黄芩素对UVB诱导HaCaT细胞光老化及相关p38信号通路的影响

Effect of Baicalein on UVB-induced HaCaT Cell Light Aging and p38 Signaling Pathway

目的:研究黄芩素对中波紫外线(UVB)诱导人皮肤角质形成细胞(HaCaT)光老化及相关p38丝裂原活化蛋白激酶(p38MAPK)信号通路的影响。方法:选择1×10^(-11),1×10^(-10),1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6),1×10^(-5),1×10^(-4),1×10^(-3)mol·L^(-1)9种浓度黄芩素作用于HaCaT细胞,噻唑蓝(MTT)法筛选出最佳有效浓度。经预实验筛选出辐射强度为0.5 mW·cm^(-2)的UVB,辐射时间为5 min建立光老化模型,筛选出最佳有效浓度作用于光老化模型,另设空白组MTT法检测各组细胞活性。超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px),过氧化氢酶(CAT)及丙二醛(MDA)试剂盒检测SOD,GSH-Px,CAT活性及MDA含量。实时定量荧光定量聚合酶连式反应(RT-PCR)及蛋白免疫印迹法(Western blot)分别检测各组细胞中mRNA及蛋白表达。结果:筛选出1×10^(-7),1×10^(-6),1×10^(-5)mol·L^(-1)黄芩素为最佳有效浓度;与空白组比较,UVB组对HaCaT细胞无明显的增殖作用。与UVB组比较,1×10^(-7),1×10^(-6),1×10^(-5)mol·L^(-1)黄芩素组对光老化模型无明显增殖作用。与空白组比较,UVB组SOD,GSH-Px及CAT活性明显的降低,MDA含量显著升高(P<0.01);与UVB组比较,1×10^(-7),1×10^(-6),1×10^(-5)mol·L^(-1)黄芩素组SOD,GSH,CAT活性明显升高,MDA含量明显降低(P<0.05,P<0.01)。与空白组比较,UVB组肿瘤坏死因子-α(TNF-α)mRNA及TNF-α,磷酸化p38(p-p38)蛋白表达显著升高(P<0.01);与UVB组比较,1×10^(-7),1×10^(-6),1×10^(-5)mol·L^(-1)黄芩素组TNF-αmRNA及TNF-α,p-p38蛋白表达明显降低(P<0.05,P<0.01)。结论:黄芩素对UVB诱导HaCaT细胞光老化保护作用主要是通过提高氧化酶活性以及阻断p38MAPK信号通路,抑制炎症因子产生。

Objective：To study the effect of baicalein on ultraviolet B（UVB） induced-human skin keratinocyte（HaCaT） cell light aging and p38 mitogen activated protein kinase（p38MAPK） signaling pathways.Method：The 1×10（-11）,1×10（-10）,1×10（-9）,1×10（-8）,1×10（-7）,1×10（-6）,1×10（-5）,1×10（-4）,1×10（-3）mol·L（-1） baicalein were used on HaCaT cells,and the best effective concentration was determined by methyl thiazolyl tetrazolium（MTT） method.In the preliminary experiments,UVB with radiation intensity of 0.5 mW·cm（-2） was selected,and with radiation time for 5 min to establish light aging models.Then the best effective concentration was used on the light aging models.MTT method was used to detect the cells activity in each group;superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,catalase（CAT） and malondialdehyde（MDA） kits were used to detect SOD,GSH-Px,CAT activity and MDA content respectively.Real-time quantitative PCR and Western blot were used respectively to detect mRNA and protein expression levels in each group.Result：1×10（-7）,1×10（-6）,1×10（-5）mol·L（-1）baicalein were selected as the best effective concentrations.As compared with the blank group,UVB had no significant proliferation effect on HaCaT cells.As compared with UVB group,1×10（-7）,1×10（-6）,and 1×10（-5）mol·L（-1）baicalein had no obvious effect on the proliferation of light aging models.As compared with the blank group,SOD,GSH-Px and CAT activities were significantly decreased while MDA contend was increased in UVB group（P〈0.01）.As compared with UVB model group,1×10（-7）,1×10（-6）,1×10（-5）mol·L（-1）baicalein could increase SOD,GSH-Px and CAT activities,and decrease MDA contend（P〈0.05,P〈0.01）.As compared with the blank group,the expression levels of tumor necrosis factor（TNF）-α mRNA as well as TNF-α and p38 protein were significantly increased in UVB group（P〈0.01）.As compared with UVB model group,1×10（-7）,1×10（-6）,1×10（-5）mol·L（-1）baicalein could decrease the expression levels of TNF-α mRNA as well as TNF-α and p38 protein（P〈0.05,P〈0.01）.Conclusion：Baicalein has the protective effect on UVBinduced cell light aging,and its mechanism is mainly associated with tenhancing GSH-Px,SOD and CAT activities,blocking p38 MAPK signaling pathway,and inhibiting inflammatory cytokines.