H2O2抑制小鼠肾小管上皮细胞Klotho的表达

H2O2 decreases Klotho expression in mouse renal tubular epithelial cells

目的 研究过氧化氢（H2O2）引起的氧化应激损伤对小鼠肾小管上皮细胞（TCMK-1）Klotho蛋白表达的影响及可能的作用机制.方法 体外培养TCMK-1细胞,以不同浓度的H2O2刺激细胞,采用流式细胞术检测活性氧族（ROS）生成;CCK-8检测细胞生存率;流式细胞术和Hoechst 33258染色检测细胞凋亡;Western印迹法检测Klotho蛋白、细胞凋亡相关蛋白和抗氧化酶的表达.结果 与正常对照组比较,H2O2刺激后TCMK-1细胞内ROS水平升高（均P＜ 0.05）,抗氧化酶锰超氧化物歧化酶（manganese superoxide dismutase,SOD2）和过氧化氢酶（catalase,CAT）表达水平降低（均P＜0.05）;Klotho蛋白的表达水平降低（均P＜0.05）;TCMK-1细胞生存率显著降低（均P＜0.05）,且呈剂量依赖（0.3～0.9 mmol/L H2O2）;TCMK-1凋亡细胞比例呈剂量依赖性增加（均P＜ 0.05）;Bax/Bcl-2表达比升高,JNK、p38蛋白磷酸化水平均升高（均P＜ 0.05）.结论 H2O2刺激TCMK-1细胞诱导氧化应激损伤,抑制Klotho蛋白的表达,而Klotho表达下降伴随着细胞凋亡的增加和p38、JNK通路的活化.

Objective To evaluate the effect of oxidative injury induced by peroxide oxidase on Klotho expression in mouse renal tubular epithelial cells （TCMK-1） and to explore the possible pathway.Methods TCMK-1 cells were exposed to H2O2 of different concentrations.Reactive oxygen species （ROS） was examined byflow cytometrry.Cell viability was assessed by CCK-8.Cell apoptosis was evaluated by flow cytometry and Hoechst 33258 staining.The expression of Klotho,apoptosis-associated proteins and anti-oxidant enzymes were determined by Western blotting.Results Compared with control group,after H2O2 stimulating TCMK-1 cell,ROS was dramatically elevated （all P 〈 0.05） and the expression of anti-oxidant enzymes,SOD2 and CAT went down （all P 〈 0.05）;the expression of Klotho was inhibited （all P 〈 0.05）;cell viability of TCMK-1 cells was decreased （all P 〈 0.05） in a dose-dependent manner （0.3 to 0.9 mmol/L）;cell apoptosis was significantly increased in TCMK-1 cells following the concentration of H2O2 （all P 〈 0.05）;Bax/Bcl-2 and the phosphororation of JNK and p38 were obviously elevated in TCMK-1 by H2O2 induction （all P 〈 0.05）.Conclusion Oxidative injuries induced by H2O2 significantly suppresses the expression of Klotho in TCMK-1 cells.And cell apoptosis was increased,p38 and JNK pathway was activated.