CN111592581B

CN111592581B - Polypeptide compound for treating polycystic ovarian syndrome and application thereof

Info

Publication number: CN111592581B
Application number: CN202010605265.XA
Authority: CN
Inventors: 韩亚光; 朱小琳; 韩亚鹏
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2021-05-28
Anticipated expiration: 2040-06-29
Also published as: CN111592581A

Abstract

The invention relates to a polypeptide compound for treating polycystic ovarian syndrome and application thereof, wherein the polypeptide compound has an amino acid sequence shown as SEQ ID NO. 1. The polypeptide can improve polycystic ovarian syndrome, is particularly suitable for regulating the sexual cycle disorder caused by the polycystic ovarian syndrome, and can effectively reverse insulin resistance.

Description

Polypeptide compound for treating polycystic ovarian syndrome and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a medicine for treating polycystic ovarian syndrome, in particular to a polypeptide compound for treating polycystic ovarian syndrome and application thereof.

Background

Polycystic ovarian syndrome (PCOS) is a common disorder of fertility in women caused by a complex endocrine and metabolic abnormality, characterized by chronic anovulation (ovulatory dysfunction or loss) and hyperandrogenism (excess production of male hormones in women), with the predominant clinical manifestations of irregular menstrual cycle, infertility, hirsutism and/or acne, being the most common endocrine disorders in women.

Current pharmacological treatment of PCOS has replaced surgical treatment as a first-line treatment, the purpose of which is primarily related to the fertility requirements of the patient. Oral Contraceptives (OCP) have been used as a traditional long-term applicable treatment for PCOS women, primarily to protect the endometrium, regulate the menstrual cycle, and improve hirsutism and/or acne by reducing androgen production by the ovaries. OCP can reduce hyperandrogenism in patients with PCOS. Among the most used OCP's to reduce hyperandrogenism are cyproterone acetate, which has progestogenic activity and can bind to ethinyl estradiol to exert an antiandrogenic effect, which also binds to dihydrotestosterone receptors in the follicular cytoplasm to block the transmission of androgen response to the nucleus, and inhibits the activity of 5 alpha reductase by inhibiting this receptor activity, resulting in reduced DHT production, reduced gonadotropin synthesis, reduced gonadotropin levels resulting in reduced steroid synthesis, increased SHBG levels and reduced gonadotropin levels. Cyproterone acetate has been the preferred method for PCOS hirsutism treatment for the last 20 years, with more than 6 consecutive cycles being effective in 60-80% of hirsutism patients. OCP is a simple, economical treatment for patients with infertility-requiring PCOS, but recent studies have shown that it may reduce insulin sensitivity and glucose tolerance in PCOS women, and that additional common side effects including headache, weight gain, mood changes, decreased libido, gastrointestinal reactions and breast pain should be given attention.

Most PCOS patients with fertility requirements can be pregnant by applying ovulation-promoting treatment, the pharmaceutical ovulation-promoting treatment of the PCOS has been greatly developed in nearly 50 years, but the curative effect of part of the patients is poor by applying a conventional method, so that the selection of a proper scheme is the key of the ovulation-promoting treatment.

Although a large number of chemical drugs such as baicalin, quercetin and the like are applied to the treatment of polycystic ovarian syndrome, the inventors of the present application tried to develop a new approach in order to obtain a short peptide compound having therapeutic activity on polycystic ovarian syndrome by screening a high-throughput polypeptide library in consideration that biological medicines are becoming the development direction in the future.

Disclosure of Invention

The invention provides a polypeptide compound and application thereof for treating polycystic ovarian syndrome. Specifically, the invention adopts the following technical scheme:

one aspect of the present invention relates to a polypeptide having an amino acid sequence as shown in SEQ ID No.1 or an amino acid sequence which is missing, substituted or added with one amino acid compared to the sequence of SEQ ID No. 1.

In another aspect, the present invention also relates to a pharmaceutical composition comprising the above polypeptide as an active ingredient, preferably, the above polypeptide as the only active ingredient.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the pharmaceutical composition is an injection, such as a liquid injection or a lyophilized powder injection.

In another aspect of the invention, the invention also relates to the application of the polypeptide in preparing a medicament for treating polycystic ovarian syndrome.

In another aspect of the invention, the invention also relates to the application of the polypeptide in preparing a medicine for reversing insulin resistance.

In another aspect, the invention also relates to the use of the above polypeptide in the preparation of a medicament for modulating the sexual cycle.

Advantageous effects

The polypeptide can improve polycystic ovarian syndrome, is particularly suitable for regulating the sexual cycle disorder caused by the polycystic ovarian syndrome, and can effectively reverse insulin resistance.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1

SD rats of 3 weeks old are selected to be used for preparing polycystic ovary syndrome insulin resistance animal models and to perform drug intervention on successful model rats.

The detection is carried out by adopting methods such as ELISA and the like, statistical software SPSS19.0 is adopted for analysis, and the treatment effect and possible action mechanism of the polypeptide Met-Trp-Trp-His-His-Met on the polycystic ovarian syndrome insulin resistance animal model are determined.

1. Material

1.1 Experimental animals

120 SPF-grade female SD rats of 3 weeks old were housed in the first hospital animal center affiliated to the university of traditional Chinese medicine in black dragon river. Feeding at constant temperature (50% humidity) of 25 deg.C for clean level, without feeding vitamin product, and periodically alternating 12h light and 12h dark.

1.2 Primary reagents

Dehydroepiandrosterone (Sigma), polypeptide Met-Trp-His-Met (synthesized in shanghai), metformin (shanghai ziyi ltd), glucometer (acuu-CHEKActive, germany), serum Insulin (INS), testosterone (T), enzyme-linked immunosorbent assay kit (novetaine, usa).

1.3 Experimental methods

1.3.1 establishment of polycystic ovarian syndrome insulin resistance animal model

The SD rat of 3 weeks old is injected with 0.6mg/100g of Dehydroepiandrosterone (DHEA) and 0.2ml of sesame oil for injection every day subcutaneously at the back of the neck, the vagina shed cytology is observed for 10 days after the continuous injection for 20 days, the rat with the vagina epithelial cells continuously in a cornification state is used as a rat polycystic ovary syndrome animal model successfully induced by the DHEA, the subsequent experiment is carried out, and atypical people are discarded without using the DHEA. Successful model mice were fasted at 8 nights and orbital veins were bled the next 8 morning for fasting glucose and insulin testing.

According to a calculation formula: the rat insulin resistance index (fasting blood glucose x fasting insulin/22.5), wherein the insulin resistance index is larger than 2.7, is used as a successful rat polycystic ovary syndrome insulin resistance animal model and enters subsequent experiments.

1.3.2 groups of experimental rats and drug intervention:

normal group: injection, normal saline-10 ml/kg

Model group: injection, normal saline-10 ml/kg

Treatment groups: injection, polypeptide (10 mug/kg) + physiological saline-10 ml/kg

Control group: injection, metformin (100mg/kg) + physiological saline-10 ml/kg

All the 4 groups were raised under the same conditions for 28 days.

2. Observation of indices and results

2.1 insulin resistance index profiles in groups of rats

According to a calculation formula: the insulin resistance index of rats (fasting blood glucose x fasting insulin/22.5) is greater than 2.8, and the insulin resistance index of the model control group is obviously higher than that of the normal control group (p <0.01) as can be seen from the following table.

TABLE 1 insulin resistance index profile in rats

2.2 estrus cycle in rats

After 28 days of drug intervention, the rat vagina abscission cytology is observed for 10 days, the rat sexual cycle change condition is observed according to the histological characteristics of the vaginal smear at each stage of the sexual cycle of the rat, the sexual cycle recovery rate reaches 70.83%, and compared with a model group without drug intervention (the sexual cycle recovery rate is 29.17%), the difference has statistical significance. The polypeptide of the invention is shown to have obvious improvement effect on the rat model cycle of polycystic ovarian syndrome, and the effect is stronger than that of the positive control drug metformin (the recovery rate of the middle and later period of the property is about 54.17%).

2.3 rat body weight and ovarian coefficient

And (5) picking the ovaries of the two sides of the rat in the operation, weighing the wet weight by an electronic balance, and calculating the ovarian coefficient. The wet weight of the ovary of the rat in the model group is obviously increased compared with that of the normal group, and the wet weight of the ovary of the treatment group is obviously lower than that of the model group. Therefore, the rat ovary volume and wet weight increase of the DHEA model successfully under the condition of basically same body weight can be obtained, and the polypeptide and the metformin medicament can relieve the condition, but have no statistical significance and have no statistical difference between the two medicaments.

2.4 morphological and pathological observation of liver, kidney and ovary of rat

2.4.1 liver

Normal and treatment groups: the lobule structure is intact, the hepatic sinus is clear, the hepatic cells are arranged radially, the size is uniform, the mononuclear and nuclear plasma ratio is normal, and the bile duct epithelium is cuboidal.

Control group: individual steatosis of hepatocytes, increased cytoplasmic transparency, unclear cytoplasm, partial nuclear disintegration, liver plate destruction, and increased thickening of the tract.

2.4.2 kidneys

Each group was normal, the renal pelvis epithelial cells were pseudostratified (meeting functional requirements), and the epithelium was intact.

2.4.3 ovaries

Normal group: the ovary is bright in color, a plurality of corpus luteum and follicles in different development stages can be seen under the microscope, and the granular cell layers are 6-8 layers, complete in shape and orderly arranged.

Model group: the ovary is pale, a plurality of cystic-expanded follicles can be seen under an endoscope, the granular cell layer is 1-2 layers, or even disappears, and the corpus luteum is reduced.

Treatment groups: the color of ovary is improved, the follicle with cystic dilatation under the microscope is reduced, the number of follicles and corpus luteum in different developmental stages is increased, and the number of granular fine cell layers is increased to 5-7 layers.

2.5 insulin Release test conditions in groups of rats

After the rat drug intervention is finished, blood is taken from the orbital veins of the rat in the prematurity stage according to the cytological examination of vaginal shedding, and the level of serum insulin is detected. As can be seen from the table below, the blood INS levels at each time point in the model control group were higher than those in the normal control group (p < 0.05); the blood INS levels in the treatment group were all lower than those in the model control group (p <0.05) for fasting, 60min after meal and 120min after meal, as shown in Table 2.

TABLE 2 insulin Release test conditions in the groups of rats

Group of	Number only	0min(mU/L)	60min(mU/L)	120min(mU/L)
					Normal group	24	7.11±0.22	35.62±2.36	13.65±1.25
Model set	24	14.62±1.59	67.58±5.68	32.76±3.57
					Treatment group	24	9.28±1.02	52.36±5.58	25.39±3.36
Control group	24	7.85±0.87	46.15±4.13	17.59±2.35

And (4) experimental conclusion: the polypeptide can improve the content of serum insulin of each time point of a polycystic ovarian syndrome insulin resistance model rat and improve the insulin resistance.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Sequence listing

<110> Heilongjiang university of traditional Chinese medicine

<120> polypeptide compound for treating polycystic ovarian syndrome and application thereof

<130> CP2020072

<141> 2020-06-29

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6

<212> PRT

<213> Unknown (Unknown)

<400> 1

Met Trp Trp His His Met

1 5

Claims

1. A pharmaceutical composition contains a polypeptide as an active ingredient, wherein the amino acid sequence of the polypeptide is the amino acid sequence shown in SEQ ID No. 1.

2. The pharmaceutical composition of claim 1, said polypeptide being the sole active ingredient.

3. The pharmaceutical composition of claim 1, comprising a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, which is an injectable formulation.

5. Use of the pharmaceutical composition of claim 1 in the manufacture of a medicament for the treatment of polycystic ovary syndrome.

6. Use of a pharmaceutical composition according to claim 1 for the manufacture of a medicament for reversing insulin resistance.

7. Use of the pharmaceutical composition of claim 1 for the manufacture of a medicament for modulating a sexual cycle disorder caused by polycystic ovary syndrome.