动物造模 z-bio 2022-07-19 300

　　Background: Primary hypogonadism is defined as ovarian insufficiency with high serum follicle-stimulating hormone (FSH) levels. In women, one of the most common forms of primary hypogonadism is premature ovarian failure (POF), also known as premature menopause. It may be due to the increased rate of follicle loss, the decrease in the number of follicles during ovarian development, or the slow response of follicles to hormonal stimulation, manifesting as amenorrhea, elevated gonadotropins, and irregular menstrual cycles. Previous studies have identified POF as a disease with a heterogeneous pathogenic background, and known causes of POF include genetic abnormalities, autoimmune diseases, and environmental insults, as well as iatrogenic injury following surgery, radiotherapy, and drug therapy. The treatment of premature ovarian failure mainly includes hormone replacement therapy and infertility treatment. HRT can compensate for estrogen deficiency in POF patients, thereby reducing menopausal symptoms. However, due to the increased risk of breast cancer, heart attack and stroke, there has been increasing interest in this therapy. Therefore, HRT is usually the last resort for premature ovarian failure, and the lowest dose should be used for the shortest period of time. In recent years, traditional Chinese medicine has attracted people's attention to female reproductive dysfunction due to its efficacy, safety, and low cost. The combination of BRT and HRT showed a significant therapeutic effect in POF patients, significantly improving menstrual status and serum sex hormone levels. BTR consists of four parts, Rehmannia glutinosa, white peony root, turtle shell turtle shell and dogwood. In vitro and in vivo studies have shown that the main bioactive compounds isolated from these components have anti-depletion or pharmacological effects on ovarian disease. More importantly, pro-angiogenic and anti-apoptotic signaling are associated with the biological activities of these compounds. Therefore, we speculate that the effects of BTR on POF may be mediated through pro-angiogenic and anti-apoptotic mechanisms.

　　To test this hypothesis, in this study, we investigated the effect of BTR on a rat model of premature ovarian failure induced by triptolide (TG).

　　Methods: Animals and induction of premature ovarian failure: All animal care and treatments were performed in strict accordance with institutional guidelines. Adult female SD rats (body weight 180-220 g, 12 weeks old) were housed in an environment of room temperature 25±1°C, humidity 50±5%, and a light/dark cycle of 12 hours. All animals had free access to food and water. Before the experiment, acclimate to the animal facility for 1 week. Fifty animals with normal estrus cycle were used as experimental subjects, and vaginal smears were analyzed every day for 10 consecutive days. The selected animals were randomly divided into five groups with ten animals each: control group, premature ovarian failure model group, BTR low-dose group, BTR medium-dose group and BTR high-dose group. Control group: 4 ml of 0.9% normal saline was administered twice a day (9:00 am and 3:00 pm) by intragastric administration for 15 consecutive days. Premature ovarian failure model group: TG 50 mg/kg at 9:00 am, TG 50 mg/kg plus 4 mL of 0.9% normal saline at 3:00 pm, for 15 consecutive days. BTR low-dose group: TG 50 mg/kg at 9:00 am, TG 50 mg/kg plus 1.88 g/kg BRT at 3:00 pm, intragastrically for 15 consecutive days. BTR medium-dose group: TG 50 mg/kg at 9:00 am, TG 50 mg/kg plus 3.75 g/kg BRT at 3:00 pm, intragastrically for 15 consecutive days. BTR high-dose group: TG 50 mg/kg at 9:00 am, TG 50 mg/kg plus 7.50 g/kg BRT at 3:00 pm, intragastrically for 15 consecutive days.

　　Estrus cycle assessment and sample collection: During the dosing period, the estrous cycle of each animal was analyzed by vaginal smear as previously described. The length of a cycle was determined as the number of days between two non-consecutive days of estrous cytology observation. A cycle of estrus ≥ 15 days was defined as cessation of the cycle. When the number of estrous cycles was ≥ 2, the average length of estrous cycles of each animal was calculated. Fifteen days after dosing, animals in heat (based on vaginal smear analysis) were sacrificed in proestrus (within 1-5 days after the last dose). Animals that stopped the estrous cycle were sacrificed the day after the last dose. The femoral arterial blood samples of animals were taken before sacrifice. After sacrifice, two ovaries were taken and weighed with an electronic balance. The ovarian index of each animal was calculated according to the following formula: ovarian index = wet weight of bilateral ovaries (mg)/body weight (g) × 100%.

　　Determination of serum E2, FSH, P, T levels: Serum E2, follicle stimulating hormone (FSH), P and T levels of each animal were determined using a commercial radioimmunoassay kit. Each assay was performed in duplicate and the mean value was calculated.

　　Histological analysis: For histochemical analysis, the left ovary of each animal was fixed in 4% neutral formaldehyde, embedded in paraffin, and cut into 3-5 μm thick. Stained with hematoxylin-eosin. Sections were observed and photographed at 100× magnification. For quantitative evaluation of apoptosis, five parts of each rat were randomly photographed under a 400× microscope. Subsequently, ten non-overlapping high-power fields (HPFS) were randomly selected to count the number of granulosa cells apoptotic. Counts were performed by two independent analysts, who were unclear about treatment groupings, and averages were recorded.

　　Immunohistochemical analysis: Immunohistochemical analysis was performed to detect the expressions of VEGF, VEGFR2, Bcl-2, Bax and Caspase 3 in ovarian sections of different treatment groups. The right ovary of each animal was fixed in 4% neutral buffered formaldehyde solution for 2 h. The fixed samples were dehydrated with graded ethanol solution, embedded in paraffin, and cut into 3-5 μm. Immunohistochemical staining was performed using commercial immunohistochemical reagents. Finally, sections were developed with DAB, HE stained, and photographed at 100×. 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 estrous cycle and improved ovarian index: During the experiment, one animal in the POF model group died for unknown reasons, and the subsequent data were discarded. We studied the estrous cycle and ovarian index of different groups of animals. After 5 days of TG induction, food intake, locomotor activity and stimulus responses were reduced in POF model group compared with control group. In the three BTR-treated groups, animals' food intake remained unchanged and no abnormal behavior was observed. Within 15 days of TG induction, most animals in the POF model group showed cessation of estrous cycles, and the remaining estrous cycles were longer than control animals. However, this TG-induced abnormal estrous cycle was clearly counteracted by BTR in a dose-dependent manner. High doses of BTR showed similar estrous cycles as control animals.

　　After the treatment, the ovaries of each group were anatomically examined, and the ovarian index of each group was calculated. The ovarian index of POF model group was significantly lower than that of control group. The TG-induced reduction in ovarian index could be counteracted by different concentrations of BTR in a dose-dependent manner. These results suggest that BTR can restore abnormal estrous cycle and ameliorate TG-induced premature ovarian failure.

　　The effect of BTR on serum E2, FSH, and P levels in TG-induced premature ovarian failure model: During premature ovarian failure, the ovary cannot function normally under appropriate gonadotropin stimulation, so it cannot 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 animals in each group were measured. To explore the effect of BTR on the secretion of these reproductive hormones. Compared with the control group, the serum E2 level of the POF model group was significantly decreased, and the FSH, P, T levels were significantly increased. These TG-induced changes were significantly prevented by BTR in a dose-dependent manner, suggesting that BTR ameliorated POF-related abnormal secretion of reproductive hormones.

　　The effect of BTR on primary follicles, growing follicles and corpus luteum function in a TG-induced premature ovarian failure model: POF manifests as a decrease in the number of developing follicles, thereby affecting reproductive activity. Histological analysis of ovarian sections was performed to investigate the effect of BTR on primary follicle, growing follicle and corpus luteum function in TG-induced premature ovarian failure model. Stained sections of the control group showed normal cortex and medulla with multiple mature follicles at different stages. The corpus luteum was visible, but there was no follicular ovarian cyst or corpus luteum hematoma. No inflammatory cell infiltration or ovarian fibrosis was found in either 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 the number of oocytes degenerated and matured follicles were less. Ovarian interstitial fibrosis, corpus luteum degeneration and necrosis, inflammatory cell infiltration, and vasodilation were also seen in the sections. For the three BTR-treated groups, slices showed that TG-induced pathological changes were significantly attenuated by BTR. The histology of BTR middle and high group was close to that of normal tissue and control group.

　　The effect of BTR on the expression of VEGF and VEGFR2 in the ovary of TG-induced premature ovarian failure model rats: Histological analysis of ovarian sections showed that the vascular morphology changed in each group. Immunohistochemical staining was performed to observe the intraovarian expression of BTR on two key pro-angiogenic factors, VEGF and VEGFR2, in a TG-induced premature ovarian failure model. Compared with the control group, samples from the POF model group showed a significant decrease in the intensity of immunohistochemical staining, and the decrease in staining was significantly recovered by BTR in a dose-dependent manner. These findings were also confirmed by the results of a semiquantitative immunohistochemical assessment. TG-induced POF reduces ovarian VEGF and VEGFR2 expression.

　　BTR protects TG-induced premature ovarian failure rat model and reduces granulosa cell apoptosis: To study the protective effect of BTR on granulosa cell apoptosis in TG-induced premature ovarian failure rat model. The ovarian staining sections of the control group showed that most of the granulosa cells were healthy and there was no sign of apoptosis. In the POF model group, the cells were dense and irregular in shape, and the nuclei were small and dense. In addition, in the late stages of apoptosis, nuclei are fragmented, resulting in vacuolization and apoptotic bodies. The BTR-treated group showed that most of the granulosa cells had intact cell membranes and clear nuclei. Quantitative analysis showed that the proportion of apoptotic cells decreased in all BTR-treated groups, especially in the BTR high-dose group. The results showed that BTR could protect TG-induced premature ovarian failure model rat ovarian granulosa cells from apoptosis. To further verify the results of histological examination, the expression of several apoptosis-related proteins Bcl-2, Bax and caspase-3 in BTR was studied. Compared with the control group, bcl-2 in the premature ovarian failure model group was significantly decreased, and bax and caspase 3 were significantly increased. However, these changes were reversed by BTR in a dose-dependent manner.

　　Conclusion: The current study shows that BTR is effective in the treatment of TG-induced premature ovarian failure in rats. Analysis of histological and immunohistochemical results indicated that promoting angiogenesis and anti-apoptosis are two mechanisms that may be affected by BTR.