Objective To observe the effects of different concentration and intervention time of triptolide(TP)on ovarian oxidative stress and mitophagy in rats.Methods Fifty 3-month-old female Sprague-Dawley rats were observed for 2 consecutive estrus cycles,and 25 rats with normal estrus cycles were selected and divided into the blank control group,experiment group 1,experiment group 2,experiment group 3 and experiment group 4 according to random number table method,with 5 rats in each group.Rats in experiment group 1 were administered intragastrically 400 μg·kg-1 TP once a day for 30 d.Rats in experiment group 2 were given 400 μg·kg-1 TP once a day for 40 d.Rats in experiment group 3 were given 500 μg·kg-1 TP once a day for 30 d.Rats in the experiment group 4 were given 500 μg·kg-1 TP once a day for 40d.Rats in the blank control group were given 10 mL·kg-1 distilled water once a day for 40 d.Serum anti-mullerian hormone(AMH),estradiol(E2)and follicle-stimulating hormone(FSH)levels were detected by enzyme-linked immunosorbent assay;hematoxylin-eosin staining was used to observe the pathological changes of ovarian tissue;malondialdehyde(MDA)level and superoxide dismutase(SOD)activity in ovarian tissue were detected by microplate reader.The morphological and structural changes of mitochondria in ovarian granulosa cells of rats in each group were observed by transmission electron microscope,and the apoptosis rate of ovarian granulosa cells was detected by flow cytometer.Results Compared with the blank control group,the levels of AMH and E2 in serum and SOD activity in ovarian tissue of rats in the experiment groups 1,2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P＜0.05).Compared with the experiment group 1,the levels of AMH and E2 in serum and SOD activity in ovarian tissue of rats in experiment groups 2,3 and 4 significantly decreased,while the levels of FSH in serum and MDA in ovarian tissue significantly increased(P＜0.05).There was no significant difference in the levels of AMH,E2 and FSH in serum,SOD activity and MDA level in ovarian tissue of rats among experiment groups 2,3 and 4(P＞0.05).The apoptosis rate of ovarian granulosa cells in experiment groups 1,2,3 and 4 was significantly higher than that in the blank control group(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 2,3 and 4 was significantly higher than that in experiment group 1(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment groups 3 and 4 was significantly higher than that in experiment group 2(P＜0.05);the apoptosis rate of ovarian granulosa cells in experiment group 4 was significantly higher than that in experiment group 3(P＜0.05).In the blank control group,the ovarian tissue capsule was intact,and the number and development of primordial,primary and secondary follicles in the cortex were normal,with rare atretic follicles and less corpus luteum.The fibrous connective tissue in the medullary area was closely arranged,and no obvious edema or necrosis was observed.In experiment groups 1,2,3 and 4,the number of follicles in ovarian tissue decreased,the number of atretic follicles increased,and necrosis and shedding of follicles in the granular layer and cystic dilatation of follicles were observed.The pathological changes of ovarian tissue in experiment group 1 were relatively light,with fewer atretic follicles and less cell necrosis in the granulosa cell layer.The degree of ovarian tissue lesions in experiment group 4 was the most severe,with more atretic follicles and cell necrosis and shedding in the granulosa cell layer.The degree of ovarian tissue lesions in experiment groups 2 and 3 was less than that in experiment group 4,with fewer atretic follicles,necrosis of granulosa cell layer and follicle cystic dilatation.In the blank control group,the ovarian granulosa cells were normal in morphology and structure,with irregular polygon nuclei,uniform distribution of chromatin,mainly euchromatin,clear and complete nuclear membrane,mitochondria and other organelles with complete and clear structure visible in the cytoplasm.Different degrees of mitophagy were observed in the cytoplasm of granulosa cells of rats in experiment groups 1,2,3 and 4;the mitophagy of rats in experiment groups 2,3 and 4 was more severe than that in experiment group 1;that in experiment groups 3 and 4 was more severe than experiment group 2;the mitophagy of rats in experiment group 3 was similar to that in experiment group 4.Under electron microscopy,more mitochondrial autophagosomes were found in the cytoplasm of ovarian granulosa cells in experiment groups 3 and 4.Most mitochondria were mildly swollen,some mitochondrial crista structures disappeared,and the rough endoplasmic reticulum showed cystic dilatation.Conclusion The ovarian dysfunc-tion model in rats can be successfully established by TP intragastric administration.The ovarian injury of rats is related to TP dose and intervention time.TP-induced ovarian oxidative stress may be an important factor triggering ovarian hypofunction,and it may work through oxidative stress affecting ovarian endocrine function and inducing granulosa cell apoptosis and other patho-physiological processes.With the aggravation of oxidative stress damage,the mitophagy increases.After reaching a certain de-gree,mitophagy will not continue to increase with the increase of TP intervention time.

Objective To study the mechanism of Yangjing Zhongyu Decoction in regulating the initiation of primordial follicles in model rats with diminished ovarian reserve(DOR)based on lncRNA.Methods Three-day-old female rats were selected and their ovaries were cultured in vitro.The blank group,model group,DHEA group and Yangjing Zhongyu Decoction high-,medium-and low-dosage groups were set.The DOR model was induced by triptolide,corresponding drug containing serum was given to culture respectively.HE staining was used to observe germ cells and follicles,Western blot was used for determining the expressions of AMH,BMP15,PTEN,MST,TGF-β1,p-Smad1 protein,RT-PCR was used to detected AMH,BMP15,PTEN,MST,LTCONS-00011173,TGF-β1,Smad1 mRNA expression.Results Compared with the blank group,the number of primordial and growing follicles in the model group rats decreased(P<0.05),the expression of AMH,BMP15,TGF-β1,p-Smad1 protein in ovarian tissue decreased(P<0.05),expressions of PTEN and MST proteins increased(P<0.05),AMH,BMP15,TGF-β1,Smad1 mRNA expression decreased(P<0.05),while the expressions of PTEN,MST,and LTCONS-00011173 mRNA increased(P<0.05).Compared with the model group,the DHEA group and Yangjing Zhongyu Decoction high-and medium-dosage groups showed an increase in the number of primordial and growing follicles(P<0.05),the expressions of AMH,BMP15,TGF-β1 and p-Smad1 protein in ovarian tissue increased(P<0.05),PTEN and MST protein expressions decreased(P<0.05),AMH,BMP15,TGF-β1,Smad1 mRNA expressions increased(P<0.05),while PTEN,MST,and LTCONS-00011173 mRNA expressions decreased(P<0.05).Conclusion Yangjing Zhongyu Decoction may mediate TGF-β1/Smad1 signaling pathway through LTCONS-00011173,regulating primordial follicle initiation in DOR model rats.