龙胆苦苷预处理对过氧化氢诱导的人脐静脉内皮细胞氧化应激损伤的影响及机制

Effect of gentiopicroside pretreatment on oxidative stress injury induced by hydrogen peroxide in human umbilical vein endothelial cells and its mechanism

目的 观察龙胆苦苷(GPS)预处理对过氧化氢(H2O2)诱导的人脐静脉内皮细胞(HUVEC)氧化应激损伤的影响,并探讨其作用机制。方法 取对数生长期的HUVEC,随机分为正常对照组、模型组和GPS干预组。模型组加入200μmol/LH2O2培养4h建立氧化应激损伤模型;GPS干预组先加入20μmol/LGPS培养12h,然后加入200μmol/LH2O2培养4h;正常对照组仅加入0.01%DMSO。采用MTT法检测各组细胞存活率,TUNEL法检测各组细胞凋亡率;ELISA法测定各组细胞中丙二醛(MDA)含量及谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性;Western blotting法检测各组细胞中PI3K/AKT信号通路蛋白PI3K、p-AKT以及凋亡相关蛋白Caspase-3、线粒体凋亡通路相关蛋白Bax、抗凋亡蛋白Bcl-2的相对表达量。结果 与正常对照组比较,模型组细胞存活率降低,细胞凋亡率升高(P均<0.01);Caspase-3、Bax蛋白表达及MDA含量升高,Bcl-2、p-AKT、PI3K蛋白表达及GSH-Px、SOD含量降低(P均<0.01)。与模型组比较,GPS干预组细胞存活率升高,细胞凋亡率降低(P均<0.01);Bcl-2、p-AKT、PI3K蛋白表达及GSH-Px、SOD含量升高,Caspase-3、Bax蛋白表达及MDA含量下降(P<0.05或<0.01)。结论 GPS可对抗H2O2造成的HUVEC氧化应激损伤,对内皮细胞具有保护作用;其机制可能为抗氧化及调控PI3K/AKT信号通路,从而抑制细胞凋亡。

Objective To observe the effect of gentiopicroside (GPS) pretreatment on oxidative stress injury of human umbilical vein endothelial cells (HUVEC) induced by hydrogen peroxide (H2O2) and to explore its mechanism. Methods HUVEC were randomly divided into thenormal control group, model group and GPS intervention group. The HUVEC in the model group were incubated with 200 μmol/L H2O2 for 4 h to establish the oxidative stress injury model;the HUVEC in the GPS intervention group were incubated with 20 μmol/L GPS for 12 h, and then followed by 200 μmol/L H2O2 for 4 h;the HUVEC in the normal control group were only incubated with 0.01% DMSO. MTT assay was used to detect cell viability, TUNEL assay was used to detect apoptosis;ELISA was used to determine the content of malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD);Western blotting was used to detect PI3K/AKT signaling pathway protein PI3K, p-AKT, apoptosis-related protein Caspase-3, mitochondrial apoptotic pathway-related protein Bax and anti-apoptotic proteinBcl-2. Results Compared with the normal control group, the cell viability and apoptotic rate decreased(all P <0.01), while the expression of Caspase-3, Bax and MDA increased, and the expression of Bcl-2, p-AKT, PI3K and the content of GSH-Px and SOD decreased in the model group (all P <0.01). Compared with the model group, the cell survival rate of the GPS intervention group increased( P <0.01), the apoptosis rate decreased, Bcl-2, p-AKT, PI3K protein expression and GSH-Px, SOD content increased, and the Caspase-3 Bax protein expression and MDA content decreased (all P <0.05). Conclusion GPS can protect endothelial cells from oxidative stress injury induced by H 2O 2, and its mechanism may be related to antioxidation and regulation of PI3K/AKT signaling pathway, and thus inhibiting the apoptosis.