Method for relieving mouse early-onset ovarian insufficiency

Abstract

The invention discloses a method for relieving early-onset ovarian insufficiency of mice. The method comprises the following steps: S11, selecting experimental animals: 8-week-old ICR mice which are female, 20-25g and clean; s12, grouping the mice and establishing a POI (Point of Interest) mouse model; s13, administration is carried out on the mice in different groups; s14, identifying the estrus cycle of each group of mice; according to the method for relieving the early-onset ovarian insufficiency of the mice, AS-IV can promote primordial follicle activation and follicle development of all levels by activating PI3K / AKt / mTOR signal channels and inhibit follicle atresia so as to play a role in relieving POI symptoms, and the effect of AS-IV with the medium dosage (80 mg / kg / d) is more obvious.

Description

Technical Field

[0001] The invention relates to the technical field of alleviating premature ovarian insufficiency in mice, and specifically to a method for alleviating premature ovarian insufficiency in mice. Background Art

[0002] Premature Ovarian Insufficiency (POI) refers to ovarian dysfunction in women before the age of 40, mainly manifested by abnormal menstruation, elevated gonadotropin levels (FSH>25U / L), and fluctuating decreases in estrogen levels. Common causes of POI include genetic factors, iatrogenic factors, and environmental factors. POI patients caused by iatrogenic factors account for 7% to 8% of the total number of patients. Studies have found that POI patients may have fertility difficulties or even infertility due to physiological or psychological factors. Therefore, in order to improve the quality of life of POI patients and maintain their fertility, it is worth studying to explore the pathogenesis of POI patients and find effective prevention and treatment methods.

[0003] Long-term hormone replacement therapy with estrogen, progesterone, melatonin and other hormones is currently a relatively effective treatment method. However, some clinical studies have found that patients receiving HRT have a longer risk period and a greater risk of breast cancer, heart disease and other disorders. Therefore, people are exploring non-hormonal treatments such as herbal medicines, traditional Chinese medicine, and stem cells.

[0004] Traditional Chinese medicine has certain advantages in improving reproductive function. Studies have shown that in addition to enhancing the body's immune function, strengthening the heart and lowering blood pressure, and anti-aging, astragaloside IV (AS-IV) is a triterpenoid saponin with the best biological activity extracted from astragalus.

[0005] Network pharmacology analysis showed that the genes targeted by astragaloside IV were mainly enriched in biological processes such as epithelial cell proliferation, inflammatory response and protein kinase B signaling. Protein kinase B, or AKt, is a serine / threonine-specific protein kinase that plays an important role in various cellular processes such as apoptosis, proliferation and migration. The study by Li Youqian et al. showed that astragaloside IV may activate the phosphatidylinositol 3-kinase / protein kinase B / mammalian target of rapamycin (PI3K / AKt / mTOR) signaling pathway to inhibit vascular smooth muscle cell autophagy to inhibit the proliferation and inflammation of the intima of the common carotid artery in rats after balloon injury. In the female reproductive system, the PI3K / AKt / mTOR signaling pathway also plays an important role in the activation of ovarian primordial follicles and the maintenance of female fertility. The two most typical substrates downstream of mTORC1 are eukaryotic translation initiation factor 4E binding protein (4E-BP1) and ribosomal protein S6 kinase (p70ribosomalproteinS6kinase, S6K). The mTORC1 complex controls translation regulation, ribosome generation, and promotes cell growth and proliferation by activating 4E-BP1 and S6K. Although current studies have shown that astragaloside IV plays a role in inhibiting inflammatory responses and oxidative stress in a variety of diseases including POI, there is a lack of research on whether astragaloside IV can relieve POI symptoms by activating primordial follicles in POI patients and the specific targeting mechanism. Therefore, whether astragaloside IV can activate primordial follicles of POI patients and promote the development of follicles at all levels through the PI3K / AKt / mTOR signaling pathway, and rescue and maintain their fertility, is a question worthy of discussion and research. To this end, we proposed a method to alleviate premature ovarian insufficiency in mice. Summary of the invention

[0006] The object of the present invention is to provide a method for alleviating premature ovarian insufficiency in mice, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for alleviating premature ovarian insufficiency in mice, characterized in that the method comprises the following steps:

[0008] S11. Selection of experimental animals: 8-week-old ICR mice, female, 20-25 g, clean grade;

[0009] S12, grouping mice and establishing POI mouse model;

[0010] S13, administering drugs to different groups of mice;

[0011] S14, identifying the estrus cycle of each group of mice;

[0012] S15. Samples were collected from each group of mice and tested.

[0013] Preferably, the method for grouping mice in S12 comprises the following steps:

[0014] S21. From the day after adaptive feeding of 8-week-old ICR female mice was completed, vaginal exfoliated epithelial cell smears were performed on all mice every other day for 15 consecutive days to clarify the changes in the estrus cycle of experimental mice.

[0015] S22. Excluding mice with abnormal estrous cycle, 60 mice with normal estrous cycle were randomly divided into normal control group, model group, high-, medium- and low-dose astragaloside IV groups and fenmorphone group, with 10 mice in each group.

[0016] Preferably, the method for establishing the POI mouse model in S12 comprises the following steps:

[0017] S31, the normal control group was given an equal volume of saline intraperitoneal injection, and the other groups were given a single intraperitoneal injection of 120 mg / kg cyclophosphamide / sodium chloride solution and 12 mg / kg busulfan / sodium chloride solution to establish the POI mouse model;

[0018] S32. From the day after the intraperitoneal injection, vaginal epithelial smear examination was performed on mice in each group once every other day for 15 consecutive days to observe the changes in the estrus cycle of the mice to determine the modeling situation. If the estrus cycle was longer than 5 days, it was considered to be an estrus cycle disorder and the POI model was successfully established.

[0019] Preferably, the method of administering the drug to different groups of mice in S13 comprises the following steps:

[0020] S41. The mice were given the drug by gavage starting from the day after the estrous cycle was identified.

[0021] S42, low, medium, and high doses of astragaloside IV groups: Astragaloside IV was dissolved in 1% sodium carboxymethylcellulose solution and intragastrically administered at doses of 40, 80, and 120 mg / kg / d for 28 days;

[0022] S43, normal control group and POI model group: given an equal volume of 1% CMC-Na solution by intragastric administration for 28 days;

[0023] S44, Fentaton group: Fentaton was dissolved in 1% CMC-Na solution, and the equivalent dose of the drug was calculated based on the body surface area of ​​humans and animals. The drug was given repeatedly in a cycle of 5 days, and the drug was administered by gavage for 28 days.

[0024] Preferably, the method for identifying the estrus cycle of each group of mice in S14 is: after the POI model is established and the next day after the end of the administration cycle, the mice are fixed, the exfoliated cells of the vaginal mucosal epithelium are taken for smear, and the estrus cycle period is observed under a microscope after Papanicolaou staining, and the estrus cycle period is determined once every other day for 15 consecutive days, and the estrus cycle disorder rate is calculated.

[0025] Preferably, the method for sampling each group of mice in S15 is: the day after the estrus cycle identification is completed, the body weight of each group of mice is weighed, blood is collected from the eyeballs after anesthesia to obtain serum, and then the mice are killed by cervical dislocation, and the bilateral ovaries are taken, one ovary is fixed with 4% paraformaldehyde, and the other ovary is frozen at -80°C.

[0026] Preferably, the testing of mouse samples in S15 includes detecting the levels of E2, FSH and AMH in serum by ELISA: using ELISA kits for mouse E2, FSH and AMH to detect the levels of E2, FSH and AMH in the serum of each group of mice, taking serum samples of 3 mice in each group, and performing duplicate wells on both the standard and the samples.

[0027] Preferably, the sampling of mice for testing in S15 includes counting the number of follicles at each level after HE staining of ovarian tissue paraffin sections: unilateral ovaries of 3 mice are taken from each group, fixed with 4% paraformaldehyde for 3 hours, dehydrated with gradient ethanol and transparent with xylene, and then embedded in paraffin. Paraffin tissue sections are sliced ​​once for each ovary at an interval of 50 μm, for a total of 10 times, with a thickness of 5 μm. 10 paraffin sections are taken and dewaxed routinely, and then HE staining is performed, and then the number of follicles at each level is counted.

[0028] Preferably, the detection of mouse samples in S15 includes detecting the expression levels of ER and FSHR in the ovaries of each group by immunofluorescence method: taking paraffin sections of ovaries from each group, after routine dewaxing, washing 3 times with 0.01 mol / L PBS, 10 min / time, covering the sections with blocking solution at 37°C for 1 hour, and then covering with rabbit FSHR antibody and incubating at 4°C overnight, the next day, covering the sections with FITC-goat anti-rabbit antibody and incubating at 37°C for 1 hour, covering the sections with 5 μg / ml Hoechst33342 and incubating at room temperature for 15 minutes to stain the nucleus to prevent quenching of the sealing agent, and finally placing the slide on the slide for observation and filming under a fluorescence microscope, observing 3 female mouse ovarian tissues in each group, and using Imagepro-Plus6.0 image analysis software to analyze the ovarian samples, selecting 3 preantral follicles and antral follicles for each ovarian section, and selecting the same area of ​​corpus luteum tissue for FSHR fluorescence OD value analysis.

[0029] Preferably, the detection of mouse samples in S15 includes the detection of FSHR, estrogen synthesis key proteins and PI3K-AKt-mTOR signaling pathway related proteins by western blotting: ovaries of each group are taken, RIPA strong lysis buffer is used to extract proteins, and the sample volume of each sample is calculated according to the protein concentration determination result. After protein loading, 240V constant voltage electrophoresis is performed for about 35 minutes, and then 400mA current wet transfer is performed for 30-60 minutes, and then 5% skim milk powder or 5% BSA is used for blocking at room temperature for 1 hour, and finally rabbit β-actin and FSH are used. R, P450scc, Aromatase, P-PI3K, P-AKT1, mTOR, P-mTOR, P-S6K and P-eIF4EBP1 antibodies were incubated at 4°C overnight. The next day, HRP-goat anti-rabbit antibody was incubated at room temperature for 40-50 min. Finally, the PVDF membrane was placed in the Tanon 5200 imaging system, covered with 200 μl of enhanced developer, and photographed using the Tanon Imaging System software. After repeating 3 times, the grayscale value of the images was analyzed using ImageJ analysis software and statistical analysis was performed.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] After the treatment cycle, the present invention detected the changes in estrus cycle and serum hormone levels in each group. The results showed that AS-IV significantly alleviated the estrus cycle disorder of POI mice and significantly improved the abnormalities of serum E2, FSH and AMH levels, indicating that AS-IV alleviated the POI symptoms of mice to a certain extent.

[0032] The AS-IV of the present invention can promote the activation of primordial follicles and the development of follicles at all levels and inhibit follicular atresia by activating the PI3K / AKt / mTOR signaling pathway, thereby alleviating POI symptoms, and the effect of a medium dose (80 mg / kg / d) of AS-IV is more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a smear of vaginal mucosal exfoliated cells at different stages of the normal mouse estrus cycle (100X) (Note: A is proestrus: mainly nucleated epithelial cells; B is estrus: keratinized anucleated cells with large and irregular shapes; C is metestrus: keratinized anucleated epithelial cells, nucleated epithelial cells and leukocytes coexist, mainly wrinkled anucleated epithelial cells; D is diestrus: mainly leukocytes.);

[0034] Figure 2 This is a smear of vaginal mucosal exfoliated cells at different stages of the estrus cycle of POI mice (100X) (Note: AD Wei estrus: mainly white blood cells and epithelial cells.);

[0035] Figure 3Serum E2, FSH and AMH levels of POI mice (Note: compared with NC group, *P<0.05; compared with POI group, #P<0.05. N=3 samples);

[0036] Figure 4 HE staining images of the ovaries of mice in each POI group (100X) (Note: A is the NC group; B is the POI group; C is the Femerton group; D is the H-AS-IV group; E is the M-AS-IV group; F is the L-AS-IV group);

[0037] Figure 5 The expression of ER in the ovaries of mice in each group of POI (200X) (AC: NC group; DF: POI group; GI: Femerton group; JL: H-AS-IV group; MO: M-AS-IV group; PR: H-AS-IV group. Compared with the NC group, * P<0.05; compared with POI group, # P < 0.05. N = 3 batches);

[0038] Figure 6 The expression of FSHR in the ovaries of mice in each group of POI (200X) (AC: NC group; DF: POI group; GI: Femerton group; JL: H-AS-IV group; MO: M-AS-IV group; PR: H-AS-IV group. Compared with the NC group, * P<0.05; compared with POI group, # P<0.05. N=3 batches. );

[0039] Figure 7 The expression of P450scc and Aromatase proteins in the ovaries of mice in each group of POI (compared with the NC group, * P<0.05; compared with POI group, # P<0.05. N=3 batches. );

[0040] Figure 8 The expression of proteins related to PI3K-AKt-mTOR signaling pathway in the ovaries of mice in each group of POI (compared with the NC group, * P<0.05; compared with POI group, # P<0.05. N=3 batches. ). DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] See also Figure 1-8 The present invention provides a technical solution: a method for alleviating premature ovarian insufficiency in mice, characterized in that the method comprises the following steps:

[0043] S11. Selection of experimental animals: 8-week-old ICR mice, female, 20-25 g, clean grade;

[0044] S12, grouping mice and establishing POI mouse model;

[0045] S13, administering drugs to different groups of mice;

[0046] S14, identifying the estrus cycle of each group of mice;

[0047] S15. Samples were collected from each group of mice and tested.

[0048] The drugs selected were: cyclophosphamide (powder, China Yuanye Company, batch number F13IS206786), busulfan (powder, China Yuanye Company, batch number A25M11H110042), fenogest (each estradiol tablet contains 1 mg of estradiol, and each estradiol dydrogesterone tablet contains 1 mg of estradiol and 10 mg of dydrogesterone, Abbott Healthcare Products BV, batch number 370364), and astragaloside standard (China Dester Biotechnology Co., Ltd., batch number DSTDH001503).

[0049] As a preferred embodiment, the method for grouping mice in S12 comprises the following steps:

[0050] S21. From the day after adaptive feeding of 8-week-old ICR female mice was completed, vaginal exfoliated epithelial cell smears were performed on all mice every other day for 15 consecutive days to clarify the changes in the estrus cycle of experimental mice.

[0051] S22. Excluding mice with abnormal estrous cycle, 60 mice with normal estrous cycle were randomly divided into normal control group (NC group), model group (POI group), high-, medium- and low-dose astragaloside IV groups (L-AS-IV group, M-AS-IV group, H-AS-IV group) and femerton group (Femerton group), with 10 mice in each group.

[0052] As a preferred embodiment, the method for establishing the POI mouse model in S12 comprises the following steps:

[0053] S31, the normal control group was given an equal volume of saline intraperitoneal injection, and the other groups were given a single intraperitoneal injection of 120 mg / kg cyclophosphamide / sodium chloride solution and 12 mg / kg busulfan / sodium chloride solution to establish the POI mouse model;

[0054] S32. From the day after the intraperitoneal injection, vaginal epithelial smear examination was performed on mice in each group once every other day for 15 consecutive days to observe the changes in the estrus cycle of the mice to determine the modeling situation. If the estrus cycle was longer than 5 days, it was considered to be an estrus cycle disorder and the POI model was successfully established.

[0055] As a preferred embodiment, the method of administering the drug to different groups of mice in S13 comprises the following steps:

[0056] S41. The mice were given the drug by gavage starting from the day after the estrous cycle was identified.

[0057] S42, low, medium, and high doses of astragaloside IV groups: Astragaloside IV was dissolved in 1% sodium carboxymethylcellulose solution and intragastrically administered at doses of 40, 80, and 120 mg / kg / d for 28 days;

[0058] S43, normal control group and POI model group: given an equal volume of 1% CMC-Na solution by intragastric administration for 28 days;

[0059] S44, Fentaton group: Fentaton was dissolved in 1% CMC-Na solution, and the equivalent dose of the drug was calculated based on the body surface area of ​​humans and animals (the daily dose for mice was equivalent to 9.01 times that of adults, who took one tablet a day with an average body weight of 70 kg, so the oral dose for mice was 1 / 70×9.01). The drug was administered repeatedly in a cycle of 5 days (estradiol tablets 0.13 mg / kg / d for 2 consecutive days, and estradiol dydrogesterone tablets 1.43 mg / kg / d from the 3rd day for 3 consecutive days), and the drug was administered by oral gavage for 28 days.

[0060] As a preferred embodiment, the method for identifying the estrus cycle of each group of mice in S14 is as follows: after the POI model is established and the next day after the end of the administration cycle, the mice are fixed, the vaginal mucosal epithelial exfoliated cells are taken for smear, and the estrus cycle period is observed under a microscope after Papanicolaou staining, and the estrus cycle period is determined once every other day for 15 consecutive days, and the estrus cycle disorder rate is calculated.

[0061] As a preferred embodiment, the method for sampling each group of mice in S15 is as follows: the day after the estrus cycle identification is completed, the body weight of each group of mice is weighed, blood is collected from the eyeballs after anesthesia to obtain serum, and then the mice are killed by cervical dislocation, and the bilateral ovaries are taken, one ovary is fixed with 4% paraformaldehyde, and the other ovary is frozen at -80°C.

[0062] As a preferred embodiment, the testing of mouse samples in S15 includes detecting the levels of E2, FSH and AMH in serum by ELISA: using ELISA kits for mouse E2, FSH and AMH to detect the levels of E2, FSH and AMH in the serum of each group of mice, taking serum samples of 3 mice in each group, and performing duplicate wells on both the standard and the samples.

[0063] As a preferred embodiment, the detection of mouse samples in S15 includes counting the number of follicles at each level after HE staining of ovarian tissue paraffin sections: unilateral ovaries of 3 mice are taken from each group, fixed with 4% paraformaldehyde for 3 hours, dehydrated with gradient ethanol and transparent with xylene, and then embedded in paraffin. Paraffin tissue sections are sliced ​​once for each ovary at an interval of 50 μm, for a total of 10 times, with a thickness of 5 μm. 10 paraffin sections are taken and dewaxed routinely, and HE staining is performed, and then the number of follicles at each level is counted.

[0064] As a preferred embodiment, the detection of mouse samples in S15 includes detecting the expression levels of ER and FSHR in the ovaries of each group by immunofluorescence method: taking paraffin sections of ovaries from each group, after routine dewaxing, washing 3 times with 0.01 mol / L PBS, 10 min / time, covering the sections with blocking solution at 37°C for 1 hour, and then covering with rabbit FSHR antibody and incubating at 4°C overnight, the next day, covering the sections with FITC-goat anti-rabbit antibody and incubating at 37°C for 1 hour, covering the sections with 5 μg / ml Hoechst33342 and incubating at room temperature for 15 minutes to stain the nucleus to prevent quenching of the sealing agent, and finally placing the slide on the slide for observation and filming under a fluorescence microscope, observing 3 female mouse ovarian tissues in each group, and using Imagepro-Plus6.0 image analysis software to analyze the ovarian samples, selecting 3 preantral follicles and antral follicles for each ovarian section, and selecting the same area of ​​corpus luteum tissue for FSHR fluorescence OD value analysis.

[0065] As a preferred embodiment, the detection of mouse samples in S15 includes the detection of FSHR, estrogen synthesis key proteins and PI3K-AKt-mTOR signaling pathway related proteins by western blotting: ovaries of each group were taken, RIPA strong lysis buffer was used to extract proteins, and the sample loading volume of each sample was calculated according to the protein concentration determination result. After the protein was loaded, 240V constant voltage electrophoresis was performed for about 35 minutes, and then 400mA current wet transfer was performed for 30-60 minutes, and then 5% skim milk powder or 5% BSA was used for blocking at room temperature for 1 hour, and finally rabbit β-actin , FSHR, P450scc, Aromatase, P-PI3K, P-AKT1, mTOR, P-mTOR, P-S6K and P-eIF4EBP1 antibodies were incubated at 4°C overnight. The next day, incubated with HRP-goat anti-rabbit antibody at room temperature for 40-50 min. Finally, the PVDF membrane was placed in the Tanon 5200 imaging system, covered with 200 μl of enhanced developer, and photographed using the Tanon Imaging System software. After repeating 3 times, the grayscale value of the images was analyzed using ImageJ analysis software and then statistically analyzed.

[0066] The identification results of the above implementation methods are:

[0067] Results of estrus identification after establishment of POI mouse model and treatment with astragaloside IV:

[0068] The estrous cycle of mice in the normal control group was normal, and there was no disorder in their estrous cycle; 42 mice in the model establishment group had disordered estrous cycle, and the disorder rate of estrous cycle was 85.59%, with prolonged estrus or proestrus. The POI mouse model was successfully established. After treatment with fentermatone and AS-IV, the disorder of estrous cycle of mice was improved. Figure 1 , Figure 2 and Table 1.

[0069] Table 1 Number of mice with estrous cycle disorders before and after treatment (n = 1)

[0070]

[0071]

[0072] AS-IV improves serum E2, FSH and AMH levels in POI mice:

[0073] Compared with the control group, the levels of E2 and AMH in the serum of the POI group were significantly decreased, and the level of FSH was significantly increased; compared with the POI group, the levels of E2 and AMH in the serum of the H-AS-IV, M-AS-IV, and L-AS-IV groups were significantly increased, and the level of FSH was significantly decreased. Figure 3 shown.

[0074] AS-IV improves follicle development in POI mice:

[0075] Compared with the control group, the number of preantral follicles, anterior antral follicles and mature follicles in the POI group was significantly decreased, and the number of primordial follicles and atretic follicles was significantly increased; compared with the POI group, the number of preantral follicles, anterior antral follicles and mature follicles in the H-AS-IV, M-AS-IV and L-AS-IV groups was significantly increased, and the number of primordial follicles and atretic follicles was significantly decreased. Figure 4 And as shown in Table 2.

[0076] Table 2 Number of follicles at different levels and atretic follicles in mice

[0077]

[0078]

[0079] AS-IV improves the expression levels of FSHR and ER in the ovaries of POI mice:

[0080] Estrogen receptor (ER) and FSH receptor (FSHR) are expressed on the cell membranes of oocytes, granulosa cells, theca cells, and luteal cells. Compared with the control group, the expression levels of ER and FSHR in the ovaries of female mice in the POI group were significantly decreased; compared with the POI group, the expression levels of ER in the ovaries of female mice in the Femerton group, H-AS-IV, M-AS-IV, and L-AS-IV groups were significantly increased, such as Figure 5 and Figure 6 shown.

[0081] AS-IV improves the expression levels of P450scc and Aromatase proteins in the ovaries of POI mice:

[0082] Compared with the NC group, the expression levels of P450scc (P450 side chain cleavage enzyme) and Aromatase (aromatase) proteins, which are key enzymes in estrogen synthesis, in the ovaries of the POI group mice were significantly decreased; compared with the POI group, the expression levels of P450scc and Aromatase proteins in the ovaries of the H-AS-IV, M-AS-IV and L-AS-IV groups were significantly increased. Figure 7 shown.

[0083] AS-IV improves the expression levels of proteins related to the PI3K-AKt-mTOR signaling pathway in the ovaries of POI mice:

[0084] Compared with those in the NC group, the expression levels of P-PI3K, P-AKT1, mTOR and P-S6K proteins in the ovaries of the POI group mice were significantly decreased, while the expression levels of P-mTOR and P-eIF4EBP1 proteins were significantly increased; compared with the POI group, the expression levels of P-PI3K, P-AKT1 and P-S6K proteins in the ovaries of the H-AS-IV, M-AS-IV and L-AS-IV groups were significantly increased, while the expression levels of P-mTOR and P-eIF4EBP1 proteins were significantly decreased. Figure 8 shown.

[0085] Although long-term hormone replacement is currently the conventional treatment for POI, clinical studies have found that patients receiving this treatment have a greater risk of developing diseases such as breast cancer and heart disease. Therefore, more and more research is focusing on non-hormonal treatments in the hope of finding other drugs that can be used in combination with or instead of hormone therapy. Traditional Chinese medicine has advantages in improving reproductive function. Astragalus, as the main medicine for nourishing the kidney, spleen and blood, is an important component of reproductive health prescriptions.

[0086] Astragaloside IV (AS-IV) is the most biologically active ingredient. Current studies have shown that it can inhibit inflammatory responses and oxidative stress in a variety of diseases including POI. The protein kinase B (AKt) signaling pathway is one of the targeted pathways of astragaloside IV that has been discovered so far. AKt is an important kinase for activating primordial follicles in the ovary. Studies have found that somatic mTORC1 signaling is essential for controlling the activation of primordial follicles and the development of dormant oocytes. Other studies have found that mTORC2 plays a key role in promoting folliculogenesis and maintaining ovarian function. These studies have emphasized the role of the mTOR signaling pathway in preventing excessive consumption of the primordial follicle pool, promoting folliculogenesis and maintaining ovarian function. Therefore, whether astragaloside IV can activate the primordial follicles or atretic follicles of POI patients through the PI3K / AKt / mTOR signaling pathway to relieve POI symptoms and save their fertility is a question worth exploring. In the present application, after the POI mouse model was established, AS-IV was given by oral gavage for 28 days at doses of 40, 80 and 120 mg / kg / d. After the treatment cycle, the changes in estrus cycle and serum hormone levels in each group were detected. The results showed that AS-IV significantly alleviated the estrus cycle disorder of POI mice and significantly improved the abnormalities of serum E2, FSH and AMH levels, indicating that AS-IV alleviated the POI symptoms of mice to a certain extent.

[0087] To explore whether AS-IV relieves POI symptoms through the PI3K / AKt / mTOR signaling pathway, the changes in the number of ovarian follicles at all levels, the expression levels of ovarian ER and FSHR, the key enzymes of ovarian E2 synthesis, and the expression levels of proteins related to the PI3K / AKt / mTOR signaling pathway were detected after treatment. The results showed that AS-IV significantly increased the expression levels of P-PI3K and P-Akt, activated the expression of mTOR, and further phosphorylated S6K to activate it. S6K is the downstream protein of mTOR. After phosphorylation, it activates the 40S ribosomal protein in the cell. Phosphorylation of Akt S6 promotes the translation of mRNA containing a 5'-end cap structure. The products of the latter translation are mainly essential components of the translation apparatus such as ribosomes, initiation factors, and elongation factors. Therefore, activation after S6K phosphorylation can promote protein synthesis, cell growth and proliferation. The present application observed that AS-IV significantly increased the number of preantral follicles, antral follicles and mature follicles in POI ovaries and significantly reduced the number of atretic follicles. It may be that the activated Akt and S6K jointly promoted the activation of POI primordial follicles, inhibited the atresia of follicles and promoted the development of follicles at all levels, but did not excessively consume primordial follicles.

[0088] After the activation of primordial follicles and the significant improvement of the development of follicles at all levels, the levels of AMH and E2 secreted by the ovaries increased significantly, which negatively fed back to the pituitary gland to reduce FSH secretion. This is consistent with the observed results of significantly increased expression of key enzymes P450scc and Aromatase proteins in E2 synthesis and significantly increased expression of ER and FSHR. Therefore, AS-IV can promote the activation of primordial follicles and the development of follicles at all levels and inhibit follicular atresia by activating the PI3K / AKt / mTOR signaling pathway, thereby alleviating POI symptoms, and the effect of a medium dose (80 mg / kg / d) of AS-IV is more obvious.

[0089] In addition, the present application also found experimental results that are contrary to those of previous studies and the present application, namely, the expression levels of P-mTOR (phosphorylation site is S2248) and P-4E-BP1 proteins. Some previous studies found that when mTOR is activated, the level of P-mTOR also increases, while in the present application, the level of P-mTOR in POI ovaries is significantly higher than that in the normal control group. Fentaton and AS-IV significantly reduced the expression level of this protein, which may be the influence of other signaling pathways, or it may be related to preventing excessive consumption of primordial follicles. The specific reasons need further study. Most studies have shown that mTOR can promote 4E-BP1 phosphorylation, while the present application found that the expression of P-4E-BP decreases after the activation of mTOR, which may also be related to the influence of signaling pathways.

[0090] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0091] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for alleviating premature ovarian insufficiency in mice, characterized in that: The method comprises the following steps: S11. Selection of experimental animals: 8-week-old ICR mice, female, 20-25 g, clean grade; S12, grouping mice and establishing POI mouse model; S13, administering drugs to different groups of mice; S14, identifying the estrus cycle of each group of mice; S15. Samples were collected from each group of mice and tested.

2. The method for alleviating premature ovarian insufficiency in mice according to claim 1, characterized in that: The method for grouping mice in S12 includes the following steps: S21. From the day after adaptive feeding of 8-week-old ICR female mice was completed, vaginal exfoliated epithelial cell smears were performed on all mice every other day for 15 consecutive days to clarify the changes in the estrus cycle of experimental mice. S22. Excluding mice with abnormal estrous cycle, 60 mice with normal estrous cycle were randomly divided into normal control group, model group, high-, medium- and low-dose astragaloside IV groups and fenmorphone group, with 10 mice in each group.

3. The method for alleviating premature ovarian insufficiency in mice according to claim 2, characterized in that: The method for establishing the POI mouse model in S12 includes the following steps: S31, the normal control group was given an equal volume of saline intraperitoneal injection, and the other groups were given a single intraperitoneal injection of 120 mg / kg cyclophosphamide / sodium chloride solution and 12 mg / kg busulfan / sodium chloride solution to establish the POI mouse model; S32. From the day after the intraperitoneal injection, vaginal epithelial smear examination was performed on mice in each group once every other day for 15 consecutive days to observe the changes in the estrus cycle of the mice to determine the modeling situation. If the estrus cycle was longer than 5 days, it was considered to be an estrus cycle disorder and the POI model was successfully established.

4. The method for alleviating premature ovarian insufficiency in mice according to claim 3, characterized in that: The method for administering drugs to different groups of mice in S13 comprises the following steps: S41. The mice were given the drug by gavage starting from the day after the estrous cycle was identified. S42, low, medium, and high doses of astragaloside IV groups: Astragaloside IV was dissolved in 1% sodium carboxymethylcellulose solution and intragastrically administered at doses of 40, 80, and 120 mg / kg / d for 28 days; S43, normal control group and POI model group: given an equal volume of 1% CMC-Na solution by intragastric administration for 28 days; S44, Fentaton group: Fentaton was dissolved in 1% CMC-Na solution, and the equivalent dose of the drug was calculated based on the body surface area of ​​humans and animals. The drug was given repeatedly in a cycle of 5 days, and the drug was administered by gavage for 28 days.

5. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: The method for identifying the estrus cycle of each group of mice in S14 is as follows: after the POI model is established and the next day after the end of the drug administration cycle, the mice are fixed, and the exfoliated cells of the vaginal mucosal epithelium are taken for smear. After Papanicolaou staining, the estrus cycle period is observed under a microscope and determined. This is done every other day for 15 consecutive days, and the estrus cycle disorder rate is calculated.

6. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: The method for sampling mice in S15 is as follows: the day after the estrus cycle identification is completed, the mice in each group are weighed, blood is drawn from the eyeballs after anesthesia to obtain serum, and then the mice are killed by cervical dislocation, and the bilateral ovaries are taken. One ovary is fixed with 4% paraformaldehyde, and the other ovary is frozen at -80°C.

7. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: The testing of mouse samples in S15 included detecting the levels of E2, FSH and AMH in the serum by ELISA: the levels of E2, FSH and AMH in the serum of each group of mice were detected using ELISA kits for mouse E2, FSH and AMH, and 3 mouse serum samples were collected from each group, and duplicate wells were performed for both the standard and the samples.

8. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: The testing of mouse samples in S15 included counting the number of follicles at each level after HE staining of ovarian tissue paraffin sections: unilateral ovaries of 3 mice were taken from each group, fixed with 4% paraformaldehyde for 3 hours, dehydrated with gradient ethanol and transparent with xylene, and then embedded in paraffin. Paraffin tissue sections were cut once every 50μm for each ovary, for a total of 10 times, with a thickness of 5μm. 10 paraffin sections were taken and dewaxed routinely, and HE staining was performed, and then the number of follicles at each level was counted.

9. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: In S15, the detection of mouse samples included the detection of ER and FSHR expression levels in the ovaries of each group by immunofluorescence method: paraffin sections of ovaries were taken from each group, and after routine dewaxing, they were washed 3 times with 0.01 mol / L PBS, 10 min / time, and covered with blocking solution at 37°C for 1 hour, and then covered with rabbit FSHR antibody and incubated at 4°C overnight. The next day, the sections were covered with FITC-goat anti-rabbit antibody and incubated at 37°C for 1 hour, and the sections were covered with 5μg / ml Hoechst33342 and incubated at room temperature for 15 minutes to stain the nucleus to prevent quenching of the sealing agent. Finally, the slides were placed on the slides for observation and filming under a fluorescence microscope. Three female mouse ovarian tissues were observed in each group, and the ovarian samples were analyzed using Imagepro-Plus6.0 image analysis software. Three preantral follicles and antral follicles were selected from each ovarian section, and the same area of ​​corpus luteum tissue was selected for FSHR fluorescence OD value analysis.

10. The method for alleviating premature ovarian insufficiency in mice according to claim 4, characterized in that: In S15, the mouse samples were tested by western blotting to detect FSHR, key proteins for estrogen synthesis, and proteins related to the PI3K-AKt-mTOR signaling pathway: ovaries from each group were taken, and proteins were extracted with RIPA strong lysis buffer. The sample volume of each sample was calculated according to the protein concentration determination results. After protein loading, 240V constant voltage electrophoresis was performed for about 35 minutes, and then 400mA current wet transfer was performed for 30-60 minutes, and then blocked with 5% skim milk powder or 5% BSA at room temperature for 1 hour, and finally rabbit β-actin, FSHR, P450scc, Aromatase, P-PI3K, P-AKT1, mTOR, P-mTOR, P-S6K and P-eIF4EBP1 antibodies were incubated at 4°C overnight. The next day, HRP-goat anti-rabbit antibody was incubated at room temperature for 40-50 min. Finally, the PVDF membrane was placed in the Tanon 5200 imaging system, covered with 200 μl of enhanced developer, and photographed using the Tanon Imaging System software. After repeating 3 times, the grayscale value of the images was analyzed using ImageJ analysis software and statistical analysis was performed.

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