CN109913541B - GPR1 target and application of antagonist thereof in infertility-related diseases - Google Patents

Abstract

The invention provides GPR1 targets and application of antagonists thereof in infertility-related diseases. The invention determines that the chemerin/GPR1 system is a novel target for regulating polycystic ovary syndrome and is closely related to mTOR signal paths related to primordial follicle activation, and is possibly involved in regulating the process of primordial follicle activation; and provides the application of the GPR1 gene or GPR1 protein in screening medicaments and diagnostic targets for treating or preventing ovulation disorder and related diseases. Also disclosed is the use of an antagonist of the GPR1 gene or GPR1 protein for the prevention or treatment of ovulation disorders and related diseases.

Description

GPR1 target and application of antagonist thereof in infertility-related diseases

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of GPR1 and an antagonist thereof in the treatment of diseases related to ovulation disorder.

Background

Infertility is a common problem caused by reproductive disorders, affecting approximately at least 10% -15% of women of childbearing age. Many influencing factors causing infertility, such as organic infertility caused by organ lesions, oogenic infertility caused by ovarian failure or premature ovarian failure, oogenic infertility caused by ovulation failure, and the like, cause serious harm to the health of females and family happiness, and therefore, have important significance in preventing and treating female infertility.

(1) Organic infertility

Organic infertility refers to infertility caused by pathological anatomical changes of genitals, such as inflammation, tumor, deformity, etc. Among them, ovarian cancer is a malignant tumor with higher incidence rate, which not only interferes with the normal secretion of ovarian hormone, but also damages most of ovarian tissues, resulting in infertility.

However, when a large portion of ovarian tissue is destroyed by a tumor or other cause resulting in a follicular dysplasia, a large number of primordial follicles are also present in the follicular cortex. Li Jing and its team have studied to activate primordial follicles by activating PI3K and mTOR signaling pathways, while our study has found that GPR1 is also closely linked to mTOR signaling pathways, and therefore GPR1 is a target that may be associated with primordial follicular activation and may be useful in the treatment of infertility caused by ovarian injury or follicular dysplasia.

(2) Infertility without ovum

With age, female ovarian function gradually fails to permanently stop menstruation, a physiological phenomenon known as menopause. Menopause can be divided into natural menopause and artificial menopause. Natural menopause refers to the exhaustion of the follicles in the ovary, or the loss of the response of the remaining follicles to gonadotrophin, the inability of the follicles to develop and secrete estrogen, and the inability to stimulate endometrial growth, resulting in menopause. Artificial menopause refers to surgical removal of bilateral ovaries or other cessation of ovarian function, such as radiation therapy and chemotherapy.

Menopause represents a decline in ovarian function, exhaustion of estrogen secretion, cessation of menstruation, and termination of reproductive function. The average natural menopausal age in women is around 50 years old and when this occurs before 40 years old it is called premature ovarian failure.

For young premature ovarian failure patients, both physiology and psychology are severely affected. Up to now, no effective therapeutic measures have been clearly demonstrated to restore or protect the ovarian function of premature ovarian failure patients. With the open second-birth policy, advanced women are more and more challenged by clinicians.

Likewise, primordial follicles are still present in the ovarian cortex of menopausal women and premature ovarian failure patients, and therefore, basic research on primordial follicle activation and its clinical application are significant.

(3) Infertility with ovum

Polycystic ovary syndrome (polycystic ovary syndrome, PCOS) is a common endocrine syndrome for women of childbearing age, the incidence rate accounts for 6-10% of women of childbearing age, and is mainly manifested by menoxenia or amenorrhea, infertility, polycystic ovary degeneration, obesity, hirsutism, hyperandrogenism and the like. Treatment for PCOS patients includes surgical treatment, medication, and assisted fertility techniques.

Surgical treatments for PCOS mainly include bilateral ovariectomy (bopr), laparoscopic ovarian electrocautery or laser drilling (Laparoscopic ovariandrilling, LOD) and transvaginal hydro-laparoscopy (THL).

Pharmaceutical treatments for PCOS have been used as a first-line treatment instead of surgical treatments, and the purpose of the treatment is mainly related to the fertility requirements of patients, such as the use of a drug for reducing hyperandrogenism, an ovulation-promoting drug, or an Insulin Sensitizer (ISD) treatment.

In addition, assisted fertility techniques for PCOS patients, particularly those who have ovulation but remain non-pregnant after treatment with a standard ovulation induction period of more than 6 months, or multiple drug ovulation induction and assisted treatment of anovulation and urgent pregnancy patients, may be selected, including in vitro fertilization (In vitro fertilization, IVF) and oocyte in vitro maturation (In vitro maturation, IVM).

To date, the pathogenesis of PCOS remains unclear, and a number of studies have shown that 40-60% of PCOS patients are obese, with insulin resistance (insulin resistance, IR) and secondary blood insulin elevation. It is well known that adipose tissue in the human body is one of the important target organs for insulin action, and thus certain pathological, physiological changes in adipose tissue will also affect the occurrence and development of PCOS IR.

Chemerin on the one hand acts as a chemokine, chemotactic dendritic cells and macrophages, bridging between immunity and adaptive immunity; on the other hand, the novel fat factor is secreted by adipose tissues, regulates the differentiation and lipolysis of fat cells, and can promote the biological effects such as insulin signal transduction in the fat cells.

GPR1 is taken as one of three receptors of Chemerin, belongs to a member of the superfamily of G Protein Coupled Receptors (GPCRs), has 7 transmembrane helix (7 TM) structures, is expressed in fat and gonadal tissues, is specifically and highly expressed in ovarian membranous cells, granulosa cells and luteal cells, and indicates that the target point of GPR1 is closely related to ovarian function. Therefore, the GPR1 is used as a target, and the screening of the novel polypeptide medicaments and the humanized antibodies for preventing and treating infertility caused by organ damage, ovarian function decline and ovulation disorder has important social significance and wide economic market.

Disclosure of Invention

The main purpose of the invention is to find a new target for treating infertility and diseases related to infertility, in particular to treat PCOS, so as to better prevent and/or treat the infertility and diseases related to infertility.

The invention determines that the chemerin/GPR1 system is a novel target for regulating infertility caused by the injury of the cytoplasm, the decline of the ovarian function and the ovulation disorder.

GPR1 (G Protein-Coupled Receptor 1, G Protein-Coupled Receptor-1), which is one of the receptors for chemokines/adipokines, belongs to the family of G Protein-Coupled receptors.

In one aspect, the invention provides the use of a GPR1 gene or GPR1 protein in screening drug targets for treating or preventing infertility and infertility-related diseases.

In another aspect, the invention provides the use of a GPR1 gene or GPR1 protein as a therapeutic target for the prevention and/or treatment of infertility and infertility-related diseases or as a diagnostic target for ovulation disorders and related diseases.

In another aspect, the invention provides the use of an antagonist of the GPR1 gene or GPR1 protein in the manufacture of a medicament for the prophylaxis or treatment of infertility and infertility-related disorders.

In a specific technical scheme of the invention, the application of the GPR1 gene or the antagonist of the GPR1 protein in preparing a medicament for preventing or treating polycystic ovary syndrome is disclosed.

In another aspect, the invention provides a use of an antagonist of GPR1 gene or GPR1 protein in the manufacture of a medicament for the prophylaxis or treatment of infertility and infertility-related disorders.

In another aspect, the invention provides the use of an antagonist of the GPR1 gene or GPR1 protein in the manufacture of a medicament for increasing estrogen levels in a subject or for activating primordial follicles in a subject.

In another aspect, the invention provides the use of an antagonist of the GPR1 gene or GPR1 protein to increase estrogen levels in a subject or to activate primordial follicles in a subject.

In the technical scheme of the invention, the infertility is selected from organic infertility, oospermless infertility or oospermic infertility; infertility-related disorders selected from premature ovarian failure, climacteric syndrome, menoxenia, ovulation failure, polycystic ovary syndrome;

preferably, the organic infertility is mechanical or pathological damage of the ovaries, but the cortex still contains primordial follicles;

the non-egg infertility refers to premature ovarian failure patients or women of overchildhood age, the ovaries have no developing follicles, and the cortex of the ovaries still contains primordial follicles.

The GPR1 antagonist is selected from GPR1 antibody, GPR1 receptor antibody, modified GPR1, partial peptide of GPR1, siRNAs, shRNAs targeting GPR1 gene sequence, antisense molecule and dnase, or an expression vector comprising siRNAs, shRNAs, antisense molecule.

The inventors found in the study that GPR1 is a novel target related to primordial follicular activation, and the use of gene knockout technology or shRNA to interfere with GPR1 gene expression, or the use of specific antibodies to interfere with GPR1 receptor effects, can effectively alleviate the symptoms of experimental mouse polycystic ovary syndrome.

In the specific experiment of the invention, the gene expression of GPR1 is interfered by a gene knockout technology, so that the symptom of experimental mouse polycystic ovary syndrome caused by Dehydroepiandrosterone (DHEA) can be effectively relieved. Further, in experiments in which ovarian granulosa cells were cultured in vitro, we found that the GPR1 target was closely related to the mTOR signaling pathway.

Accordingly, the present invention provides a method for preventing and/or treating infertility, the method comprising: targeting GPR1, reducing the level of expression of GPR1 and/or antagonizing GPR1 to prevent and/or treat infertility. Specifically, it is possible to reduce the expression level of GPR1 (knock-out GPR1 gene or knock-down GPR1 gene expression level) and/or antagonize the effect of GPR1 using an antagonist against GPR 1.

In the present invention, the terms "antagonist" and "inhibitor" are used in the same sense and refer to any agent that reduces the level of expression of GPR1, antagonizes the effects of GPR1, and inhibits the binding of GPR1 to a ligand as described for GPR 1. Such antagonists accomplish this by a variety of means. One class of antagonists will bind GPR1 protein with sufficient affinity and specificity to neutralize the biological effects of GPR1 protein. Such molecules include antibodies and antibody fragments. Another class of antagonists comprises fragments of proteins, muteins or small organic molecules, i.e. mimetic peptides, which will bind to GPR1 or GPR1 binding partners, e.g. compounds or small molecule polypeptides having a blocking effect on the binding of chemokines to GPR1, thereby inhibiting the biological activity of GPR 1. The GPR1 antagonist may be of any of these classes, as long as it is a substance that inhibits the biological activity of GPR 1. GPR1 antagonists include GPR1 antibodies, GPR1 receptor antibodies, modified GPR1 and partial peptides of GPR 1. Another class of GPR1 antagonists includes siRNAs, shRNAs, antisense molecules and DNases disclosed herein that target GPR1 gene sequences as known in the art. Such agents are available to those skilled in the art in accordance with the prior art and may be any antagonist known in the art that reduces the level of expression of GPR1 and/or antagonizes the effects of GPR1, as such, or may be agents modified based on this formula that still have the function of reducing the level of expression of GPR1 and/or antagonizing the effects of GPR 1.

According to a specific embodiment of the invention, the antagonist against GPR1 is any agent which reduces the expression level of GPR1 and/or antagonizes the effect of GPR 1. Agents having such a function may include, for example, siRNA, shRNA, antisense RNA, antibodies, or combinations thereof. Such agents are available to those skilled in the art in accordance with the prior art and may be any antagonist known in the art that reduces the level of expression of GPR1 and/or antagonizes the effects of GPR1, as such, or may be agents modified based on this formula that still have the function of reducing the level of expression of GPR1 and/or antagonizing the effects of GPR 1.

According to a specific embodiment of the present invention, the prevention and/or treatment of infertility according to the present invention comprises: modulating primordial follicular activation processes, and/or alleviating symptoms of polycystic ovary syndrome.

The GPR1 (G protein-coupled receptor 1) gene is a known gene in the prior art and is unique.

In combination, the invention proves that GPR1 is a novel target for preventing and/or treating infertility caused by organ injury, ovarian function decline and ovulation disorder, is closely related to mTOR signaling pathway, possibly participates in regulating the primordial follicular activation process, and can effectively relieve the symptoms of polycystic ovary syndrome by interfering GPR1 gene expression or interfering the action of GPR1 by any technology.

Drawings

Fig. 1: effect of GPR1 gene deletion on changes in ovarian phenotype caused by dehydroepiandrosterone. Wherein, WT, wild type: wild-type mice. Gpr1-/-: GPR1 gene-deleted mice. Control (Ctl): control group. DHEA, dehydroepiandrosterone: the experimental group of dehydroepiandrosterone was subcutaneously injected.

Fig. 2: effects of GPR1 gene deletion on hormone levels in mouse serum caused by dehydroepiandrosterone. Wherein, testosterone is Testosterone and Estradiol is Estradiol.

Fig. 3: effect of GPR1 gene deletion on the amount of weight gain in mice due to dehydroepiandrosterone.

Fig. 4: effect of GPR1 on estrogen-related synthetase expression by dehydroepiandrosterone through the mTOR signaling pathway. Aromatase, aromatase; HSD17B7:17B hydroxysteroid dehydrogenase 7; GPR1 Ab, GPR1 antibody; mTOR Ih, mTOR signaling pathway inhibitor. DHEA concentration 10 ^-5 M, GPR1 antibody concentration 0.5. Mu.g/ml, mTOR signaling pathway inhibitor concentration 10 ^-3 nM

Detailed Description

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

Example 1, interfering with GPR1 gene expression, or antagonizing GPR1 effects, can be effective in alleviating the symptoms of experimental mouse polycystic ovary syndrome caused by DHEA.

In this example, it was verified that the effect of interfering with the gene expression of GPR1, or antagonizing GPR1, can effectively alleviate the symptoms of experimental mouse polycystic ovary syndrome caused by DHEA. Wherein, an experimental mouse polycystic ovary syndrome model caused by high androgen DHEA is established. However, the symptoms of polycystic ovary syndrome in GPR1 knockout mice were significantly alleviated compared to wild-type mice, including: the estrogen level in the serum rises and there is still luteal production on the ovaries.

The method comprises the following steps:

(1) DHEA experimental mouse polycystic ovarian syndrome model

GPR1 knockout mice with a C57BL/6J background were purchased from Deltagen Inc. and supplied by ZASE doctor laboratories, stanford university. C57BL/6J wild female mice were from the Guangdong provincial medical laboratory animal center.

Animals were kept in a light-dark cycling feeding chamber for 12 hours at constant temperature and humidity. The feed and water can be eaten freely. All animal procedures were performed following procedures approved by the animal welfare ethics committee.

Wild type mice: female mice were randomized on postnatal day 25 (D25) into: control group, DHEA group, at least 6 mice per group.

GPR1 gene deleted mice: female mice were randomized on postnatal day 25 (D25) into: control group, DHEA group, at least 6 mice per group.

DHEA group: the mice were continuously subcutaneously injected with DHEA (opening tylosin limited of dan, hubei province) for 21 days, 6mg/100g, the powder was dissolved in 100ul of alcohol, and 900 ul of sesame oil was added.

Control group: mice were continuously subcutaneously injected with 100 μl of alcohol without DHEA plus 900 μl of sesame oil.

The mice were sacrificed at the end of the molding period, 21 days, with body weights taken every three days. In addition, at the end of the 21D molding period, blood samples and tissues were collected. Mice were anesthetized with isoflurane, and blood was collected by eye blood sampling. Ovary and uterus, gonadal fat were isolated and fixed overnight in 4% paraformaldehyde fluid. In addition, ex vivo tissue was rapidly frozen in liquid nitrogen until further processing.

FIG. 1 shows the effect of a deletion of the GPR1 gene on dehydroepiandrosterone-induced changes in ovarian phenotype.

As shown in fig. 1A, wild-type mice (WT-control) have follicles at different stages of development in their ovaries, and the corpus luteum is present; whereas many small follicles and large cystic follicles were present in wild-type mice (WT-DHEA) ovaries treated with Dehydroepiandrosterone (DHEA) for 21 days, the corpus luteum disappeared. Dehydroepiandrosterone severely affects normal ovulation in mice.

In FIG. 1B, GPR1 gene deleted mice (Gpr 1-/-control) had more luteal in the ovary. There was still luteal presence after treatment of GPR1 gene deleted mice with dehydroepiandrosterone (Gpr 1-/-DHEA). Indicating that the mice still had an ovulation process.

FIG. 2 shows the effect of deletion of the GPR1 gene on hormone levels in serum of mice caused by dehydroepiandrosterone.

The testosterone (T) content in serum was compared to the WT-Control, WT-DHEA, gpr1-/-Control, gpr1-/-DHEA of group 4 mice, where the testosterone content in the WT-Control group was around 0.3 ng/mL. In both the WT-DHEA and Gpr1-/-DHEA treated groups, testosterone levels in the serum increased to about 1.2ng/mL, demonstrating successful modeling of dehydroepiandrosterone treated groups.

The level of estrogen (estradiol) in the serum was then measured separately: treatment with DHEA had no significant effect on estrogen levels in the serum of mice, whereas for Gpr 1-/-mice, estrogen levels in the serum were significantly elevated compared to wild-type mice. P <0.05 represents statistically significant differences.

From the results of FIGS. 1 and 2, it was found that the deletion of GPR1 gene could affect the estrogen level and up-regulate the estrogen level, thus indicating that it could restore the ovulation process in mice.

Figure 3 shows that the body weight gain of mice of different groups was significantly increased after DHEA modeling, while the body weight gain after GPR1KO mice DHEA modeling was significantly reduced compared to WT mice DHEA treatment.

Because PCOS patients mostly have an obese phenotype, GPR1 antagonists may be targeted for treatment of obese PCOS.

Example 2: GPR1 regulates estrogen-related synthetase expression caused by dehydroepiandrosterone through mTOR signaling pathway

C57BL/6J wild female mice were from the Guangdong provincial medical laboratory animal center.

Animals were kept in a light-dark cycling feeding chamber for 12 hours at constant temperature and humidity. The feed and water can be eaten freely. All animal procedures were performed following procedures approved by the animal welfare ethics committee.

Mice were intraperitoneally injected with 5IU PMSG (pregnant horse serum gonadotropin) at day 21, followed by 48 hours later by intraperitoneal injection of 5IU hCG (human chorionic gonadotropin), 7-12 hours later by sacrifice of mice, ovaries removed, ovaries punctured, oocytes removed by three 70um sieves, centrifugation at 1000g for 5 minutes, supernatant discarded, resuspended in serum-free DMEMF12+3% BSA+ITS incubator 37 ℃,5% CO ₂ Culturing.

Control group: 1. granulosa cells in culture;

2. adding DHEA at a concentration of 10 ^-5 M；

3. Addition of mTOR signalling pathway inhibitor, concentration 10 ^-3 nM；

4. Simultaneous addition of DHEA and mTOR signaling pathway inhibitors

GPR1 antibody group: GPR1 antibody was pre-added at a concentration of 0.5ug/ml 1-2 hours in advance and incubated.

1. Granulosa cells in culture;

2. adding DHEA at a concentration of 10 ^-5 M；

3. Addition of mTOR signalling pathway inhibitor, concentration 10 ^-3 nM；

4. Simultaneous addition of DHEA and mTOR signaling pathway inhibitors

After 24 hours of incubation, supernatants were collected, hormone levels were measured, granulosa cells were collected, and qPCR was performed to detect the expression levels of estrogen-related synthetases.

Figure 4 shows the effect of GPR1 on dehydroepiandrosterone-induced estrogen-related synthetase expression via the mTOR signaling pathway. As shown, in the DHEA-treated group, estrogen-related synthetase expression was significantly elevated after mTOR signaling was inhibited, while this was inhibited after GPR1 antibody addition. GPR1 is shown to regulate DHEA-induced changes in estrogen levels through mTOR signaling.

While the invention has been illustrated and described with reference to certain specific embodiments, it is not intended to be limited to the details shown. Rather, the invention relates to GPR1 antagonist polypeptides, polynucleotides, antibodies, apparatus and kits disclosed herein and uses thereof, as well as to control GPR1 levels, and various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the spirit of the invention.

Claims (2)

1. Use of an antagonist of GPR1 protein in the manufacture of a medicament for the treatment of infertility;

wherein the infertility is polycystic ovary syndrome; the polycystic ovary syndrome is phenotyped by obesity of a patient;

wherein the antagonist of GPR1 protein is selected from GPR1 antibody, siRNAs, shRNAs targeting GPR1 gene sequence, antisense molecule, or expression vector comprising siRNAs targeting GPR1 gene sequence, antisense molecule.

2. Use of an antagonist of GPR1 protein in the manufacture of a medicament for the treatment of infertility;

wherein the infertility is polycystic ovary syndrome; the polycystic ovary syndrome is phenotyped by obesity of a patient;

wherein the antagonist of GPR1 protein is an expression vector comprising shRNAs targeting the GPR1 gene sequence.