抑制Keap1基因对染砷HaCaT细胞蛋白底物的影响

The effect of inhibition of Keap1 gene on the metabolism enzyme expression in HaCaT cells pre-treated with arsenic

目的探讨抑制Keap1基因对染砷人永生化皮肤角质(HaCaT)细胞砷蛋白底物的影响。方法将人源性正常HaCaT细胞作为实验的空白对照组,Keap1抑制HaCaT细胞作为染毒组和阴性对照组,培养8、24、48、72h,对染毒组给予无机砷混合物进行染毒,分为高、中、低浓度组,各浓度组的染毒浓度为LC50的1/10(29μmol/L)、1/50(5.8μmol/L)和1/100(2.9μmol/L);采用酶联免疫吸附试验(ELISA)检测砷处理细胞内同型半胱氨酸(HCY)、S-腺苷甲硫氨酸(SAM)、三价砷甲基转移酶(As3MT)N-6-腺嘌呤DNA甲基转移酶1(N6AMT1的蛋白含量。结果染砷8h,与阴性对照组比较,空白对照组、高浓度组SAM蛋白表达下调,低、中浓度组表达上调;低、中浓度组HCY蛋白表达上调,AS3MT蛋白在空白对照组、低浓度组表达上调,N6AMT1蛋白在空白对照组下调,低、中浓度组表达上调(P<0.012 5)。染砷24h,与阴性对照组比较,As3MT蛋白在低、中、高3个浓度组表达上调,SAM蛋白在空白对照组表达下调,低浓度组表达上调,高浓度组表达下调,HCY在空白对照组、低浓度组表达上调(P<0.012 5)。染砷48h,与阴性对照组比较,SAM、HCY的蛋白在低、中浓度组表达上调;N6AMT1的蛋白在中浓度组表达上调,高浓度组表达下调(P<0.012 5)。染砷72h,与阴性对照组比较,SAM蛋白在空白组和高浓度组表达下调,低浓度组表达上调,AS3MT的蛋白在空白对照组、低和中浓度组表达上调;N6AMT1的蛋白在空白对照组、高浓度组表达下调,在高浓度组SAM、As3MT、N6AMT1蛋白表达均下调(P<0.012 5)。结论 SAM和HCY蛋白表达在砷甲基代谢中表达有一致性;砷甲基化代谢中N6AMT1与AS3MT共同作用,其中N6AMT1起辅助作用;在高浓度、长时间的砷暴露中造成蛋白底物的耗竭,砷代谢发生障碍。

Objective To study the inhibition of Keap1 gene on the expression of metabolism enzyme in HaCaT cells pre-treated with arsenic.Methods Human resource derived normal HaCaT cells were used as blank control group,Keap1 inhibition in HaCaT cells were used as treatment group.The treatment group was treated with inorganic arsenic compounds for 8h,24 h,48hand 72 h.In terms of dose given,the treatment group was further subdivided into three group that termed high,medium and low concentration group,respectively.The concentration of each group was 1/10（29μmol/L）,1/50（5.8μmol/L）and 1/100（2.9μmol/L）of LC50.HCY,SAM,As3 MT,6AMT1protein content were detected with enzymelinked immunosorbent assay（ELISA）.Results At the time of treatment for 8h,in comparison with negative control group,the content of SAM was shown to be decreased in high concentration of arsenic group whereas to be increased in low and medium concentration groups;the level of HCY was found to be upregulated in low and medium concentration of arsenic groups;AS3MT was observed to be up-regulated in blank control group and low concentration of arsenic group;N6AMT1was shown to be down-regulated in blank control group whereas shown to be pronouncedly up-regulated in low and medium concentration of arsenic groups（P 0.012 5）.At the time of treatment for 24 h,compared with negative control group,As3 MT was found to be increased in the low,medium and high concentration of arsenic groups;SAM was shown to be decreased in blank control group and high concentration group whereas displayed to be increased in low concentration group;HCY was presented to be remarkably increased in blank control group and low concentration group（P 〈0.012 5）.At the time of treatment for 48 h,in comparison with negative control group,both SAM and HCY were shown to be up-regulated in low and medium concentration group;N6AMT1 was shown to be up-regulated in medium concentration group while to be markedly down-regulated in high concentration group（P 〈0.012 5）.At the time of treatment for 72 h,compared with negative control group,SAM was presented to be down-regulated in blank control and high concentration groups but shown to be up-regulated in low concentration group;As3MT was found to be increased in blank control,low and medium concentration groups;N6AMT1 was displayed to down-regulated in both blank control and high concentration groups whereas;in high concentration group,both SAM,As3 MT and N6AMT1 were all shown to be significantly down-regulated（P 〈0.012 5）.Conclusion Expression of SAM and HCY was shown to be consistent in the metabolism of arsenic;N6AMT1and As3 MT could collaborate in the metabolism of arsenic,during which N6AMT1 could comparatively play an auxiliary role;in the presence of high concentration of arsenic for long time of exposure,which may lead to the exhaustion of protein substrate mentioned above,thereby contributing to the dysbolism of arsenic.