紫外线对人皮肤成纤维细胞中Hrd1及Nrf2表达的影响

目的探讨紫外线(UV)照射对人皮肤成纤维细胞中羟甲基戊二酰辅酶A还原酶降解蛋白(Hrd)1及核因子E2相关因子(Nrf)2表达的影响及可能机制。方法共收集12份人皮肤组织标本,曝光及非曝光部位标本各6份,免疫组化检测2组Hrd1及Nrf2的表达。将体外培养人皮肤成纤维细胞分为对照组、UVA组、UVB组,照光后用Western blot法检测各组成纤维细胞中Hrd1及Nrf2的表达。应用Hrd1-siRNA转染体外培养人皮肤成纤维细胞,下调细胞中Hrd1的表达后,Western blot检测成纤维细胞Nrf2的表达。应用免疫荧光分析检测细胞中Hrd1与Nrf2在细胞中的共定位情况,应用免疫共沉淀检测体外培养人成纤维细胞中Hrd1与Nrf2是否存在内源性结合。结果临床标本实验中,曝光部位皮肤组织中Hrd1表达(0.4756±0.0785)明显高于避光部位(0.1270±0.0253,t=7.317,P<0.05),曝光部位皮肤组织中Nrf2表达(0.1190±0.0217)明显低于避光部位(0.2629±0.0263,t=7.306,P<0.05)。体外培养成纤维细胞实验中,经紫外线照射后,UVA组与对照组相比,Hrd1蛋白表达水平明显增高(t=7.227,P<0.05),Nrf2蛋白表达水平明显下降(t=6.456,P<0.05);同样,UVB组与对照组相比,Hrd1蛋白表达水平明显增高(t=2.980,P<0.05),Nrf2蛋白表达水平明显下降(t=13.52,P<0.05)。应用Hrd1-SiRNA下调体外培养人纤维细胞中Hrd1的表达后,无论是经UVA照射还是UVB照射,被下调Hrd1组细胞内Nrf2的表达较未下调组明显升高。免疫荧光分析结果显示Hrd1与Nrf2在体外培养人皮肤成纤维细胞中的均有表达,并且在细胞中存在Hrd1与Nrf2共定位。免疫共沉淀验证了Hrd1与Nrf2在体外培养人皮肤成纤维细胞中存在内源性结合。结论Hrd1与Nrf2在人皮肤成纤维细胞中存在结合,紫外线照射可通过增加细胞中Hrd1的表达从而抑制Nrf2的表达。

Effects of Ultraviolet on the Expression of Hrd1 and Nrf2 in Human Skin Fibroblasts

Objective In order to investigate the effects of ultraviolet irradiation on the expression of 3-hydroxy-3-methylglutaryl reductase degradation(Hrd)1 and nuclear factor-E2-related factor(Nrf)2 in human skin fibroblasts and its possible mechanism.Methods A total of twelve human skin tissue samples were collected,including six from sun-exposed and six from sun-protected sites.Immunohistochemical analysis was performed to determine the expression of Hrd1 and Nrf2 in the above groups.Human skin fibroblasts cultured in vitro were divided into control group,UVA group and UVB group,and western blotting was performed to determine the expression of Hrd1 and Nrf2 in the above groups.Hrd1 siRNA was used to downregulate Hrd1 expression in fibroblasts and western blot was performed to determine the expression of Nrf2.The co-localization of Hrd1 and Nrf2 in cells was detected by immunofluorescence analysis,and the endogenous binding of Hrd1 and Nrf2 in cultured human fibroblasts was detected by immunoprecipitation.Results In clinical specimen experiment,the expression of Hrd1(0.4756±0.0785)was significantly higher in the sun-exposed area than in the sun-protected area(0.1270±0.0253,t=7.317,P<0.05)and the expression of Nrf2(0.1190±0.0217)was significantly lower in the sun-exposed area than in the sun-protected area(0.2629±0.0263,t=7.306,P<0.05).In vitro,after ultraviolet irradiation,the expression level of Hrd1 protein in UVA group was significantly higher than that in control group(t=7.227,P<0.05),and the expression level of Nrf2 protein was significantly lower(t=6.456,P<0.05).Similarly,the expression level of Hrd1 protein in UVB group was significantly higher than that in control group(t=2.980,P<0.05),and the expression level of Nrf2 protein was significantly lower(t=13.52,P<0.05).Knockdown of Hrd1 could increase Nrf2 expression in fibroblasts exposed to UV radiation.Immunofluorescence analysis showed that both Hrd1 and Nrf2 were expressed in human skin fibroblasts,and there were Hrd1 and Nrf2 co-localization in cells.Immunocoprecipitation showed that there was endogenous binding between Hrd1 and Nrf2 in cultured human skin fibroblasts.Conclusion The combination of Hrd1 and Nrf2 exists in human skin fibroblasts.Ultraviolet irradiation can inhibit the expression of Nrf2 by increasing the expression of Hrd1 in human skin fibroblasts.