CN111455040A

CN111455040A - New application of polypeptide Humanin

Info

Publication number: CN111455040A
Application number: CN202010281795.3A
Authority: CN
Inventors: 饶猛; 王莹莹
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-04-11
Filing date: 2020-04-11
Publication date: 2020-07-28
Anticipated expiration: 2040-04-11
Also published as: CN111455040B

Abstract

The invention discloses a new application of polypeptide Humanin, namely the application of polypeptide Humanin gene as an action target point in screening and preparing a medicine for treating polycystic ovarian syndrome, or the application of polypeptide Humanin in preparing a medicine for treating polycystic ovarian syndrome, and experimental results show that the exogenous supplementation of Humanin polypeptide can obviously reduce the weight of a PCOS rat, reduce the androgen level of the PCOS rat, improve the ovulation state, reduce the oxidative stress level of a PCOS rat system and a part, and the exogenous supplementation of Humanin polypeptide can obviously reduce the fasting blood glucose and the fasting insulin level of the PCOS rat, reverse the abnormal conduction of a glucose metabolism pathway IRS1/PI3K/AKT and the abnormal protein expression of G L UT4 in ovary granular cells of the PCOS rat.

Description

New application of polypeptide Humanin

Technical Field

The invention relates to a new application of polypeptide Humanin, namely an application of the polypeptide Humanin in treating polycystic ovarian syndrome.

Background

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic abnormality syndrome of women of childbearing age, is mainly characterized by chronic rare ovulation, anovulation and hyperandrogenemia, is often accompanied by insulin resistance and metabolic abnormality, and has clinical manifestations of irregular menstrual cycle, hirsutism, acne, infertility and the like; the incidence rate is 5% -15%, and the drug accounts for about 75% of patients with infertility caused by ovulation failure.

The pathogenesis of polycystic ovarian syndrome is complex and is not completely understood at present. There is increasing evidence that chronic inflammation, an imbalance in oxidative stress, both systemic and local to the ovary is associated with polycystic ovary syndrome and its metabolic abnormalities. Chronic inflammation interacts with oxidative stress, exacerbating the endocrine abnormalities of polycystic ovarian syndrome. Factors that contribute to the PCOS system and local oxidative stress include hyperglycemia, hyperlipidemia, insulin resistance, androgen excess, abdominal obesity, and obesity, suggesting that oxidative stress plays an important regulatory role in the pathogenesis of polycystic ovary syndrome; it is therefore necessary to understand the mechanisms underlying and regulating oxidative stress in PCOS in order to prevent and intervene early.

Insulin Resistance (IR) means that the efficiency of glucose uptake and utilization is reduced, and the body secretes too much insulin for compensation to produce hyperinsulinemia, so as to maintain the stability of blood sugar. Insulin resistance is predisposed to metabolic syndrome and type 2 diabetes. It was found that patients with PCOS are more prone to develop impaired glucose tolerance, IR, and type 2 diabetes mellitis (T2 DM) than normal women. IR is a phenomenon in 50-70% of PCOS patients. Studies have shown that obese women with polycystic ovarian syndrome have significantly elevated blood glucose levels in the Oral Glucose Tolerance Test (OGTT) compared to normal control women of comparable age and weight. However, the glucose tolerance-glucose response in obese ovulatory hyperandrogenism women was similar to control women, suggesting that dysbiosis is a feature of the anovulatory polycystic ovary syndrome phenotype. In recent years, it has been found that PCOS patients have IR in the ovary part in addition to IR in the classical target tissues of insulin action, such as skeletal muscle, fat and liver, and even PCOS patients with normal glucose tolerance have abnormal glucose uptake in ovarian granulosa cells.

Humanin (HN) is a linear polypeptide which is discovered in 2001 by Hashimoto of Japan scholaria in the undamaged brain area of occipital cortex of Alzheimer ' S disease patient, and is a 24-amino acid peptide coded by mitochondrial gene and coded by mitochondrial genome 16S ribosomal RNA gene MT-RNR2, HN can inhibit β amyloid peptide-induced neuronal apoptosis of Alzheimer ' S disease and a plurality of Alzheimer ' S disease-related gene mutations, Humanin polypeptide is expressed in various tissues and comprises brain, retinal pigment epithelium, blood vessels and testis, and other functions of Humanin polypeptide include neuroprotection, anti-inflammation, anti-apoptosis, anti-fibrogenesis and the like through interaction with BAX, insulin-like growth factor binding protein 3 and a trimer receptor composed of gp130, WSX1 and CNTFR and the like.

There is currently no report of any role of the human polypeptide in the treatment of polycystic ovarian syndrome.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a new application of polypeptide Humanin, namely the application of the polypeptide Humanin as an action target in screening and preparing a medicine for treating polycystic ovarian syndrome, or the application of the polypeptide Humanin in preparing the medicine for treating polycystic ovarian syndrome.

The polypeptide Humanin is a linear polypeptide coded by 16S ribosomal RNA gene MT-RNR2 of mitochondrial genome and consists of 24 amino acids; according to the invention, follicular fluid and ovarian granulosa cells of a PCOS patient and a population with normal ovarian function are collected, the expression level of a Humanin gene in the cells is detected by adopting a qRT-PCR method, and experiments show that the expression level of polypeptide Humanin in the follicular fluid and the ovarian granulosa cells of the PCOS patient is lower than that of the population with normal ovarian function; taking a PCOS rat model as a research object, and observing the changes of the weight and the ovarian weight of the rat, the ovarian morphology, the hormone level of the rat, the serum of the rat and the local oxidative stress index of the ovary by exogenously supplementing polypeptide Humanin (HNG);

meanwhile, the invention carries out exogenous polypeptide Humanin supplementation on PCOS model rats, detects the blood sugar level and the glucose concentration of the rats, detects the expressions of IRS1/PI3K/AKT and G L UT4 in ovarian granular cells at the gene level, detects the expressions of IRS1, PI3K, AKT and G L UT4 in the ovarian granular cells at the protein level, observes and verifies the influence of the Humanin expression level on the glucose metabolism capability through experiments, and observes the improvement of the glucose metabolism capability of the ovarian granular cells through exogenous polypeptide Humanin supplementation.

The invention has the following advantages and technical effects:

the polypeptide Humanin is supplemented to a PCOS rat model based on that the expression level of the polypeptide Humanin in follicular fluid and ovarian granulosa cells of patients with polycystic ovarian syndrome is lower than that of normal ovarian functional population, and the polypeptide Humanin is found to be capable of obviously reducing the weight of a rat, reducing the androgen level of the PCOS rat, improving the ovulation state of the rat and reducing the oxidative stress level of the system and the local part of the PCOS rat; through a cell oxidative stress injury model, the polypeptide Humanin is proved to relieve oxidative stress injury caused by vitK3 by regulating a Keap1/Nrf2 signal channel and promote the expression of genes and proteins related to the Keap1/Nrf2 signal channel, namely the polypeptide Humanin plays an important role in the regulation of the oxidative stress of PCOS.

The exogenous polypeptide Humanin can obviously reduce the level of fasting blood glucose and fasting insulin of a PCOS rat, reverse the signal transduction abnormality of a carbohydrate metabolism pathway IRS1/PI3K/AKT and the protein expression abnormality of G L UT4 in ovary granular cells of the PCOS rat, and in a Humanin low expression model constructed by an ovary granular cell line COV434, the fact that the glucose uptake capacity of COV434 cells of the low expression Humanin is obviously reduced is found, the exogenous polypeptide Humanin can improve the glucose uptake of the COV434 cells, and reverse the expression abnormality of genes and proteins such as the carbohydrate metabolism pathway IRS1/PI3K/AKT signal transduction abnormality and G L UT4, the expression and transposition of G L UT4 in COV434 cells and primary rat granular cells are promoted, the polypeptide Humanin can improve the insulin resistance of the PCOS rat by targeting IRS1/PI3K/AKT pathways, and the polypeptide Humanin can be used as a target point for treating polycystic ovarian syndrome, and can be used as a medicine for developing a medicine for treating polycystic ovarian syndrome.

Drawings

FIG. 1 is a diagram showing the results of expressing polypeptide Humanin in follicular fluid and ovarian granulosa cells of patients with PCOS and in persons with normal ovarian function, wherein A is the concentration of polypeptide Humanin in follicular fluid, and B is the result of expressing the Humanin gene in ovarian granulosa cells;

FIG. 2 is a graph showing the results of the weight of rats (panel A) and the weight of ovaries (panel B) in each group in example 2;

FIG. 3 is a graph showing HE staining patterns of rat ovarian tissues in each group of example 2, wherein Conrol is a blank control group, PCOS is a PCOS rat model group, and the rest is a PCOS model + (2.5mg/kg, 5mg/kg, 10mg/kg) dose Humanin polypeptide group;

FIG. 4 is a graph showing the statistics of the number of primordial follicles (panel A) and antral follicles (panel B) in rats in each group in example 2;

FIG. 5 shows statistics of the number of corpus luteum (panel A), atretic follicle (panel B) and antral follicle (panel C) in rats in each group of example 2;

FIG. 6 shows the measurement results of the hormones in rats in the groups of example 2, wherein the A diagram shows the measurement results of the estrogen in the serum of the rat, and the B diagram shows the measurement results of the dehydroepiandrosterone in the serum of the rat;

FIG. 7 is a graph showing the results of detecting oxidative stress indicators in ovarian tissues of rats in each group in example 2; wherein, the A picture is the result of superoxide dismutase, the B picture is the result of catalase, and the C picture is the result of lipid peroxidation product malondialdehyde;

FIG. 8 is a graph showing the results of detecting oxidative stress indicators in rat sera in each group in example 2; wherein, the A picture is the result of superoxide dismutase, the B picture is the result of catalase, and the C picture is the result of lipid peroxidation product malondialdehyde;

FIG. 9 is a graph showing the identification of the ovarian granulosa cell line COV434 and primary rat granulosa cells and the verification of the expression of Humanin in example 3, wherein the first row 2 is a graph showing the identification of the ovarian granulosa cell line COV434 and the verification of the expression of Humanin in COV434, and the next row 2 is a graph showing the identification of rat primary granulosa cells and the verification of the expression of Humanin in rat, FSHR is a molecular Marker on the surface of ovarian granulosa cells, and Rattin is a homolog of Humanin in rats, namely the Humanin gene is called Rattin in rats;

FIG. 10 is a graph showing the results of the change in the cell viability of the ovarian granulosa cells COV434 caused by varying concentration gradients of vitK3 in example 3;

FIG. 11 is a graph showing the results of cytotoxicity of the polypeptide Humanin on COV434 cells in example 3;

FIG. 12 is a graph showing the results of the effect of exogenous complement polypeptide Humanin on the reduction in cell viability induced by vitK3 in example 3;

FIG. 13 is a graph showing the effect of the polypeptide Humanin on the activity of SOD induced by vitK3 (graph A) and the concentration of MDA (graph B) in example 3;

FIG. 14 is a graph showing the expression results of Kea1 and Nrf2 mRNAs in the white control group, the vitK3 group, the vitK3+ HNG group, and example 3;

FIG. 15 is a Western Blotting protein band diagram of Keap1, Nrf2, HO-1 and NQO1 proteins in the white control group, vitK3 group and vitK3+ HNG group in example 3;

FIG. 16 is a graph showing the results of Western Blotting expression quantification of Keap1 (Panel A), Nrf2 (Panel B), HO-1 (Panel C), and NQO1 proteins (Panel D) in the blank control group, vitK3 group, vitK3+ HNG group, and example 3;

FIG. 17 is a graph showing the results of the polypeptide Humanin in example 3 promoting the expression of Nrf2 in COV434 and translocation from cytoplasm to nucleus;

FIG. 18 is a graph showing the results of the polypeptide Humanin in example 3 promoting the expression of Nrf2 in primary rat granulocytes and translocation from the cytoplasm to the nucleus;

FIG. 19 is a graph showing the results of the fasting blood glucose level (panel B) and the change in insulin concentration (panel A) in PCOS rats after exogenous supplementation of the polypeptide Humanin in example 4;

FIG. 20 is a graph showing that IHC verifies expression of a glycometabolism pathway-associated protein in rat ovarian granulosa cells in example 4;

FIG. 21 is a graph showing the results of Western Blotting method in example 4 for detecting the expression of IRS1/PI3K/AKT and G L UT4 in ovarian granulosa cells;

FIG. 22 is a western blotting quantitative statistical chart of the expression of IRS1/PI3K/AKT and G L UT4 proteins in rat ovarian granulosa cells in example 4;

FIG. 23 is a graph of transfection efficiency of knockdown Humanin in COV434, wherein the left graph is a siRNA negative control graph with red fluorescent protein, and the right graph is a white light graph;

FIG. 24 is a graph of qRT-PCR validation of the knockdown efficiency of human in COV434 for 12h (left panel), 24h (middle panel) and 48h (right panel) knockdown;

FIG. 25 is a graph showing that Western Blotting verifies the knockdown effect of knockdown Humanin in COV 434;

FIG. 26 shows that knocking down the expression of Humanin in COV434 causes the glucose metabolism ability to be decreased, and exogenous HNG supplementation can increase the glucose metabolism uptake of ovarian granulosa cells, wherein a represents that the difference is statistically significant compared with the 10ng/m L insulin group, p is less than 0.05, a represents that the difference is statistically significant compared with the control group, p is less than 0.05, a represents that the difference is statistically significant compared with the siRNA1 knocking down 10ng insulin group, p is less than 0.05, & represents that the difference is statistically significant compared with the siRNA2 knocking down 10ng/m L insulin group, p is less than 0.05, a represents that the difference is statistically significant compared with the 10ng/m L insulin +1 μ g/m L HN group, and p is less than 0.05;

FIG. 27 shows the results of expression of genes involved in the sugar metabolism pathway after knocking down the Humanin gene, wherein in Panel A, a statistic significance is shown for the difference from the control group, # a statistic significance is shown for the difference from the HN-siRNA1 group, # a statistic significance is shown for the difference from the control group, # a statistic significance is shown for the difference from the HN-siRNA1 group, # a statistic significance is shown for the difference from the 10 nginsun group, # a statistic significance is shown for the difference from the control group, & a statistic significance is shown for the difference from the 10 nginsun group, # a statistic significance is shown for the difference from the 10ng nsulin +5 μ g/m L HN group;

FIG. 28 shows the results of experiments on exogenous supplementation of polypeptide Humanin to promote the expression of G L UT4 in COV434 and translocation to cell membrane;

FIG. 29 shows the results of exogenous polypeptide supplementation Humanin promoting the expression of G L UT4 in primary rat granulosa cells and translocation to the cell membrane;

the same symbols in the above figures indicate that the difference is not statistically significant, p < 0.05.

Detailed Description

The substance of the present invention will be further described below by way of examples, but the substance of the present invention is not limited thereto, and the methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional time-sold reagents or reagents formulated according to conventional methods unless otherwise specified.

Example 1: concentration of polypeptide Humanin in follicular fluid of PCOS patients and population with normal ovarian function and expression of polypeptide Humanin in ovarian granulosa cells

Collecting follicular fluid and ovarian granulosa cells of PCOS patients undergoing IVF/ICSI assisted pregnancy and patients (population with normal ovarian function) undergoing assisted pregnancy by non-ovarian factors by adopting assisted reproductive technology; patients should first be fully informed and informed consent written prior to collection of samples by ethical committee approval.

1. The PCOS group patients are selected from ① women, 22-35 years old, ② diagnosis PCOS according to the Pyrola standard, ③ patients have insulin resistance, the exclusion standard is ① patients with other endocrine diseases such as hyperthyroidism and hypothyroidism, ② patients with other ovarian diseases such as chocolate cyst, and the control group is patients with non-ovarian factors assisted by assisted reproductive technology (infertility patients caused by oviduct factors).

2. Collecting follicular fluid: collecting follicular fluid during ovum collection, centrifuging at 4 deg.C for 10min at 1000g, and collecting supernatant and storing at-80 deg.C.

3. Extracting and culturing primary ovarian granulosa cells, namely collecting 23 cases of follicular fluid of patients in a PCOS group and a control group, centrifuging for 10min at 25 ℃ and 1200r, discarding supernatant, resuspending with a DMEM/F12 culture medium and hyaluronidase with the mass concentration of 50-100 mu L% and 1%, whipping and uniformly mixing, digesting for 10min in water bath at 37 ℃, adding a DMEM/F12 culture medium containing 15% FBS to stop digestion, whipping suspension for 2min, centrifuging for 5min at 800r, washing cell sediment with PBS buffer solution, centrifuging for 5min at 800r again, removing supernatant to remove hyaluronidase, adding erythrocyte lysate with the volume being 3-5 times of that of the sediment into the cell sediment, whipping the sediment, centrifuging to remove a small amount of erythrocytes, washing and centrifuging with the PBS buffer solution, and taking the sediment for subsequent experiments.

4. The polypeptide Humanin in the follicular fluid is measured by a commercial E L ISA kit (Wuhan Huamei biology), the measuring method is carried out according to the kit operation instructions, and the expression of the polypeptide Humanin in the ovarian granulosa cells is measured by a qRT-PCR method, wherein the method comprises the following steps:

4.1 extraction of Total RNA from human Primary granulosa cells

(1) Adding 1m L Trizol into the cell sediment, gently blowing and uniformly mixing, and standing on ice for 15 min;

(2) transferring the lysate into an EP tube, adding 200 mu L chloroform, reversing and uniformly mixing for several times, and standing on ice for 10 min;

(3) centrifuging at 4 deg.C and 12000g for 15min, and observing that the liquid is divided into three layers, wherein the lowest layer red liquid is phenol chloroform phase, the middle layer is relatively thin milky white organic phase, and the upper layer is colorless and is RNA dissolved water phase;

(4) placing the upper water phase in a new EP tube to avoid organic phase pollution, adding isopropanol with the same volume, turning upside down and mixing uniformly to obtain a small amount of flocculent precipitate, standing on ice for 10min, centrifuging at 12000g for 10min at 4 ℃ again, and observing the formation of a small amount of white precipitate at the bottom of the EP tube after centrifugation to obtain RNA precipitate;

(5) removing supernatant, adding 75% alcohol prepared by RNase free into the precipitate, blowing the precipitate with 200 μ L gun head, centrifuging at 7500g and 4 deg.C for 5 min;

(6) removing alcohol, removing the alcohol solution as much as possible by using a pipette, and drying by blowing in a super clean bench (the drying cannot be completely carried out, so that the reduction of RNA solubility is avoided);

(7) adding a proper amount of RNase free water to dissolve the precipitate, measuring the concentration and the purity of the RNA by using the Nanodrop2000, wherein the OD260/280 ratio is more than 2.0 (between 2.0 and 2.2), which indicates that the RNA purity is better;

4.2 removal of DNA contamination by impurities in RNA

(1) Reagents were added according to the experimental procedures of the Takara kit instructions;

(2) mixing, centrifuging and putting into a PCR instrument, wherein the reaction conditions are as follows: 42 ℃ for 2 min; infinity at 4 ℃;

4.3 reverse transcription into DNA

(1) Preparing reaction liquid according to the steps of the specification

(2) Mixing, centrifuging and putting into a PCR instrument, wherein the reaction conditions are as follows: 15min at 37 ℃; 5min at 85 ℃; infinity at 4 ℃;

4.4 fluorescent quantitative PCR

(1) Primer sequences of the Humanin, Keap1 and Nrf2 genes are designed and synthesized in advance, and the specificity of the primer sequences is detected

(2) Performing fluorescent quantitative PCR on a PCR amplification instrument by adopting SYBR Green I fluorescent dye, wherein the reaction solution is prepared as follows:

the reagents are mixed evenly, centrifuged and placed in a PCR instrument in a dark place, and the reaction steps are as follows:

① pre-denaturation at 95 deg.C for 30s, denaturation at ② at 95 deg.C for 5s, annealing at 60 deg.C for 30s, elongation at 72 deg.C for 30s for 40 s

③ dissolution curve, 95 ℃, 15s, 60 ℃, 60s, 95 ℃ and 1 s;

the experimental result is shown in figure 1, and the expression of the polypeptide Humanin gene in the follicular fluid and ovarian granulosa cells of PCOS patients is obviously lower than that of the normal ovarian function population.

Example 2: establishment of PCOS rat model and experiment for influence of exogenous supplementary polypeptide Humanin (HNG) on oxidative stress, endocrine and ovulation states of PCOS rat

1. Construction of PCOS rat model

Adopting DHEA (dehydroepiandrosterone) to establish a rat PCOS model, selecting 21-day-old female SD rats (about 45 g), injecting DHEA subcutaneously with injection amount of 6mg/100g body weight, dissolving DHEA with 0.2m L injection oil, and continuously injecting for 20 days;

2. 60 SD rats of 21 days old were purchased from the university of science and technology laboratory animals center in Huazhong, and the rats were randomly divided into 5 groups of 12 rats each, and the specific groups were as follows:

A. PCOS rat model group

B. PCOS model + Low dose HNG group

C. PCOS model + medium dose HNG group

D. PCOS model + high dose HNG group

E. Blank control group

The rats in groups A-D are PCOS rat models, wherein the rats in groups B-D are injected with HNG intraperitoneally every day for two weeks (namely one week after the model construction is finished) continuously from one week before the model construction is finished, wherein the injection amount of the rats in group B every day is 2.5mg/kg of body weight, the injection amount of the rats in group C is 5mg/kg of body weight, and the injection amount of the rats in group D is 10mg/kg of body weight; group E is blank.

3. Measurement of rat body weight and ovarian weight

After HNG intervention is finished, weighing each group of rats, recording the weight of the rats in each group, then killing the rats, collecting venous blood and taking out ovarian tissues, and weighing and recording the ovarian tissues;

the results are shown in FIG. 2, and it can be seen that exogenous supplementation with the polypeptide Humanin significantly reduces the weight of PCOS rats and the weight of ovaries, as compared to the PCOS rat model group.

4. Observation of ovarian morphology, and enumeration of primordial, Antral (Antral), atretic (atretic), luteal and cystic (cystic) follicles

(1) Fixing: fixing the ovary tissues collected in the step (3) in paraformaldehyde solution, and standing at 4 ℃ for 12 hours;

(2) tissue gradient alcohol dehydration: then, sequentially placing the fixed ovarian tissues in 70%, 75% and 80% ethanol solutions of mass concentration for respectively treating for 8 hours, and sequentially placing in 95% and 100% ethanol for respectively dehydrating for 2 hours;

(3) after dehydration, placing the tissue in xylene and completely soaking for 5min, and then placing the specimen in new xylene again;

(4) embedding: placing the ovarian tissue specimen in paraffin of 54 ℃, soaking the ovarian tissue specimen in the paraffin for 2 hours, then taking out the ovarian tissue specimen, and forming the ovarian tissue specimen at normal temperature;

(5) slicing: cutting the wax block into slices with the thickness of 4-5 mu m, fixing the slices on a glass slide, and putting the slices in an oven at 60 ℃ overnight;

(6) performing HE staining on the section, and performing follicle counting under a microscope;

(7) counting follicles by using 3 sections of each ovary, and counting 6 ovaries in each group;

the HE staining results of rats in each group are shown in figure 3, the counting results are shown in figures 4 and 5, and it can be seen that exogenous supplementary polypeptide Humanin can increase the proportion of follicle in corpus luteum and reduce the proportion of atretic follicle and follicle; the exogenous supplementary polypeptide Humanin can promote the follicular development of PCOS rats and promote ovulation.

5. Hormone level determination in rats of each group

(1) Collecting venous blood of a rat, standing the venous blood at 37 ℃ for 30min, placing the venous blood in a refrigerator at 4 ℃ for 1h after the blood is coagulated, and collecting serum;

(2) centrifuging collected serum at 4 deg.C for 10min at 10000g, and removing insoluble substances;

(3) placing the serum in a refrigerator at-80 deg.C for use;

(4) measuring the hormone content in rat serum by adopting an estrogen and dehydroepiandrosterone E L ISA kit (Wuhan Gene Mei);

the results are shown in FIG. 6, which shows that exogenous supplementation of polypeptide Humanin at 5mg/kg and 10mg/kg can reduce androgen level in PCOS rats, and shows that exogenous supplementation of polypeptide Humanin can improve endocrine state of PCOS rats and improve hyperandrogenism of PCOS rats.

6. Determination of serum and ovary local oxidative stress indexes of rats in each group

(1) Taking the serum collected in the step 5, and preparing ovarian tissue homogenate according to the kit instruction;

(2) detecting Superoxide Dismutase (SOD), Catalase (CATalase, CAT) activity and Malondialdehyde (MDA) concentration of lipid peroxidation product in homogenate of rat serum and ovarian tissues by using a kit purchased from Nanjing Biochemical Limited company, carrying out experimental operation according to the kit specification, and finally reading an experimental result by using a 96-hole enzyme labeling instrument;

the results are shown in FIGS. 7 and 8, and it can be seen that exogenous supplementation with the polypeptide Humanin reduces the systemic and local oxidative stress level of PCOS rats, indicating that exogenous supplementation with the polypeptide Humanin can relieve the oxidative stress imbalance of PCOS rats.

Example 3: construction of a cell oxidative stress injury model, a molecular mechanism experiment 1 for relieving oxidative stress by polypeptide Humanin, identification of an ovary granular cell line COV434 and primary rat granular cells, and a verification experiment of Humanin in expression thereof

1) Preparation of COV434 cell slide

a. Digesting COV434 cells by a pancreatin infiltration method, namely removing the culture medium, washing for 2 times by PBS, adding pancreatin of 300 mu L into a cell culture bottle, completely infiltrating the cells, sucking out the pancreatin by a pipette gun, removing the pancreatin, and adding the complete culture medium to stop digestion when the cells are digested into a sandy state;

b. inoculating the cells into a 24-hole culture dish containing a cell slide, and culturing in an incubator until the cells grow to a required experimental density;

2) extraction of rat primary granular cells and preparation of cell slide

a. Injecting PMSG 15U into abdominal cavity of SD rat of 26 days old, killing rat after 48h, taking off bilateral ovaries from cell to cell, putting ovaries into a culture dish of sterile PBS to wash away bloodstain, transferring ovaries into IMDM complete medium containing 10% serum and 1% double antibody, puncturing follicles with a needle of a 1m L syringe under a microscope, releasing ovary granular cells, and removing ovary impurities and small ovaries;

b. collecting IMDM complete culture medium containing ovarian granulosa cells in a culture dish into a centrifuge tube with the length of 15m L, centrifuging for 5min at the speed of 1000g, and adding a proper amount of complete culture medium for resuspension;

c. inoculating primary rat granular cells into a 24-hole plate containing a cell slide, placing the plate in an incubator for culturing for 48 hours, changing the liquid, continuing culturing the cells until the cells reach the density required by the experiment, and taking out the slide for carrying out the subsequent experiment;

3) immunofluorescence

a. Taking out the cell slide from the 6-well plate, and gently washing the slide for 2 times by using PBS;

b. fixing the slide in 4% paraformaldehyde for 15min, and washing with PBS twice;

c. breaking the membrane with 0.1% of Ttiton100 for 20min, and washing with PBS twice;

d. blocking with 5% BSA at room temperature for 30 min;

e. adding diluted primary antibody, placing in a wet box at 4 deg.C in a refrigerator overnight, and washing with PBS for 5min for 4 times;

f. adding diluted fluorescent secondary antibody, incubating at room temperature in dark for 30min, washing with PBS for 4 times, each for 5 min;

g. sealing the anti-fluorescence quencher and taking a picture;

the results are shown in FIG. 9, from which it can be seen that the polypeptide Humanin is expressed in both the ovarian granule cell line COV434 and rat primary ovarian granule cells.

2. Construction of cell oxidative stress injury model

Treating the ovary granular cell line COV434 cells with 0, 20, 40, 60, 80 and 120 mu mol/L menadione (VitK3), placing the treated cells into an incubator to be cultured for 24 hours, measuring the cell viability by using a CCK8 kit (according to the kit operation instructions), and taking the concentration of vitK3, of which the cell viability is about 50% of the cell viability of a control group, as the concentration for subsequent experiments;

(1) per hole 10⁴Inoculating 96-well plates at the density of each cell, and culturing for 24h until 70-80% of cells are combined;

(2) adding VitK3(0, 20, 40, 60, 80, 120 μmol/L) with different concentrations into cells, at least 4 multiple wells each, culturing for 24h, removing cell culture medium containing VitK3, and washing twice with sterile PBS;

(3) mixing DMEM complete culture medium and CCK8 reagent in the volume ratio of 9 to 1, and adding into 96-well plate with each well being 100 mu L;

(4) continuously placing the 96-well plate in an incubator for culturing for 1-3h, measuring the absorbance value of each hole at 450nm by using a 96-well microplate reader, and calculating the corresponding cell activity;

the results are shown in FIG. 10, from which it can be seen that the cell viability of COV434 decreases with increasing concentration of VitK3, and about 50% when the concentration of VitK3 is 80. mu. mol/L.

3. Cytotoxicity test of polypeptide Humanin on COV434

After treating the cells of the nest granular cell line COV434 with 0, 5, 10, 20, 40, 80 and 160 mu mol/L of polypeptide Humanin for 24h, measuring the cell viability by using a CCK8 kit;

(1) per hole 10⁴Inoculating 96-well plates at the density of each cell, and culturing the cells to reach 70-80% union after 24-cell culture;

(2) adding Humanin (0, 5, 10, 20, 40, 80, 160 μ g/m L) with different concentrations into cells, at least 4 wells for each concentration, removing cell culture medium containing VitK3 after 24h of culture, and washing twice with sterile PBS;

the results are shown in FIG. 11, from which it can be seen that COV434 does not increase in cell viability with increasing concentrations of Humanin in the concentration range of 0 to 160. mu. mol/L, indicating that Humanin has no toxic effect on cells in this concentration range.

4. Experiment on influence of exogenous supplemented polypeptide Humanin on COV434 cell viability caused by vitK3

Treating COV434 cells with polypeptide Humanin with the concentrations of vitK3 and vitK3 causing cell viability to be reduced by about 50% and 0, 2.5, 5, 10, 15 and 20 mu g/m L respectively, and measuring cell viability after 24 hours;

(1) per hole 10⁴Density of individual cellsPlanting 96-well plate, culturing for 24h, and culturing to obtain 70-80% confluency;

(2) adding 80 μmol/L and Humanin (0, 2.5, 5, 10, 15, 20 μ g/m L) with different concentrations into cells, each concentration at least 4 multiple wells, removing the cell culture medium containing VitK3 after culturing for 24h, and washing twice with sterile PBS;

the results are shown in FIG. 12, from which it can be seen that the exogenous supplement polypeptide Humanin can significantly alleviate the cell viability decrease caused by VitK3, indicating that the exogenous supplement polypeptide Humanin can significantly alleviate oxidative stress injury caused by VitK 3.

5. Experiment for relieving COV434 oxidative stress injury caused by vitK3 by polypeptide Humanin through regulating Keap1/Nrf2 signal channel

5.1 Effect experiment of polypeptide Humanin on SOD activity and MDA concentration caused by vitK3

(1) After being trypsinized, COV434 cells are inoculated in a culture dish with the diameter of 6cm, and 3 grouped blank control groups, a vitK3 group and a vitK3+ HNG group are arranged;

(2) when the cell density reaches 70-80% confluency, treating the cells respectively with complete culture medium, complete culture medium containing 80 mu mol/L of vitK3 and complete culture medium containing 80 mu mol/L of vitK3 and 5 mu g/m L of HNG for 24 h;

(3) collecting cells, and measuring the activity of SOD and the concentration of MDA according to the operation steps on the instruction manual of an SOD and MDA measuring kit (Nanjing construction);

the results are shown in fig. 13, from which it can be seen that HNG is able to reverse the decrease in SOD activity and MDA concentration caused by vitK 3.

5.2 qRT-PCR test for Keap1 and Nrf2 mRNA expression in three groups

Respectively extracting RNA of the three groups of cells, performing reverse transcription to form cDNA, and performing the same experimental steps as example 1 by using qRT-PCR to measure the expression of Keap1 and Nrf2 mRNA in the three groups;

the experimental result is shown in fig. 14, and it can be seen from the figure that exogenous complementary polypeptide humain can significantly inhibit the expression of Keap1mRNA and can significantly improve the expression of Nrf2 mRNA.

5.3, Weastern Blotting detecting the expression conditions of pathway-associated proteins Keap1, Nrf2, HO-1 and NQO1 in the three groups;

(1) protein extraction, namely inoculating cells into a 6-well plate, carrying out VitK3 or VitK3 HNG treatment when the cells reach 70-80% of union, collecting the cells after 24h treatment, discarding a culture medium in the 6-well plate, washing 3 times by PBS, adding 100 mu L RIPA lysate containing 1 mu L PMSF and 1 mu L phosphatase inhibitor into the culture medium, scraping the cells by the cells, placing the cells on ice for lysis for 10min, centrifuging at 12000rpm at 4 ℃ for 15min, taking supernatant into a clean EP tube, taking out 2-4 mu L for measuring the concentration of the protein on the Nanodrop2000, and storing the cells in a refrigerator at-80 ℃;

(2) protein concentration by BCA method: drawing a standard curve according to the operation steps of the Byunnan BCA protein concentration detection kit, and calculating the concentration of the extracted protein according to the standard curve;

(3) protein denaturation, namely uniformly mixing the protein liquid and 5 × protein loading buffer solution according to the volume ratio of 4:1, putting into a water bath kettle at 97 ℃ for boiling for 10min, and preserving the denatured protein at-80 ℃;

(4) preparing glue: preparing concentrated gel and separation gel by adopting a Biyuntian SDS-PAGE polyacrylamide gel electrophoresis kit;

(5) adding sufficient electrophoretic liquid (prepared from Biyuntian electrophoretic buffer solution powder) into an electrophoretic bath, pulling out comb teeth to ensure that the sample loading amount of each hole is the same, adding protein indication markers 5 mu L at two ends of a sample lane, connecting a power supply of an electrophoresis device, then, running the gel to the bottom of concentrated gel at constant voltage of 80V, wherein the bromophenol blue lines of all samples are flush for about 30min, then adjusting the voltage to 120V, running the separated gel at constant voltage until the bromophenol blue moves to the bottom of the separated gel, and the markers indicate that the separation is complete, namely, the power supply can be turned off for about 60-90 min;

(6) film transfer: preparing a membrane conversion solution, placing the membrane conversion solution in a refrigerator for precooling for standby at 4 ℃, cutting a PVDF membrane according to the size of gel, then placing the PVDF membrane in methanol for activation for 2-3min, taking off a glass plate, cutting the gel according to Marker instructions to ensure the wetting and integrity of the gel, pouring a membrane conversion buffer solution prepared in advance into a tray, and fully soaking filter paper and sponge; placing a film transferring clamp to enable the black surface of the film transferring clamp to face downwards, and sequentially placing a film transferring sponge cushion, filter paper, a target and internal reference colloid, a PVDF film, the filter paper and the sponge cushion from bottom to top; putting the film transferring clamp into a film transferring groove, adding the precooled film transferring liquid, and transferring the film for 60min on ice with the voltage adjusted to 280 mA;

(7) and (3) sealing: after the membrane conversion is finished, putting the PVDF membrane into pre-prepared 5% skimmed milk, and sealing on a shaking table at room temperature for 1 h;

(8) applying a first antibody: diluting the primary antibody with 5% skimmed milk, placing the PVDF membrane in the primary antibody diluent, and refrigerating at 4 deg.C overnight;

(9) washing the membrane and applying a secondary antibody, namely washing the membrane for 3 times by using the freshly prepared TBST, diluting the secondary antibody by using 5% skimmed milk according to the proportion of 1:5000 for 10min each time, then placing the membrane in a secondary antibody diluent for sealing, incubating for 1-2h at room temperature, washing the membrane for 3 times × 10min in a shaking table by using the same TBST, and then soaking in a TBST solution for subsequent exposure;

(10) exposure, namely preparing EC L luminous liquid, uniformly mixing the liquid A and the liquid B in the Biyunyan EC L chemiluminescence kit according to the proportion of 1:1 and placing the mixture in a dark place, putting a PE glove with a PVDF membrane into a cassette of an exposure instrument, adding a proper volume of luminous liquid on the PVDF membrane, fully covering the membrane and standing for 3min, then opening an exposure program of Bio-Rad ChemiDox XRS to expose the PVDF membrane, collecting the gray value of a strip, taking GAPDH as an internal reference, and analyzing the expression amount of protein;

the experimental results are shown in fig. 15 and 16, and it can be seen from the graphs that exogenous addition of the polypeptide Humanin can inhibit the expression of the Keap1 protein and improve the expression of Nrf2, HO-1 and NQO1 proteins, which indicates that exogenous addition of the polypeptide Humanin can relieve oxidative stress injury caused by VitK3 by regulating a Keap1/Nrf2 signal channel.

5.4, the immunofluorescence verification polypeptide Humanin can activate the expression of Nrf2 in COV434 and primary rat granulosa cells and promote the transfer of Nrf2 from cytoplasm to nucleus

(1) The preparation of COV434 cell slide, the extraction of rat primary granular cells, the preparation of slide and the immunofluorescence experiment method are the same as those in step 1 of example 3;

the experimental results are shown in fig. 17 and 18, and it can be seen from the graphs that the fluorescence intensity of Nrf2 is further enhanced after exogenous polypeptide human is supplemented, that is, the exogenous polypeptide human can further enhance the expression of Nrf2 in ovarian granulosa cells, and the fluorescence intensity of Nrf2 is obviously enhanced in the nucleus part after HNG is added, compared with the other two groups, which indicates that the polypeptide human can obviously promote the translocation of Nrf2 to the nucleus.

Example 4: experiment on influence of exogenous supplemented polypeptide Humanin on PCOS rat insulin resistance

1. Constructing a PCOS rat model by the same method as the construction of the PCOS rat model in the example 2;

after the completion of the HNG pretreatment, the rats were sacrificed and venous blood was collected to collect ovarian tissue.

2. Measurement of fasting plasma glucose level and fasting insulin concentration in rats

(1) The steps of collecting rat serum were the same as those of the rat hormone level measurement in example 2;

(2) detecting the fasting blood glucose level (Wuhan gene American organism) of each group of rats by adopting a glucose oxidase method, and carrying out experimental operation according to the instruction of the kit;

(3) detecting the fasting insulin level of the serum of each group of rats (Beijing prilley gene technology, Inc.) by applying an ISA method of a rat serum insulin determination kit E L, and carrying out experimental operation according to the instruction of the kit;

the experimental result is shown in FIG. 19, from which it can be seen that the exogenous polypeptide Humanin can obviously reduce the fasting blood glucose and fasting insulin level of PCOS rats, which indicates that the exogenous polypeptide Humanin can improve the carbohydrate metabolism of PCOS rats.

3. IHC detects the expression of IRS1/PI3K/AKT and G L UT4 in ovarian granulosa cells

(1) Dewaxing, namely sequentially putting the slices obtained in the step 4 in the example 2 into a container ①②③ containing xylene, soaking for 10min each time, sequentially putting the slices into 100%, 95%, 90%, 80% and 75% alcohol for treatment for 10min each time, and finally washing with PBS for 3 times, 5min each time;

(2) removal of endogenous peroxidase: with 3% of H₂O₂Soaking the slide or slide in the solution for 20min, and washing with PBS for 5min for 3 times;

(3) preparing 0.01 mol/L citrate buffer solution, putting the slices into a microwave oven, heating and boiling for 15min after the slices are completely immersed, wherein the antigen repairing solution is enough to avoid drying the slices;

(4) the slides were cooled at room temperature and then washed 3 times with PBS, 10min each;

(5) and (3) sealing: dripping 5% BSA onto the slices or slide, sealing at room temperature for 30min, and washing with PBS for 5min for 3 times;

(6) primary antibody incubation: diluting the primary antibody with 5% BSA, dripping the primary antibody on the slice, putting the slice into a wet box, incubating the slice overnight in a refrigerator at 4 ℃, and setting a corresponding negative control;

(7) taking out the product the next day, rewarming at room temperature for 1h, washing with PBS for 3 times, 5min each time;

(8) and (3) secondary antibody incubation: diluting the horseradish complex labeled secondary antibody with 5% BSA to a corresponding concentration, dripping the secondary antibody on the slice, placing the slice in a wet box, incubating for 1h at room temperature, washing for 3 times with PBS (phosphate buffer solution) for 5min each time, and not adding the corresponding secondary antibody for a negative control;

(9) color development: dyeing with freshly prepared DAB for about 3min, re-dyeing with hematoxylin dye for 2min, washing with PBS for 3 times (1 min each time) after washing off the dye with tap water, and soaking in pure water to turn blue;

the experimental results are shown in FIG. 20, and it can be seen that exogenous supplementation with the polypeptide Humanin can promote protein expression of sugar metabolism pathways IRS1/PI3K/AKT and G L UT4 in PCOS rat ovarian granulosa cells.

4. Western Blotting method for detecting protein expression in ovarian granulosa cells

(1) Collecting ovary tissue of each group of rats, washing with sterile PBS buffer solution, placing into culture dish containing 10% serum and 1% double-antibody IMDM complete culture medium, penetrating follicle with 1m L syringe needle under microscope, removing ovary debris and small follicle, collecting granulosa cells, centrifuging for 5min, discarding supernatant, washing cells with PBS twice

(2) Adding 200 μ L RIPA lysate containing protease inhibitor and phosphatase inhibitor to the precipitate to detect the expression of IRS1, PI3K, AKT, G L UT4 (see 5.3 in example 3 for details)

The experimental results are shown in FIGS. 21 and 22, and it can be seen from the graphs that the exogenous supplementation of the polypeptide Humanin can improve the protein expression of PCOS rat ovary granular cell carbohydrate metabolism pathways IRS1/PI3K/AKT and G L UT4, which indicates that the polypeptide Humanin can relieve local insulin resistance of the PCOS rat ovary.

Example 5: effect of exogenous supplement of polypeptide Humanin on glucose metabolism ability of ovarian granule cell line COV434 cells 1, knocking down expression of Humanin in COV434 granule cells

(1) After COV434 cell pancreatin digestion, inoculating the cell to a 6-well plate, putting the cell into an incubator to culture until the cell density reaches 50-60% and starting a transfection experiment, respectively setting a siRNA-NC control group with red fluorescence and two pairs of interference sequences of Humanin, an HN-siRNA1 group and an HN-siRNA2 group, and adding a Mock transfection group (Mock group) only adding a transfection reagent L ipo3000 when determining the change of the protein level after transfection;

the interfering sequences are as follows: siRNA1 r (CCAGUGAAAUUGACCUGCC) d (TT)

siRNA2:r(GGGUUCAGCUGUCUCUUAC)d(TT)

a. 10 μ L siRNA (20 μ M) per well was mixed with 200 μ L serum-free diluent (Opti-MEM) and mixed well;

b. adding 200 mu L serum-free diluent (Opti-MEM) into lipo3000 of 4 mu L per well, mixing, and standing for 5 min;

c. uniformly mixing the diluted siRNA with the diluted L ipo3000, and standing for 15min to form a transfection mixture;

d. the transfection mixture of 414 μ L was added to a 6-well plate well containing 1586 μ L complete medium to a final volume of 2m L at a final concentration of 100 nM;

e. after 6h of transfection, the cell status was observed, and the transfection efficiency of the siRNA-NC negative control with red fluorescent protein (i.e., cells with red fluorescence, which account for the proportion of total cells seen under white light) was observed and photographed under a fluorescence microscope;

f. continuously culturing for 12-48h, and detecting the knockdown efficiency by using a qRT-PCR method;

g. protein is extracted at the time point with the best knockdown efficiency, and protein level knockdown is detected by Western Blotting;

the results are shown in FIGS. 23, 24 and 25, and it can be seen from FIG. 23 that the transfection efficiency of siRNA can reach more than 95%, and from FIG. 24 that the knockdown efficiency of siRNA is highest after 24h transfection and reaches more than 70%, and from FIG. 25 that the expression of polypeptide Humanin is obviously reduced in the siRNA1 and siRNA2 groups after 24h transfection.

2. Experiment for influence of Humanin gene expression down-regulation and exogenous HNG supplementation on glucose metabolism capability

(1) After passage of COV434 cells, the cells were pressed 10⁴Inoculating the cells into 96-well plate at density of each well, culturing for 24 hr until cell density reaches 70-80%, discarding culture medium, gently washing with PBS for 3 times, and grouping according to different intervention experiments on cells as follows

A. Control group COV 434;

B. control group COV434+10ng/m L insulin;

C. control group COV434+10ng/m L insulin + various concentrations of HNG (1, 2.5, 5. mu.g/m L);

D. COV434 of low expression humanin

E. COV434+10ng/m L insulin with low expression of humanin

F. COV434+10ng/m L insulin + different concentrations of HNG (1, 2.5, 5. mu.g/m L) with low expression of humanin

(2) siRNA interfered the expression of Humanin in COV434 cells, namely diluting the siRNA with DEPC water into a 20 mu M stock solution, washing the cells after 24-year culture with PBS, adding 0.5 mu L of siRNA and 0.25 mu L of lipo3000 into each hole according to the density of 100nM siRNA, and adding 100 mu L of DMEM complete culture medium into the non-Humanin low expression group;

(3) changing the liquid after siRNA interference for 6 hours, respectively adding 10ng/m L insulin or/and HNG with different concentrations according to groups, continuously culturing for 24 hours, collecting the culture medium, and measuring the glucose concentration in the culture medium by using a glucose oxidase method;

(4) the glucose consumption of each group for 24h can be calculated by setting a complete medium control well without cells to calculate the total glucose content in the medium minus the measured glucose amount in the medium.

The results are shown in FIG. 26, which shows that the down-regulation of the gene expression of Humanin can cause the reduction of the glucose metabolism, and the exogenous HNG supplementation can improve the glucose metabolism of ovarian granulosa cells and increase the consumption of glucose.

3. Experiment for influence of knock-down Humanin gene on expression of genes related to glycometabolism pathway

Collecting culture medium to measure glucose consumption, collecting cell RNA, reverse transcribing into cDNA, and measuring the mRNA expression level of IRS1, AKT and G L UT4 in the cell by qRT-PCR (the specific experimental method of qRT-PCR is the same as that of measuring Humanin protein in follicular fluid and ovarian granulosa cells in example 1);

the results are shown in FIG. 27, from which it can be seen that exogenous HNG supplementation promotes the mRNA expression levels of the sugar metabolism pathway IRS1/PI 3K/AKT-related gene and G L UT4 in PCOS rat ovarian granulosa cells;

4. exogenous supplement polypeptide Humanin promotes expression of G L UT4 in COV434 and translocation experiment to cell membrane

The procedures of preparing COV434 cell slide and immunofluorescence were the same as those of the identification of the ovary granular cell line COV434 and primary rat granular cells and the verification experiment of humanin in its expression in example 3

The results are shown in FIG. 28, from which it can be seen that exogenous HNG supplementation promotes the expression of G L UT4 in COV434 and translocation to the cell membrane.

5. The steps of the preparation of the cell climbing sheet of the primary rat granular cell and the immunofluorescence of the exogenous supplementary polypeptide Humanin on the expression of G L UT4 in the primary rat granular cell and the cell membrane translocation experiment are the same as the steps of the identification of the ovary granular cell line COV434 and the primary rat granular cell and the verification experiment of the expression of the Humanin in the example 3

The results are shown in FIG. 29, from which it can be seen that exogenous supplementation with the polypeptide Humanin can promote the expression and translocation of G L UT4 to the cell membrane of primary rat granulosa cells.

In conclusion, exogenous supplementation of polypeptide Humanin can obviously reduce the fasting blood glucose and fasting insulin levels of PCOS rats and promote the protein expression levels of sugar metabolism pathways IRS1/PI3K/AKT and G L UT4 in ovary granular cells of the PCOS rats, and in a Humanin low-expression cell model constructed by ovary granular cell line COV434 cells, the fact that the glucose uptake capacity of COV434 cells with low expression of the Humanin is reduced, exogenous supplementation of HNG can improve the glucose uptake of COV434 cells, promote the expression of genes related to the sugar metabolism pathways IRS1/PI3K/AKT and G L UT4 and promote the expression and cell membrane translocation of G L UT4 in COV434 cells and primary rat granular cells is found that the polypeptide Humanin can improve the insulin resistance of the PCOS rats by targeting IRS1/PI3K/AKT pathways.

Sequence listing

<110> rich and lively

New application of <120> polypeptide Humanin

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>21

<212>DNA

<213> Artificial sequence (Artificial)

<400>1

tcagcgacgg aaagagtatg a 21

<210>2

<211>22

<212>DNA

<213> Artificial sequence (Artificial)

<400>2

ccactggttt ctgactggat gt 22

<210>3

<211>22

<212>DNA

<213> Artificial sequence (Artificial)

<400>3

attcagctga gtgttactac cc 22

<210>4

<211>22

<212>DNA

<213> Artificial sequence (Artificial)

<400>4

cagcatagat acagttgtgc ag 22

<210>5

<211>22

<212>DNA

<213> Artificial sequence (Artificial)

<400>5

ctgaatggct ccacgagggt tc 22

<210>6

<211>22

<212>DNA

<213> Artificial sequence (Artificial)

<400>6

cctgcgggcc tatttggtct tg 22

<210>7

<211>21

<212>DNA

<213> Artificial sequence (Artificial)

<400>7

ccagugaaau ugaccugcct t 21

<210>8

<211>21

<212>DNA

<213> Artificial sequence (Artificial)

<400>8

ggguucagcu gucucuuact t 21

Claims

1. The application of the polypeptide Humanin gene as an action target in screening and preparing the medicine for treating polycystic ovarian syndrome.

2. Application of polypeptide Humanin in preparing medicine for treating polycystic ovarian syndrome.