黄芩素对中波紫外线诱导角质形成细胞凋亡及相关细胞因子表达的影响

目的:研究黄芩素对中波紫外线(UVB)诱导角质形成细胞凋亡及相关细胞因子表达的影响。方法:体外培养人皮肤角质形成细胞(HaCaT),取对数生长期的细胞,随机分为空白组、模型组和黄芩素组,并用30 mJ·cm~(-2)的UVB照射模型组和黄芩素组,建立光老化模型。噻唑蓝(MTT)比色法筛选黄芩素的有效安全浓度,并测定黄芩素对光老化HaCaT细胞增殖率的影响;活性氧自由基(ROS)试剂盒测定各组细胞中ROS含量;Annexin V-FITC细胞凋亡检测试剂盒测定各组细胞凋亡率;蛋白免疫印迹法(Western blot)测定各组细胞中B淋巴细胞瘤-2(Bcl-2),半胱氨酸天冬氨酸-3(Caspase-3)及Caspase-9蛋白表达水平。结果:筛选出1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素为最佳有效安全浓度。与空白组比较,模型组ROS含量和细胞凋亡率显著升高(P0.01),Bcl-2蛋白表达水平显著降低(P0.01),Caspase-3及Caspase-9蛋白表达水平显著升高(P0.01)。与模型组比较,1×10~(-7),1×10~(-6),1×10~(-5)mol·L~(-1)黄芩素组ROS含量和细胞凋亡率显著降低(P0.05,P0.01),Bcl-2蛋白表达水平显著升高(P0.05,P0.01),Caspase-3及Caspase-9蛋白表达水平显著下降(P0.05,P0.01)。结论:黄芩素可以通过降低细胞ROS含量和凋亡率,调控Bcl-2家族中抗凋亡因子Bcl-2蛋白的表达,降低凋亡相关细胞因子Caspase-3和Caspase-9的表达水平,抑制UVB诱导的细胞凋亡。

Effect of Baicalein on Ultraviolet B-induced Apoptosis and Expression of Related Cytokines of Human Keratinocytes

Objective: To study the effects of baicalein on ultraviolet B (UVB)-induced apotosis and the expression of related cytokines of human keratinocytes (HaCaT cells). Method: The HaCaT cells were cultured in vitro, and the logarithm of the cells was taken. Cells were randomly divided into blank control group,model group and baicalein group. The irradiation of UVB was given at irradiation dose of 30 mJ · cm^-2 to establish photoaging model for model group and baicalein group. The method of methyl thiazolyl tetrazolium(MTT) was used to screen the effective and safe concentration of baicalein and screen the effects of baicalein on the proliferation rate of photoaging HaCaT cells; the content of reactive oxygen species(ROS) in each group was determined by reactive oxygen free radical (ROS) kit;the apoptosis of HaCaT cells was determined by the Annexin V-FITC apoptosis detection kit; and Western blot method was used for detecting the effects of baicalein on B-cell lymphoma-2 (Bcl-2),cysteinyl aspartate-3(Caspase-3) and Caspase-9 protein expression. Result: The best safe and effective concentrations of baicalein were 1×10^-7, 1×10^-6, 1×10^-5 mol · L^-1.As compared with the blank group,the content of ROS and the apoptosis rate of the model group were significantly increased(P<0.01);the expression of Bcl-2 protein was significantly decreased (P<0.01); and the expression levels of Caspase-3 and Caspase-9 protein were significantly increased (P<0.01).As compared with the model group, the content of ROS and the apoptosis rate in 1×10^-7,1×10^-6, 1×10^-5 mol · L^-1 baicalein groups were significantly decreased (P<0.05,P<0.01); the expression of Bcl-2 protein was significantly increased (P<0.05, P<0.01); and the expression levels of Caspase-3 and Caspase-9 protein were significantly decreased (P<0.05,P<0.01). Conclusion: Baicalin can reduce the content of ROS and the apoptosis rate of HaCaT cells, regulate the expression of Bcl-2 protein in Bcl-2 family,decrease the expression of related cytokines Caspase-3 and Caspase-9, and inhibit the apoptosis induced by UVB.