陈氏培元汤含药血清对卵巢颗粒细胞自噬的调控作用

Regulatory Effect of Chen's Peiyuan Decoction Medicated Serum on Autophagy of Ovarian Granulosa Cells

关键词:  陈氏培元汤  含药血清  卵巢颗粒细胞  自噬

Key words:  Chen's Peiyuan decoction  medicated serum  ovarian granulosa cells  autophagy

摘要:

目的 研究陈氏培元汤含药血清对卵巢颗粒细胞（ovarian granulosa cells，OGCs）自噬的影响。方法 用SD大鼠制备含药血清，分为正常组大鼠血清和陈氏培元汤组大鼠血清；分离OGCs细胞并分为正常组、模型组、陈氏培元汤组和信号通路阻滞剂组，除正常组外，其余3组通过雷公藤甲素制作免疫性卵巢早衰模型，正常组和模型组加入正常组大鼠血清，陈氏培元汤组加入陈氏培元汤含药血清，信号通路阻滞剂组加入陈氏培元汤含药血清和信号通路阻滞剂；ELISA试剂盒测各组培养液中雌二醇（E₂）和孕酮（P）的浓度，免疫荧光染色分析LC3II、m-TOR、p62表达，透射电镜观察各组OGCs细胞自噬情况，Western blotting测定各组细胞Beclin-1、LC3II、m-TOR、p62蛋白的相对表达。结果 与正常组相比，模型组和信号通路阻滞剂组的E₂浓度明显降低（P<0.05）；陈氏培元汤组E₂浓度比模型组和信号通路阻滞剂组高（P<0.05）；与正常组相比，模型组、陈氏培元汤组和信号通路阻滞剂组P的浓度差异无统计学意义。正常组和陈氏培元汤组LC3II免疫荧光表达较弱，m-TOR、p62荧光免疫表达增强；模型组和信号通路阻滞剂组LC3II免疫荧光表达增强，m-TOR、p62荧光免疫表达减弱；模型组和信号通路阻滞剂组OGCs细胞内可见大量的自噬小体，正常组和陈氏培元汤组OGCs细胞内偶见自噬小体。与模型组和通路阻滞剂组相比，陈氏培元汤组OGCs细胞中的LC3II和Beclin-1蛋白的相对表达水平明显降低，m-TOR和p62蛋白的相对表达水平明显升高（P<0.05）。结论 陈氏培元汤含药血清可以有效抑制OGCs细胞的过度自噬，从而发挥对卵巢的保护作用。

Abstract:

OBJECTIVE To study the regulatory effect of Chen's Peiyuan decoction medicated serum on autophagy of ovarian granulosa cells(OGCs). METHODS The SD rats were divided into normal group and Chen's Peiyuan decoction group, then collected the drug medicated serum. The isolated OGCs were divided into normal group, model group, Chen's Peiyuan decoction group and signal pathway blocker group. Without the normal group, the other three groups were made immune premature ovarian failure models through tripterygium glycosides. The normal and model groups were added with normal group rat serum, Chen's Peiyuan decoction group was added with Chen's Peiyuan decoction medicated serum, and the signal pathway blocker group was added Chen's Peiyuan decoction medicated serum and signal pathway blocker. The E₂ and P in group culture medium were determined by ELISA. The expression of LC3II, m-TOR, p62 in cells were analyzed by immunofulourescence staining. The autophagy of OGCs were observed by transmission electron microscope. The relative expression of Beclin-1, LC3-II, m-TOR and p62 were measured by Western blotting. RESULTS Compared with the normal group, the E₂ concentration in the model group and signal pathway blocker group were obviously decreased(P<0.05). The E₂ concentration in Chen's Peiyuan decoction group was higher than model group and signal pathway blocker group(P<0.05). There was no significant difference in the concentration of P in the normal group, model group, Chen's Peiyuan decoction group and signal pathway blocker group. In normal group and Chen's Peiyuan decoction group, the LC3II had weak immunofluorescence expression, but m-TOR and p62 had enhanced immunofluorescence expression. In model group and signal pathway blocker, the LC3II had enhanced immunofluorescence expression, but m-TOR and p62 had weak immunofluorescence expression. A large number of autophagy bodies were observed in granulosa cells in model and signal pathway blocker groups while the normal and Chen's Peiyuan decoction groups were seldom. Compared with model group and pathway blocker group, the relative expression levels of LC3-II and beclin-1 in Chen's Peiyuan decoction group were decreased, while the relative expression level of m-TOR and p62 protein increased(P<0.05). CONCLUSION The Chen's Peiyuan decoction can effectively inhibit the transitional autophagy of OGCs and protect ovarian.