摘 要: 目的 探讨缝隙连接蛋白43(connexin 43,Cx43)在长波紫外线(ultraviolet A,UVA)诱发人皮肤光老化中的作用。方法(1)体外培养原代人皮肤成纤维细胞(human dermal fibroblasts,HDFs),分为对照组、UVA照射组,采用CCK-8实验筛选出安全条件后,使用5 J/cm2 UVA照射实验组细胞1 h;在照射后0 h、6 h、12 h、24 h分别收集细胞及培养上清液,采用ELISA法检测细胞上清中基质金属蛋白酶-1(matrix metalloproteinases-1,MMP-1)和MMP-9的水平;采用qRT-PCR法和Western blot法检测细胞内Cx43 mRNA和蛋白的表达水平。(2)瞬时转染Cx43-siRNA,采用ELISA法检测细胞上清中MMP-1和MMP-9的水平。(3)将HDFs分为对照组、UVA组、UVA+TNF受体1中和抗体组,经UVA照射后,采用qRT-PCR和ELISA法检测细胞中Cx43 mRNA表达和上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和MMP-1水平。结果(1)与对照组相比,UVA组MMP-1和MMP-9水平呈时间依赖性上升(P<0.001),同时Cx43 mRNA和蛋白表达水平均明显下降(P<0.01);(2)Cx43-siRNA显著下调Cx43表达后,MMP-1和MMP-9水平显著上升(P<0.001);(3)与对照组相比,UVA组TNF-α和MMP-1水平显著上升(P<0.01),同时UVA+TNF受体1中和抗体组Cx43和MMP-1水平无明显升高。结论 Cx43可能参与介导了UVA通过诱导TNF-α信号通路引发的皮肤光老化过程,这种介导作用可能是通过Cx43对皮肤中MMP-1的调控得以实现。 |
关键词: 缝隙连接蛋白43(Cx43) 长波紫外线(UVA) 肿瘤坏死因子-α(TNF-α) 光老化 |
中图分类号: R758
文献标识码: A
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基金项目: 国家自然科学基金(31400706);辽宁省科技厅科学技术计划项目(20141098);辽宁省教育厅科学技术研究项目(SYYX202001) |
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Role of connexin 43 in photoaging of human skin induced by ultraviolet A |
SHI Zheng-han,LOU Wei-zeng,REN Qun-xiang,HUANG Tao
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Shenyang Medical College,Shenyang 110034,China
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Abstract: Objective To investigate the role of connexin 43(Cx43)in skin photoaging induced by ultraviolet A(UVA). Methods (1)Primary human dermal fibroblasts(HDFs)cultured in vitro,were divided into control group and experiment(UVA-irradiated)group,the HDFs of experiment group were irradiated with UVA(5 J/cm2)for 1 h,after screening out the safe conditions by CCK-8 experiment,then the cells and culture supernatants were collected at 0 h,6 h,12 h and 24 h,respectively after irradiated,detect the levels of MMP-1 and MMP-9 in supernatants by ELISA,measure the expression of Cx43 mRNA and the content of protein in HDFs by qRT-PCR and Western blot.(2)After transient transfection with Cx43-siRNA,the levels of MMP-1 and MMP-9 in supernatants were detected by ELISA. After UVA irradiation,the expressions of Cx43 mRNA in the HDFs from control,UVA and UVA+TNF receptor 1 neutralizing antibody groups,and the levels of TNF-α,and MMP-1 in cell supernatant were detected by qRT-PCR and ELISA,respectively. Results The results showed that(1)Compared with control group,the levels of MMP-1 and MMP-9 in UVA group increased in a time -dependent manner(P<0.01),while the expression of mRNA and protein of Cx43 were decreased significantly(P<0.01).(2)The levels of MMP-1 and MMP-9 were significantly increased(P<0.01)after Cx43 expression was down-regulated by Cx43-siRNA.(3)Compared with control group,the levels of TNF-α and MMP-1 in UVA group were significantly increased(P<0.01),meanwhile,the levels of Cx43 and MMP-1 in UVA+TNF receptor 1 neutralizing antibody group were not increased. Conclusion The results suggested that Cx43 may be involved in the photoaging process of skin induced by UVA via the induction of TNF-α signaling pathway,and this mediating effect may be achieved through the regulation of MMP-1 in skin by Cx43. |
Keywords: connexin 43(Cx43) ultraviolet A(UVA) tumor necrosis factor-α(TNF-α) photoaging |