谷精草提取物对H2O2诱导人晶状体上皮细胞氧化损伤的保护作用

The Protective Effect of Eriocaulon Buergerianum Extract(EBE)on Oxidative Damage of Human Lens Epithelial Cells Induced by H2O2

目的:探讨谷精草提取物在人晶状体上皮细胞(SRA01/04)中的抗氧化作用及其抗氧化的分子机制。方法:SRA01/04细胞培养于DMEM/F12培养基中,内含10%FBS及100 U·mL^-1青霉素和100 mg·L^-1链霉素,置于37℃、体积分数5%CO_2饱和湿度的培养箱中培养。通过100μmol·L^-1H2O2诱导制成细胞氧化损伤模型,采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,采用Annexin V/PI法检测凋亡情况,采用ELISA检测谷胱甘肽(glutathione,GSH),采用定量PCR和免疫印迹法检测Keap1、Nrf2 mRNA和蛋白的表达。结果:谷精草提取物处理后SRA01/04细胞生长情况明显改善,比单纯H2O2损伤后存活率高22%(P<0.01);谷精草提取物处理组的SRA01/04细胞凋亡比例较单纯H2O2损伤组下降约10%。同时,在谷精草提取物处理的SRA01/04细胞中可以检测到GSH升高,且呈浓度依赖性。在100 mg·L^-1谷精草提取物处理后,SRA01/04细胞中KEAP1蛋白表达降低、Nrf2蛋白表达升高,其各自的mRNA表达分别降低了2.28倍、升高了3.56倍。结论:谷精草提取物在SRA01/04细胞中具有明显的抗氧化作用,可降低细胞凋亡,诱导细胞内GSH的增多,其机制可能与调节Keap1-Nrf2信号通路有关。

Objective:To investigate the antioxidative effect of the eriocaulon buergerianum extract on human lens epithelial cells(SRA01/04)and its molecular mechanism.Methods:SRA01/04 cells were cultured in DMEM/F12 medium,containing 10%FBS,100 U·mL-1 penicillin and 100 mg·L^-1 streptomycin.The cells were cultured in a 37℃,5%CO2 saturated humidity incu-bator.The oxidative damage model was induced 100μmol·L^-1 H2O2.Cell proliferation was detected by methyl thiazolyl tetrazoli-um(MTT)method.Apoptosis was detected by Annexin V/PI method.Glutathione(GSH)was detected by ELISA.Keap1 and Nrf2 mRNA and protein expression were detected by quantitative PCR and immunoblotting.Results:The growth of SRA01/04 cells was significantly improved after being treated with eriocaulon buergerianum extract,and the survival rate was 22%higher than that after being injured by H2O2 alone(P<0.01);the apoptotic rate of SRA01/04 cells in the eriocaulon buergerianum extract group was about 10%lower than that in the H2O2 group.At the same time,GSH could be detected in SRA01/04 cells treated with erio-caulon buergerianum extract in a concentration dependent manner.After treated with 100 mg·L^-1 eriocaulon buergerianum ex-tract,the expression of KEAP1 protein decreased and Nrf2 protein increased in SRA01/04 cells,and their mRNA expression decreased by 2.28 times and increased by 3.56 times respectively.Conclusion:Eriocaulon buergerianum extract has obvious antioxi-dant effect in SRA01/04 cells,which can reduce apoptosis and induce the increase of GSH in cells.The mechanism may be to reg-ulate Keap1-Nrf2 signal pathway.