人参皂苷Rg_(1)对顺铂损伤大鼠卵巢颗粒细胞的保护作用及其分子机制

Protective effect and molecular mechanism of ginsenoside Rg_(1) on the senescence of rat ovarian granulosa cells induced by cisplatin

目的探究人参皂苷Rg_(1)对顺铂损伤大鼠卵巢颗粒细胞的保护作用及分子机制。方法选取22~24 d SD的大鼠提取卵巢颗粒细胞进行原代培养,应用顺铂诱导建立卵巢早衰模型。设置正常组,模型组,人参皂苷Rg_(1)低浓度组、中浓度组、高浓度组和雌二醇(E2)组。HE染色及免疫细胞化学法进行颗粒细胞鉴定;CCK8法检测人参皂苷Rg_(1)作用24 h、48 h对顺铂损伤颗粒细胞的保护作用;Hoechst 33258染色检测细胞凋亡;Western blot法检测FSHR、PI3K、p-AKT、AKT、Bcl-2和Bax的蛋白表达。结果细胞形态观察和胞质FSHR表达检测结果均表明所提取细胞为卵巢颗粒细胞;顺铂处理12 h后,颗粒细胞增殖率呈剂量依赖性下降,而给予人参皂苷Rg_(1)后,细胞增殖率下降被显著抑制,且呈剂量和时间依赖性;与模型组比较,人参皂苷Rg_(1)和E2处理后卵巢颗粒细胞中FSHR、PI3K、p-AKT/AKT及Bcl-2/Bax蛋白表达水平显著上升,而PI3K抑制剂干预后,Bcl-2蛋白表达显著下降。结论人参皂苷Rg_(1)对顺铂损伤的卵巢颗粒细胞具有保护作用,该保护作用可能通过激活FSHR/PI3K/AKT通路,抑制卵巢颗粒细胞凋亡实现。

Objective To determine the effect and molecular mechanism of ginsenoside Rg_(1)on the senescence of rat ovarian granulosa cells induced by cisplatin.Methods Firstly,primary granulosa cells extracted from 22-24 d SD rats were cultured.Premature ovarian failure(POF)cell model induced by cisplatin was applied for the following experiments.Granulosa cells were divided into a normal group,a model group,ginsenoside Rg_(1)low,medium,high concentration groups,and an estradiol(E2)group.The primary granulosa cells were identified by HE staining and immunocytochemistry.CCK8 assay was used to detect the protective effect of ginsenoside Rg_(1)on the granulosa cells injured by cisplatin at 24 h and 48 h.The cell apoptosis was detected by Hoechst 33258 staining.The protein expression of FSHR,PI3K,p-AKT,AKT,Bcl-2 and Bax were examined by Western blot.Results The expression of FSHR in the cytoplasm and cell morphology indicated that the extracted cells were granulosa cells.The proliferation rate of granulosa cells decreased in a dose-dependent manner after 12 h of cisplatin treatment,while the decrease in proliferation rate of granulosa cells was inhibited in a dose and time-dependent manner after ginsenoside Rg_(1)treatment.Compared with the model group,the protein expression of FSHR,PI3K,p-AKT/AKT and Bcl-2/Bax in the ovarian granulosa cells increased significantly after ginsenoside Rg_(1)and E2 treatment,while the protein expression of Bcl-2 decreased significantly after PI3K inhibitor intervention.Conclusion Ginsenoside Rg_(1)can protect cisplatin-injured granulosa cells by activating FSHR/PI3K/AKT pathway and inhibiting the apoptosis of granulosa cells.