活血消异方影响过氧化氢诱导的氧化应激卵巢颗粒细胞自噬的机制研究

Mechanism of Huoxue Xiaoyi Prescription in Regulating Autophagy of Ovarian Granulosa Cells Exposed to Hydrogen Peroxide-Induced Oxidative Stress

目的 探讨氧化应激对卵巢颗粒细胞自噬流的影响及活血消异方的干预作用。方法 提取大鼠原代卵巢颗粒细胞进行体外培养,过氧化氢(H_(2)O_(2))诱导子宫内膜异位症氧化应激颗粒细胞模型,颗粒细胞培养箱孵育48 h后,分为未干预组、氧化应激组(H_(2)O_(2)组)、JNK抑制剂组、活血消异方含药血清组(中药组),分别加入完全培养基、10%正常大鼠血清、含800 nm SP600125的10%正常大鼠血清、10%活血消异方含药血清。自噬双标腺病毒(mRFP-GFP-LC3)感染标记,倒置激光共聚焦显微镜观察各组细胞内自噬流的变化。结果 mRFP-GFP-LC3感染复数(MOI)为100时,颗粒细胞感染效率高达87%,呈梭形铺开,生长状态良好,可用于后续实验。H_(2)O_(2)可引起颗粒细胞自噬体和自噬溶酶体数量减少,细胞皱缩,死亡增加,与未干预组比较,差异有统计学意义(P<0.05)。JNK抑制剂组和中药组自噬体和自噬溶酶体均显著增多,与H_(2)O_(2)组比较,差异具有统计学意义(P<0.05)。结论 mRFP-GFP-LC3对体外大鼠卵巢颗粒细胞的MOI为100。氧化应激状态导致卵巢颗粒细胞自噬流水平降低,细胞死亡增多,ROS-JNK介导这一进程,活血消异方可启动保护性自噬,减少颗粒细胞氧化应激损伤。

Objective To investigate the effect of oxidative stress on the autophagy flow of ovarian granulosa cells and the intervention of Huoxue Xiaoyi Prescription.Methods The primary ovarian granulosa cells of rats were cultured in vitro, and the ovarian granulosa cell model of endometriosis was established by hydrogen peroxide-induced oxidative stress.After being cultured for 48 h, the ovarian granulosa cells were assigned into blank group, model(hydrogen peroxide-induced oxidative stress)group, JNK inhibitor group, and Huoxue Xiaoyi Prescription group.The media of the above groups were then supplemented with complete medium, 10% normal rat serum, 10% normal rat serum containing 800 nm SP600125,and 10% rat serum containing Huoxue Xiaoyi Prescription, respectively.The autophagy was detected via mRFP-GFP-LC3 adenovirus transfection, and the autophagy flow changes were observed under an inverted laser scanning confocal microscope.Results When the multiplicity of infection(MOI)of mRFP-GFP-LC3 adenovirus was 100,87% of granulosa cells were infected, showing spindle shape and good growth state, which could be used for subsequent experiments.Compared with the blank group, hydrogen peroxide reduced the autophagosomes and autophagolysosomes in granulosa cells, led to cell shrinkage, and increased the cell death(P<0.05).The number of autophagosomes and autophagolysosomes in JNK inhibitor group and Huoxue Xiaoyi Prescription group was higher than that in the model group(P<0.05).Conclusion The appropriate MOI of mRFP-GFP-LC3 adenovirus for infection to rat ovarian granulosa cells in vitro is 100.Oxidative stress leads to decreased autophagy flow and increased death of ovarian granulosa cells, which is mediated by ROS-JNK.Huoxue Xiaoyi Prescription can activate protective autophagy and alleviate oxidative stress of granulosa cells.