通过p53/AMPK信号通路研究归术益坤方对多囊卵巢综合征大鼠颗粒细胞自噬的影响

Effects of Guizhu Yikun Formula on autophagy in granulosa cell of rats with polycystic ovarian syndrome through p53/AMPK pathway

目的:研究归术益坤方治疗多囊卵巢综合征(PCOS)的作用机制。方法:通过体外培养大鼠卵巢颗粒细胞,以丙酸睾丸酮(TP)、自噬诱导剂C2-神经酰胺作用颗粒细胞24h,建立大鼠PCOS颗粒细胞的自噬模型;实验分为空白组,模型组,归术益坤方低、中、高剂量组及自噬阻断剂3MA组。通过Western blot法检测各组颗粒细胞中LC3Ⅱ、p53、p-m TOR、p-AMPK的表达。结果:与空白组比较,模型组LC3Ⅱ的表达显著增加(P<0.01);与模型组比较,归术益坤方高、中、低剂量组表达降低(P<0.01)。与空白组比较,模型组p53、p-m TOR显著降低(P<0.01),p-AMPK升高(P<0.01);与模型组比较,高剂量归术益坤方可以上调p53和p-m TOR的表达(P<0.01,P<0.05);高、中剂量归术益坤方可下调p-AMPK的表达(P<0.01)。结论:归术益坤方可以通过调控p53/AMPK信号通路来抑制自噬,这可能是归术益坤方治疗PCOS的新机制。

Objective: To explore the molecular mechanism of Guizhu Yikun Formula(GZYKF) in treating polycystic ovarian syndrome(PCOS). Methods: Granulosa cells(GCs) in PCOS rats were cultured and treated with testosterone propionate(TP) and C2-ceramide for 24 h to establish PCOS cell model in vitro. GCs were divided into blank group, model group, 3 MA group, GZYKF high, middle and low dose groups. The expression of LC3 II, p53, p-m TOR, and p-AMPK were observed by Western blot. Results: Compared with blank group, the expression of LC3 II was significantly increased in model group(P<0.01).Compared with model group, the expression of LC3 II in the high, middle, low dose GZYKF group decreased(P<0.01). Compared with the blank group, the expression of p53, p-m TOR in the model group decreased(P<0.01), the expression of p-AMPK increased(P<0.01). Compared with model group, high dose GZYKF could increase the expression of p53 and p-m TOR(P<0.01, P<0.05), and high, middle dose GZYKF could decrease the expression of p-AMPK(P<0.01). Conclusion: The effects of GZYKF on mediating the p53/AMPK pathway could inhibit GC autophagy which suggesting a possible novel mechanism by GZYKF's treatment of PCOS.