动物实验,卵巢早衰 z-bo 2020-09-03 394

　　Background: Primary hypogonadism is defined as abnormal ovarian function and high levels of follicle stimulating hormone (FSH). In women, one of the most common forms of primary hypogonadism is premature ovarian failure (POF), also known as early menopause. This is due to the increased rate of follicular development, the decrease in the number of follicles during ovarian development or amenorrhea, the increase of gonadotropins, and the slow response of follicles to hormone stimulation revealed by irregular menstrual cycles. possible. Previous studies believed that POF is a disease with different pathogenic backgrounds. Known causes of POF include genetic abnormalities, autoimmune diseases, environmental aggression, and iatrogenic damage after surgery, radiation therapy, and medication. The treatment of premature ovarian failure mainly includes hormone replacement therapy and infertility treatment. HRT supplements estrogen deficiency in patients with POF and relieves menopausal symptoms. However, due to the increased risk of breast cancer, heart attack and stroke, people are paying more and more attention to this therapy. Therefore, HRT is usually the last resort for early ovarian failure, and the shortest and lowest dose should be used. In recent years, herbal medicine has caused people's attention to female reproductive dysfunction due to its effectiveness, safety and low cost. The combination of BRT and HRT has a significant therapeutic effect on POF patients, and can significantly improve menstrual status and serum sex hormone levels. BTR consists of four parts: Rehmannia glutinosa, Rehmannia glutinosa, grass and dogwood. In vitro and in vivo studies have shown that the main biologically active compounds isolated from these components have anti-destructive or pharmacological effects on ovarian diseases. More importantly, pro-angiogenesis and anti-apoptotic signals are related to the biological activities of these compounds. Therefore, it is speculated that the effect of BTR on POF may be mediated through pro-angiogenesis and anti-apoptotic mechanisms. To test this hypothesis, this study investigated the effect of BTR on a rat model of early ovarian failure induced by Tripterygium Glycoside (TG). Methods: Inducing animals and premature ovarian failure: All animals will be cared for and treated strictly in accordance with institutional guidelines. Adult female SD rats (body weight 180-220g, 12 weeks old) were reared in a light/dark cycle environment at room temperature 25±1°C and humidity 50±5% for 12 hours. All animals can eat and drink freely. Adapt to the animal facility one week before the experiment. Fifty animals with a normal estrus cycle were tested, and vaginal smears were analyzed daily for 10 days. The selected animals were randomly divided into 5 groups, each with 10 animals: control group, early ovarian failure model group, BTR low-dose group, BTR medium-dose group, and BTR high-dose group. Control group: 4 ml of 0.9% saline was administered twice a day (9:00 am and 3:00 pm) for 15 consecutive days. Premature ovarian failure model group: TG50 mg/kg at 9 am, TG50 mg/kg at 3 pm, 4 mL 0.9% saline, gavage for 15 days. BTR low-dose group: TG50 mg/kg at 9 am, TG50 mg/kg + 1.88 g/kg BRT at 3 pm, oral gavage for 15 consecutive days. BTR medium dose group: TG50 mg/kg at 9:00 in the morning, TG50 mg/kg + BRT 3.75 g/kg at 3:00 in the afternoon, oral gavage for 15 consecutive days. BTR high-dose group: TG50 mg/kg at 9 am, TG50 mg/kg + BRT 7.50 g/kg at 3:00 pm, oral gavage for 15 consecutive days. Estrus cycle assessment and sample collection: During the administration period, the estrus cycle of each animal was analyzed by vaginal smear according to the above method. The cycle length is determined as the number of days between two discrete days between cytological observations of estrus. An estrus cycle of ≥15 days is defined as a cycle stop. If the number of estrus cycles is 2 or more, please calculate the average length of the estrus cycle for each animal. Fifteen days after administration, the animals in estrus were sacrificed (by vaginal smear analysis) in the pre-estrus period (1-5 days after the last administration). The animals that stopped the female cycle were sacrificed the next day after the last administration. Collect animal femoral artery blood samples before running. After execution, collect two ovaries and weigh them on an electronic balance. Calculate the ovarian index of each animal according to the following formula: ovarian index = bilateral ovarian wet weight (mg)/body weight (g) x 100%. Measurement of serum E2, FSH, P, T levels: Use a commercially available radioimmunoassay kit to measure the serum E2, Follicle Stimulating Hormone (FSH), P and T levels of each animal. Repeat each test and calculate the average. Histological analysis: For histochemical analysis, the left ovary of each animal was fixed with 4% neutral formaldehyde, embedded in paraffin and cut into a thickness of 3-5 μm. Stain with hematoxylin-eosin. Observe the cross section at 100x magnification and take pictures. In order to quantitatively evaluate cell apoptosis, five sections of each rat were randomly taken under a 400x microscope. Subsequently, 10 non-overlapping high power fields (HPFS) were randomly selected to calculate the number of granular cell apoptosis. Two independent analysts who did not know the treatment group counted and recorded the average value. Immunohistochemical analysis: Perform immunohistochemical analysis to detect the expression of VEGF, VEGFR2, Bcl-2, Bax and Caspase 3 in ovarian slices of different treatment groups. The right ovary of each animal was fixed with 4% neutral buffered formaldehyde solution for 2 hours. The fixed sample was dehydrated in a gradient ethanol solution, embedded in paraffin, and cut into 3-5 μm. Use commercially available immunohistochemical reagents for immunohistochemical staining. Finally, the sections were developed in DAB, stained with HE, and photographed at 100x. Scoring criteria: 0, no staining, 1, weak staining, 2, moderate staining, 3, strong staining.

　　Results: TG-induced premature ovarian failure rat model BTR restored abnormal ovarian cycle and improved ovarian index: During the experiment, one animal in the POF model group died of unknown reasons and abandoned. We studied the estrus cycle and ovaries of different animal groups index. Five days after TG induction, the POF model group had reduced food intake, spontaneous activity and stimulus response compared with the control group. In the three BTR treatment groups, there was no change in animal food intake and no abnormal behavior was observed. Within 15 days of TG induction, most animals in the POF model group showed that the estrus cycle was blocked, and the rest of the estrus cycle was longer than the control animals. However, this abnormal TG-induced estrus cycle was clearly offset by BTR in a dose-dependent manner. High-dose BTR showed a similar estrus cycle to control animals. After treatment, anatomical examination was performed on each group of ovaries and the ovarian index of each group was calculated. The ovarian index of the POF model group was significantly lower than that of the control group. The decrease in ovarian index induced by TG was offset by different concentrations of BTR in a dose-dependent manner. These results indicate that BTR can restore abnormal estrus cycles and improve TG-induced premature ovarian failure. In the TG-induced early ovarian failure model, the effect of BTR on serum E2, FSH and P levels: In early ovarian failure, under appropriate gonadotropin stimulation, the ovaries cannot function normally and produce normal sex hormones. Previous studies have reported that POF is associated with decreased serum E2 levels and increased serum P, FSH and T levels. The serum E2, FSH, P and T levels of each group were measured. Study the effect of BTR on the secretion of these reproductive hormones. Compared with the control group, the serum E2 level of the animals in the POF model group was significantly reduced, and the FSH, P and T levels were significantly increased. These TG-induced changes were significantly prevented by BTR in a dose-dependent manner, indicating that BTR can improve the abnormal secretion of POF-related reproductive hormones. The effect of BTR on the function of primary follicles, developing follicles and corpus luteum in the TG-induced early ovarian failure model: POF shows that the number of developing follicles is reduced, which affects reproductive activities. Histological analysis of ovarian sections was performed to study the effect of BTR on primary follicles, developing follicles and corpus corpus luteum in TG-induced early ovarian failure models. The stained sections of the control group showed normal cortex and medulla, with multiple mature follicles at different stages. The corpus luteum is visible, but there is no follicular ovarian cyst or luteal hematoma. No inflammatory cell infiltration or ovarian fibrosis was found in the cortex or medulla. In the POF model group, the histological abnormalities of the cortex and medulla, the number of primordial follicles and primary follicles were significantly reduced, and oocyte maturation and maturation were reduced. Ovarian interstitial fibrosis, luteal degeneration and necrosis, inflammatory cell infiltration and vasodilation were also observed in these sections. In the three BTR treatment groups, the slices showed that the pathological changes caused by TG caused by BTR could be significantly reduced. The histology of BTR middle-high group is similar to that of normal group and control group. The effect of BTR on the expression of VEGF and VEGFR2 in the ovaries of rats with early ovarian failure induced by TG: Histological analysis of ovarian slices showed changes in the morphology of blood vessels in each group. Immunohistochemical staining was performed to observe the ovarian BTR expression of the two main pro-angiogenic factors VEGF and VEGFR2 in the TG-induced early ovarian failure model. Compared with the control group, the immunohistochemical staining intensity of the samples in the POF model group was significantly reduced, and BTR significantly restored the staining reduction in a dose-dependent manner. The results of semi-quantitative immunohistochemical evaluation also support these findings. TG-induced POF reduces ovarian VEGF and VEGFR2 expression.

　　BTR protects TG-induced early ovarian failure rat model and reduces granulosa cell apoptosis: To study the protective effect of BTR on TG-induced early ovarian failure rat model granulosa cell apoptosis. The stained sections of the control ovary showed that most granulosa cells were healthy and showed no signs of apoptosis. In the POF model group, the cells are irregular and dense, and the nuclei are small and compact. In addition, the cell nucleus is broken in the late stage of apoptosis, leading to vacuolization and apoptotic bodies. The BTR treatment group showed that most granulosa cells had complete cell membranes and unique nuclei. Quantitative analysis showed that the proportion of apoptotic cells in all BTR treatment groups (especially the high-dose BTR group) was reduced. it is. The results show that BTR can protect the ovarian granulosa cells in TG-induced early ovarian failure model rats from apoptosis. In order to further verify the results of histological examination, we examined the expression of several apoptosis-related proteins Bcl-2, Bax and Caspase-3 in BTR. Compared with the control group, the bcl-2 of the early ovarian failure model group was significantly reduced, while the bax and caspase3 were significantly increased. However, BTR reversed these changes in a dose-dependent manner.

　　Conclusion: The current research shows that BTR can effectively treat TG-induced premature ovarian failure in rats. The analysis of histological and immunohistochemical results showed that promoting angiogenesis and anti-apoptosis are two mechanisms affected by BTR.