菟丝子黄酮对H2O2诱导的COV434人卵巢颗粒细胞氧化应激的影响  

The protective effect of flavonoids from semen cuscutae on the oxidative damage of COV434 human ovarian granulosa cells

作　　者：班兆乔 李慕军[1] BAN Zhao-qiao;LI Mu-jun(Reproductive Medicine Center,the First Affiliated Hospital of Guangxi Medical University,Nanning 537300,China)

机构地区：[1]广西医科大学第一附属医院生殖医学中心,南宁537300

出　　处：《妇产与遗传（电子版）》2023年第1期18-24,共7页Obstetrics-Gynecology and Genetics (Electronic Edition)

基　　金：2020年广西科技计划项目重点研发计划,桂科AB20238002。

摘　　要：目的研究菟丝子黄酮(flavonoid from semen cuscutae,FSC)对过氧化氢(H_(2)O_(2))诱导的人卵巢颗粒细胞氧化应激的影响以及可能相关的作用机制。方法使用不同浓度H_(2)O_(2)处理人卵巢颗粒细胞(COV434)2 h,用CCK-8检测法评估细胞增殖活力,以确定构建细胞外氧化应激模型的H_(2)O_(2)刺激浓度;检测不同浓度的FSC对细胞的毒性作用;使用25μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL的FSC处理氧化应激模型组细胞,CCK-8法检测FSC对细胞增殖活力的影响。根据处理条件将细胞分为四组:Control组,FSC组,H_(2)O_(2)组,FSC+H_(2)O_(2)组,使用CCK-8检测细胞增殖率,活性氧(ROS)试剂盒测定细胞内ROS含量,采用Western blot测定AMPK、P-AMPK、Bax和P53蛋白的表达量。结果0.5 mmol/L的H_(2)O_(2)处理2 h为构建COV434细胞氧化应激模型最佳条件。同时,100μg/mL FSC处理24h为最佳预处理条件。H_(2)O_(2)处理后细胞增殖活力下降,ROS水平上升,FSC处理可以降低ROS水平。Western Blot结果表明,H_(2)O_(2)处理后P-AMPK蛋白表达下调,P53和Bax表达升高,而FSC可以逆转这一趋势。结论H_(2)O_(2)可能通过抑制AMPK通路进而影响下游P53、Bax的表达,FSC可逆转H_(2)O_(2)所致的COV434细胞氧化损伤这一作用。Objective To investigate the protective effects and mechanisms of Flavonoids from Semen Cuscutae(FSC)on oxidative injury of human ovarian granulosa cells(COV434)induced by H_(2)O_(2).Methods To establish an extracellular oxidative stress model,human ovarian granulosa cells(COV434)were treated with H_(2)O_(2) to test cell morphology and growth at different concentrations,and cell proliferation viability was assessed using the CCK-8 assay to determine the appropriate H_(2)O_(2) stimulation concentration.Next,to test the toxic effects of the FSC on cells,COV434 cells were pretreated with H_(2)O_(2) and then treated with 25μg/mL,50μg/mL,100μg/mL,200μg/mL,and 400μg/mL of FSC for 24 h.The optimal concentration of FSC for cell protection against oxidative damage was screened.Then the cells were divided into four groups:Control group,FSC group,H_(2)O_(2) group and FSC+H_(2)O_(2) group,and the corresponding cell proliferation rate was measured using a cell counting kit(CCK-8),intracellular ROS content was measured using a reactive oxygen species(ROS)kit,and AMPK,P-AMPK,Bax and P53 proteins were measured using Western blot.Results The 0.5 mmol/L H_(2)O_(2) for 2 h was the best pretreatment condition for establish the H_(2)O_(2)-induced oxidative stress model in COV434 cells.Meanwhile,100μg/mL FSC for 24 h was the best pretreatment condition.cell proliferation viability decreased and ROS level increased after H_(2)O_(2) treatment,and FSC treatment could reduce ROS level.Western blot results showed that P-AMPK protein expression was down-regulated and P53 and Bax expression increased after H_(2)O_(2) treatment,and post-treatment with FSC could reverse this trend.Conclusions H_(2)O_(2) may affect the expression of downstream P53 and Bax by inhibiting AMPK pathways,and FSC may reverse this effect of H_(2)O_(2)-induced oxidative damage in COV434 cells.

关 键 词：卵巢功能不全 菟丝子黄酮 氧化应激 

分 类 号：R711.75[医药卫生—妇产科学]