its analogs. One or more excipients acceptable in product production may be added to improve the absorption effect of the active ingredients or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms are prepared.

[0008] The present invention uses a 36-week-old naturally aged mouse animal model, divides it into a control group and an HNG administration group, and administers intraperitoneal injection of normal saline or 10 mg / kg HNG three times a week for a total of 8 weeks, collects ovarian tissue and serum, uses HE staining to evaluate morphological changes and the number of follicles in the ovary, uses an oxidative stress kit and a TUNEL staining kit to evaluate oxidative stress and apoptosis in ovarian tissue, and detects blood lipids and blood sugar in serum to evaluate the health status of mice. At the same time, the present invention conducts ovulation induction experiments and reproduction experiments on naturally aged mice to observe the number and quality of ovulation, pregnancy rate and litter size of mice.

[0009] The advantages and technical effects of the present invention are as follows: Based on the anti-aging and antioxidant effects of Humanin polypeptide, the present invention supplements naturally aged mice with Humanin polypeptide, and finds that Humanin polypeptide can significantly improve the ovarian function of mice, increase the number of ovarian follicles in mice and reduce ovarian cell apoptosis, improve serum sex hormones, oxidative stress and health parameter levels, and improve the fertility of naturally aged mice, and shows that Humanin polypeptide can activate the ovarian AMPK / PGC-1α signaling pathway. The present invention proves the anti-aging and antioxidant effects of Humanin polypeptide in physiological ovarian dysfunction, and provides a new direction for the prevention and treatment of ovarian dysfunction in elderly women of childbearing age in clinical diagnosis and treatment. Therefore, Humanin polypeptide can also be used as an active ingredient in drugs for treating ovarian dysfunction in elderly women. The present invention provides the possibility for the future development of drugs for ovarian dysfunction in elderly women of childbearing age based on Humanin polypeptide, and the present invention has great application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a schematic diagram of the weight trend change results of mice in each experimental group during the drug administration period; Figure 2 The figure is a schematic diagram of the test results of neutral hormones in the serum of mice in each experimental group, wherein Figure A represents the test results of estradiol (E2), Figure B represents the test results of anti-Mullerian hormone (AMH), and Figure C represents the test results of follicular estrogen (FSH); Figure 3 Schematic diagram of HE staining of mouse ovarian tissue in each experimental group, where Control is the control group and HNG is the HNG supplemented mouse model group; Figure 4 The statistical results of follicles at all levels in mice in each experimental group, among which Figure A represents the statistical results of primordial follicles, Figure B represents the statistical results of preantral follicles, Figure C represents the statistical results of antral follicles, Figure D represents the statistical results of corpus luteum, and Figure E represents the statistical results of atretic follicles; Figure 5 The statistical graphs of mouse ovulation induction in each experimental group, where Figure A represents the representative pictures of oocytes in each group of mice, Figure B represents the number of oocytes in each group of mice, and Figure C represents the proportion of abnormal oocytes in each group of mice; Figure 6 Schematic diagram of the breeding results of mice in each experimental group, where Figure A represents the number of pregnant mice in each group after mating, Figure B represents the average number of litters born in each group, and Figure C represents the average birth weight of offspring in each group; Figure 7The statistical results of blood health parameter levels of mice in each experimental group, where Figure A represents random blood glucose of mice, Figure B represents serum triglyceride (TG) of mice, Figure C represents serum low-density lipoprotein (LDL-C) of mice, Figure D represents serum high-density lipoprotein (HDL-C) of mice, Figure E represents serum TG / HDL-C ratio of mice, and Figure F represents serum total cholesterol (TC) of mice; Figure 8 The results of the determination of ovarian oxidative stress levels in mice in each experimental group, where A represents superoxide dismutase (SOD), B represents catalase (CAT), C represents reduced glutathione (GSH), and D represents malondialdehyde (MDA); Fig. 9 Representative images of TUNEL staining of mouse ovaries in each experimental group, where green fluorescence indicates apoptotic cells; In the above figure P <0.05 indicated statistical difference. DETAILED DESCRIPTION

[0011] The essential contents of the present invention are further illustrated by the following examples, but the contents of the present invention are not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the reagents used are conventionally available reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Construction of HNG administration model in naturally aged mice The mice used in this example were 36-week-old female C57 mice purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.; they were specifically divided into the following two groups: (1) Elderly control group (Control): mice were intraperitoneally injected with normal saline three times a week for 8 weeks; (2) HNG treatment group (HNG): Mice were intraperitoneally injected with 10 mg / kg body weight of HNG, 3 times a week for 8 weeks.

[0012] Example 2: Mouse body weight During the modeling period, the mice were weighed once a week and the weights of the mice in the two groups were recorded. The results showed that the weight gain trend of the mice treated with HNG was slower than that of the control group ( Figure 1 ).

[0013] Example 3: Determination of sex hormones in mouse serum (1) After modeling, the mice were anesthetized, blood was collected through the medial canthal vein, and the blood was placed in layers at room temperature; (2) Centrifuge at 3000 g for 8 minutes, collect serum and use ELISA kits (Wuhan Elaruite Biotechnology) to detect hormone levels, including estradiol (E2), anti-Mullerian hormone (AMH), and follicle-stimulating hormone (FSH). The detection method was carried out according to the instructions of the kit; Results Figure 2 After HNG treatment, the levels of E2 and AMH in the serum of mice were significantly increased, while there was no significant difference in the level of FSH, indicating that HNG treatment can delay the decline of ovarian reserve function in aged mice.

[0014] Example 4: Ovarian morphological observation (1) After modeling, anesthetize the mouse, disinfect the skin on the lower abdomen, cut open the abdomen, find the reproductive organs (ovaries are below the kidneys), carefully remove the required tissues and put them into the fixative. Remember not to break the ovaries to avoid affecting the results of subsequent experiments. (2) Embedding: Dehydrate the fixed tissue according to the gradient alcohol dehydration method (30% → 50% → 70% → 95% → 95% → 100%, 30 minutes each time), and then use xylene to make it transparent (50% alcohol + 50% xylene → xylene 1 → xylene 2 → xylene 3, 15 minutes each time); melt the paraffin and keep it at about 55°C. Dip the paraffin three times, the first time for 15 minutes, the second time for 30 minutes, and the third time for 60 minutes; place the tissue block in a wax-containing mold for embedding, and cool it for later use; (3) After the ovaries were embedded in paraffin, they were serially sectioned, and one section was taken every two sections for H&E staining. A total of five sections were taken, and the total number of follicles at different stages in the five sections was counted; Ovarian tissue staining Figure 3 The statistical results of follicle numbers are shown in Figure 4 The results showed that compared with the elderly control group, the number of growing follicles at all levels was significantly improved after HNG treatment (primordial follicles: P =0.016; Preantral follicle: P =0.009; antral follicles: P =0.003; Corpus luteum: P =0.015), the number of atretic follicles decreased significantly ( P =0.010), the results showed that HNG supplementation could delay the depletion of follicle pool in aged mice.

[0015] Example 5: Mouse oocyte collection After the modeling was completed, 5 mice in each group received an intraperitoneal injection of 5 IU PMSG (pregnant mare serum gonadotropin), and 48 hours later, 5 IU hCG (human chorionic gonadotropin) was injected again; 12-14 hours later, the mice were killed and oocytes were collected from the ampulla of the oviduct.

[0016] Results Figure 5 After HNG supplementation, the number of oocytes in mice after ovulation induction did not change significantly, but the proportion of abnormal oocytes decreased significantly.

[0017] Example 6: Mouse fertility assessment Female mice in each group were mated with male mice of normal fertility at a ratio of 2:1 for 2 months, and the number of pregnant mice, the number of litters born, and the birth weight of offspring were observed.

[0018] Results Figure 6 Supplementation of HNG increased the pregnancy rate of mice and significantly increased the average litter size, but had no significant effect on the birth weight of offspring.

[0019] Example 7: Determination of blood lipids and blood sugar in mice (1) Take the serum in Example 3; (2) The levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum samples were detected using kits purchased from Nanjing Jiancheng Bioengineering Institute according to the instructions of the reagent manufacturer. Blood samples were collected from the tail vein of mice, and random blood glucose levels were measured using a Yuyue blood glucose meter.

[0020] Results Figure 7 HNG treatment significantly reduced the levels of serum TC, TG and random blood glucose in mice, and significantly increased the content of serum HDL-C. At the same time, the serum TG / HDL-C ratio of mice in the HNG treatment group was significantly reduced. This indicates that HNG supplementation can significantly improve the health status of mice.

[0021] Example 8: Determination of oxidative stress level in mouse ovaries (1) Collect ovarian tissues from each group of mice; (2) Ovarian homogenates were prepared and the supernatants were collected and then analyzed using oxidative stress-related kits (Nanjing Jiancheng Bioengineering Institute, China) including superoxide dismutase (SOD) activity, catalase (CAT) activity, malondialdehyde (MDA) level, and glutathione (GSH) content; Results Figure 8 ,As can be seen from the figure, supplementation of HNG can significantly reduce the level of oxidative stress in the ovarian tissue of mice.

[0022] Example 9: Mouse ovarian cell apoptosis assay Analysis of ovarian cell apoptosis was performed using a TUNEL staining kit (Elabscience, China) according to the manufacturer's instructions. Briefly, 100 μL of TdT equilibration buffer was added to each sample (paraffin section) and equilibrated in a humidified chamber at 37°C for 20 minutes. Then the TdT calibration buffer was discarded, 50 μL of labeling working solution was added to each sample, and the samples were placed in a humidified chamber in the dark for 60 minutes, and then washed three times with PBS for 5 minutes each time, and then stained with DAPI and mounted for observation.

[0023] Results Fig. 9 , the green fluorescence of TUNEL staining decreased after HNG supplementation, which indicated that HNG could reduce the apoptosis of ovarian cells in aged mice.

[0024] In summary, with the development of society, the demand for fertility at an advanced age is increasing, and the ovarian function of women after the age of 35 has obviously accelerated its decline. Currently, there is still a lack of means to preserve the fertility of elderly women. This study found that after supplementing HNG, the ovarian reserve function of elderly mice was significantly delayed, the number of growing follicles was increased, the consumption of follicles was reduced, and the fertility of mice was improved, showing a broad prospect for clinical application.

Claims

1. Application of Humanin polypeptide and its analogs in the preparation of drugs for the treatment of physiological ovarian dysfunction.

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