羊毛硫氨酸合成酶C样蛋白2在细胞抗氧化中的作用及机制研究

Effect and Mechanism of Lanthionine Synthetase C-Like Protein 2(LanCL2)on Cellular Antioxidation

目的探讨羊毛硫氨酸合成酶C样蛋白2(LanCL2)在细胞抗氧化过程中的作用及相关机制。方法采用不同浓度(0、30、75、100、150、200μmol/L)的过氧化氢(H_(2)O_(2))诱导处理人胚胎肾细胞(HEK293T)24 h以诱导氧化应激,通过蛋白质印迹技术检测LanCL2的表达;分别转染GFP、GFP-LanCL2过表达质粒至HEK293T细胞,在200μmol/L H_(2)O_(2)刺激条件下,通过PI/Hoechst 33342染色检测细胞死亡;向HEK293T细胞中转染Myc、Myc-LanCL2、Myc-GSTP1、Myc-LanCL2-His和GSTP1-His质粒,分别作为Myc组、Myc-LanCL2组、Myc-GSTP1组、Myc-LanCL2-His组(诱导纯化后)和GSTP1-His组(诱导纯化后),将未经任何处理的细胞作为Ctrl组,采用紫外分光光度计检测各组谷胱甘肽硫转移酶(GST)活性及Myc组与Myc-LanCL2组的谷胱甘肽过氧化物酶(GPx)活性。结果与无H_(2)O_(2)刺激相比,在100、200μmol/L H_(2)O_(2)刺激后HEK293T细胞中LanCL2相对表达量升高(P<0.05);与GFP组相比,GFP-LanCL2组在200μmol/L H_(2)O_(2)刺激后细胞死亡率下降(P<0.01);Myc-LanCL2组GST活性与Myc组及Myc-GSTP1组相比,差异无统计学意义(P>0.05);与Ctrl组相比,GSTP1-His组具有较高的GST活性(P<0.001);而Myc-LanCL2-His组GST活性与Ctrl组相比,差异无统计学意义(P>0.05);Myc-LanCL2组GPx活性与Myc组相比,差异无统计学意义(P>0.05)。结论LanCL2能被氧化应激诱导,并减轻氧化应激介导的细胞死亡,但LanCL2不具有GSH依赖的抗氧化酶(GST和GPx)活性。

Objective To investigate the role and mechanisms of lanthionine synthetase C-like protein 2(LanCL2)in the cellular antioxidant defense system.Methods Human embryonic kidney 293T(HEK293T)cells were treated with different concentrations of hydrogen peroxide(H_(2)O_(2))(0,30,75,100,150,and 200μmol/L)for 24 hours to induce oxidative stress.The expression of LanCL2 was detected by Western blotting.The overexpression plasmids of green fluorescent protein(GFP)and GFP-LanCL2 were transfected into HEK293T cells respectively.Cell death was detected by PI/Hoechst 33342 staining under the stimulation of 200μmol/L H_(2)O_(2).HEK293T cells were transfected with overexpression plasmids of Myc,Myc-LanCL2,Myc-GSTP1,Myc-LanCL2-His and GSTP1-His,respectively,to be Myc group,Myc-LanCL2 group,Myc-GSTP1 group,Myc-LanCL2-His group(after induction and purification)and GSTP1-His group(after induction and purification).HEK293T cells without any treatment were included as the control(Ctrl)group.Ultraviolet(UV)spectrophotometry was performed to measure the glutathione S-transferase(GST)activity in each group and the glutathione peroxidase(GPx)activity in Myc and Myc-LanCL2 groups.Results The relative expression level of LanCL2 was higher in HEK293T cells induced by H_(2)O_(2)(100 and 200μmol/L)than that in HEK293T cells not induced by H_(2)O_(2)(P<0.05).The cell mortality after H_(2)O_(2)(200μmol/L)induction was lower in GFP-LanCL2 group than that in GFP group(P<0.01).There was no significant difference in GST activity between Myc-LanCL2 group and Myc and Myc-GSTP1 groups(P>0.05).The GST activity was higher in GSTP1-His group than that in Ctrl group(P<0.001).There was no significant difference in GST activity between Myc-LanCL2-His group and Ctrl group(P>0.05).There was no significant difference in GPx activity between Myc-LanCL2 group and Myc group(P>0.05).Conclusion LanCL2 can be induced by oxidative stress and alleviate oxidative stress-mediated cell death,but it does not have glutathione(GSH)dependent antioxidant enzyme(GST or GPx)activities.