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目的:探索表儿茶素对H2O2引起的Huh7细胞氧化应激的作用及其机制。方法:对H2O2和表儿茶素干预培养的Huh7细胞,应用MTT法检测细胞存活率,荧光探针DCFH-DA测定细胞内活性氧(ROS)生成量,免疫印迹测定丝氨酸/苏氨酸蛋白激酶(Akt),增殖细胞核抗原(Proliferating Cell Nuclear Antigen,PCNA),磷酸化应激激活的c-Jun蛋白水平。结果:0.8mmol/LH2O2孵育1h可诱导显著Huh7细胞损伤,细胞存活率下降到(40±3.2)%,ROS生成量比未处理细胞多5.4倍。细胞经150μmol/L表儿茶素与H2O2共孵育后,细胞存活率提高到(94.5±9.84)%;表儿茶素能显著抑制H2O2引起的Huh7细胞ROS生成,ROS生成下降60%(P<0.01)。表儿茶素抑制H2O2引起Huh7细胞死亡和ROS生成随剂量增加抑制作用加强。表儿茶素抑制H2O2激发Huh7细胞磷酸化c-Jun表达,提高细胞Akt、PCNA水平。结论:表儿茶素抑制H2O2引起的Huh7细胞氧化应激损伤而导致的细胞死亡,其作用机制是通过减少细胞内活性氧生成,抑制H2O2激活磷酸化c-Jun,提高细胞Akt,PCNA水平。
Objective: To explore the effect of epicatechin on H2O2-induced oxidative stress in Huh7 cells and its mechanism. METHODS: H2O2 and epicatechin were used to intervene the cultured Huh7 cells. The cell viability was detected by MTT assay. The amount of reactive oxygen species (ROS) produced in the cells was determined by fluorescent probe DCFH-DA. Western blot was used to determine serine/threonine protein kinases. (Akt), Proliferating Cell Nuclear Antigen (PCNA), phosphorylated stress-activated c-Jun protein levels. RESULTS: Incubation of 0.8mmol/L H2O2 for 1h could induce significant Huh7 cell injury. The cell survival rate was decreased to (40±3.2)%, and ROS production was 5.4-fold higher than that of untreated cells. After the cells were co-incubated with 150μmol/L epicatechin and H2O2, the cell survival rate was increased to (94.5±9.84)%; Epicatechin could significantly inhibit the generation of ROS in Huh7 cells induced by H2O2, and the production of ROS decreased by 60% (P< 0.01). Epicatechin inhibited H2O2 induced Huh7 cell death and ROS production enhanced with dose inhibition. Epicatechin inhibited the phosphorylation of c-Jun in Huh7 cells induced by H2O2, and increased Akt and PCNA levels. CONCLUSION: Epicatechin inhibited the cell death caused by H2O2-induced oxidative stress in Huh7 cells. The mechanism of action was to reduce the generation of reactive oxygen species in cells, inhibit the activation of phosphorylated c-Jun by H2O2, and increase the levels of Akt and PCNA in cells.