哈氏育阴汤调控RIP1/RIP3/MLKL通路对卵巢储备功能减退大鼠颗粒细胞凋亡的影响OA

Objectives:To investigate the effects of Ha's Yuyin decoction on granulosa cell apoptosis and the receptor-interacting protein kinase 1(RIP1)/receptor-interacting protein kinase 3(RIP3)/mixed lineage kinase domain-like protein(MLKL)pathway in rats with diminished ovarian reserve(DOR). Results:Compared with the control group,the model group exhibited a significant reduction in primary and secondary ovarian follicles,along with inflammatory cell infiltration.Serum levels of follicle-stimulating hormone(FSH),interleukin-6(IL-6),and tumor necrosis factor-alpha(TNF-α)were significantly increased(P＜0.05).Ovarian tissue malondialdehyde(MDA)content,granulosa cell apoptosis rate,and phosphorylation levels of RIP1/RIP3/MLKL pathway proteins were also significantly elevated(P＜0.05).Conversely,serum levels of anti-Müllerian hormone(AMH),estradiol(E),and interleukin-4(IL-4)were significantly decreased(P＜0.05),and ovarian tissue superoxide dismutase(SOD)and catalase(CAT)activities were markedly reduced(P＜0.05).Compared with the model group,the low-dose TCM group,high-dose TCM group,and inhibitor group showed a significant increase in the number of ovarian follicles at various growth stages(P＜0.05)and a significant decrease in atretic follicles(P＜0.05);Serum levels of FSH,IL-6,and TNF-α were significantly reduced(P＜0.05);Ovarian tissue MDA content,granulosa cell apoptosis rate,and phosphorylation levels of RIP1/RIP3/MLKL pathway proteins were also significantly decreased(P＜0.05);Serum levels of AMH,E2,and IL-4,as well as ovarian tissue SOD and CAT activities,were significantly increased(P＜0.05).Compared with the high-dose TCM group,the administration of rRIP1 in the activator group significantly reversed the ameliorative effects of high-dose Ha's Yuyin decoction on ovarian histopathological damage(P＜0.05). Conclusions:Ha's Yuyin decoction alleviates oxidative stress and inflammatory damage in ovarian tissue,inhibits granulosa cell apoptosis,and improves ovarian function in DOR rats.Its mechanism of action may involve the RIP1/RIP3/MLKL pathway. Methods:The DOR model was established by a single intraperitoneal injection of cyclophosphamide(75 mg/kg).The model rats were divided into the model group,Ha's Yuyin decoction low-dose group[low-dose traditional Chinese medicine(TCM)group,intragastric administration of 11.30 g/kg Ha's Yuyin decoction],Ha's Yuyin decoction high-dose group(high-dose TCM group,intragastric administration of 22.60 g/kg Ha's Yuyin decoction),pathway inhibitor group(inhibitor group,intraperitoneal injection of 2.45 mg/kg Necrostatin-1),and Ha's Yuyin decoction high-dose+pathway activator group(activator group,intragastric administration of 22.60 g/kg Ha's Yuyin decoction plus tail vein injection of 8 μg/kg rRIP1),with 10 rats in each group.Another 10 healthy rats were taken as the control group.Rats in the control group and model group were given an equal volume of normal saline by intragastric administration.Serum levels of sex hormones and inflammatory factors were measured using enzyme-linked immunosorbent assay(ELISA).Ovarian tissues from each group were collected to assess pathological changes(hematoxylin-eosin staining),oxidative stress factor levels(ELISA),granulosa cell apoptosis(TUNEL staining),and protein expression levels of the RIP1/RIP3/MLKL pathway(Western blot).