过表达Grx1抑制HEK293T细胞中H2O2诱导的p38MAPK信号通路

Grx1 Overexpression Inhibited Activation of P38 MAPK Signal Transduction Pathway Induced by H_2O_2 in HEK293T Cells

谷氧还蛋白1(glutaredoxin1,Grx1)是细胞内一种重要的巯基-二硫键氧化还原酶,在细胞内氧化还原状态的调控及抵抗氧化应激损伤过程中发挥重要作用.为进一步探讨Grx1的抗氧化机制,本实验将重组质粒pcDNA3.1(+)-hGrx1瞬时转染HEK293T细胞,经RT-PCR和Western印迹验证,细胞转染后实现了Grx1的过表达;以不同浓度H2O2为损伤因素,建立细胞氧化应激模型,检测过表达Grx1后细胞存活率,丙二醛(MDA)含量,超氧化物歧化酶(SOD)活力和乳酸脱氢酶(LDH)漏出率的变化,观察过表达Grx1后细胞的抗氧化能力;用终浓度100μmol/L H2O2作用于细胞,利用Western印迹检测120 min内HEK293T细胞中p38MAPK磷酸化水平.实验结果表明,HEK293T细胞过表达Grx1后,缓解了细胞的氧化应激损伤;转染空载体组细胞p38MAPK磷酸化水平在H2O2刺激后5min开始升高,15min达到最高值,并可维持至120min左右;而过表达Grx1组细胞p38MAPK磷酸化水平在H2O2刺激后各时间段没有明显改变,提示Grx1通过抑制H2O2诱导的p38MAPK信号通路激活发挥其抗氧化作用.

Glutaredoxin1（Grx1） is an important thiol-disulfide oxidoreductase in the cells,and it plays a crucial role in the maintenance of cellular redox homeostasis and the defense against oxidative stress.In order to study the anti-oxidative mechanism of Grx1,a recombinant expression vector pcDNA3.1（＋）-hGrx1 was transfected into HEK293T cells by transient transfection.The level of transient expressed recombinant human Grx1 was assayed by RT-PCR and Western blot analysis.The oxidation damage cell model was established by treating the HEK293T cells with H_2O_2.The viability and antioxidant effect of the cells were evaluated by MTT assay,LDH release assay,MDA contents measurement and the SOD activity test.Western blot analysis was also used to estimate p38MAPK activation following 100 μmol/L H_2O_2 exposure in HEK293T cells during 120 min.The results showed that Grx1 overexpression was able to relieve the H_2O_2-induced cellular oxidative stress in HEK293T cells.The level p38MAPK phosphorylation was increased at 5 min,reached the maximum at 15 min and lasted until 120 min in pcDNA3.1（＋） transfected control group,whereas the p38MAPK phosphorylation level remained stable in pcDNA3.1（＋）-hGrx1 pre-transfected group.These results suggested that Grx1 exerted an anti-oxidation activity by inhibiting the p38MAPK pathway.