甘草苷对中波紫外线诱导人皮肤角质形成细胞凋亡的影响

目的:研究甘草苷对中波紫外线(UVB)诱导人皮肤角质形成细胞(Ha Ca T)凋亡的影响。方法:以不同浓度的甘草苷作用于Ha Ca T细胞,噻唑蓝(MTT)比色法筛选甘草苷的有效安全浓度;用30 m J·cm-2的UVB作用于Ha Ca T细胞,建立光老化模型,MTT比色法检测光老化Ha Ca T细胞增殖率;流式细胞术检测各组细胞中活性氧自由基(ROS)含量和总凋亡率;实时荧光定量PCR(Real-time PCR)检测光老化Ha Ca T细胞中半胱氨酸天冬氨酸-3(Caspase-3),半胱氨酸天冬氨酸-9(Caspase-9)mRNA表达水平;蛋白免疫印记法(Western blot)检测光老化Ha Ca T细胞中B淋巴细胞瘤-2(Bcl-2),Caspase-3,Caspase-9蛋白表达量。结果:筛选出甘草苷的最佳有效安全浓度为1×10-7,1×10-6,1×10-5mol·L-1。与空白组比较,UVB组细胞增殖率显著降低(P0.01),细胞中ROS含量和总凋亡率显著升高(P0.01),Caspase-3,Caspase-9 mRNA表达水平显著升高(P0.01);Bcl-2蛋白表达量显著降低(P0.01),Caspase-3,Caspase-9蛋白表达量显著升高(P0.01)。与UVB组比较,1×10-7,1×10-6,1×10-5mol·L-1甘草苷+UVB组细胞增殖率显著升高(P0.01);细胞中ROS含量和总凋亡率显著降低(P0.05,P0.01),Caspase-3,Caspase-9 mRNA表达水平显著降低(P0.05,P0.01);Bcl-2蛋白表达量显著升高(P0.05,P0.01),Caspase-3,Caspase-9蛋白表达量显著降低(P0.05,P0.01)。结论:甘草苷能通过促进抗凋亡因子Bcl-2蛋白的表达,减少促凋亡相关细胞因子Caspase-3,Caspase-9 mRNA和蛋白的表达,从而抑制UVB诱导的Ha Ca T细胞凋亡。

Effect of Liquiritin on UVB-induced Apoptosis of Human Keratinocytes

Objective: To investigate the effects of liquiritin on ultraviolet B (UVB)-induced apoptosis of human keratinocytes (HaCaT cells).Method: Different concentrations of liquiritinwere used to treat HaCaT cells, and the methyl thiazolyltetrazolium(MTT) assay was used to screen the effective and safe concentration of liquiritin;light aging model was set up by the irradiation of ultraviolet radiation B (UVB) at irradiation dose of 30 mJ · cm^-2. Then the activity of cells was determined by MTT method;the content of reactive oxygen species (ROS) and the apoptosis of HaCaT cells were respectively measured by flow cytometry;the expression levels of cysteinyl aspartate-3(Caspase-3) and cysteinyl aspartate-9(Caspase-9) mRNA were determined by Real-time PCR method; and Western blot method was used to detect the effects of liquiritin on B-cell lymphoma 2(Bcl-2), Caspase-3 and Caspase-9 protein expression.Result: The optimal safe and effective concentrations of liquiritin were 1×10^-7, 1×10^-6, and 1×10^-5 mol · L^-1. As compared with the blank group,the cell proliferation rate was significantly decreased in the UVB group (P<0.01);the content of ROS and the total apoptosis rate of cells were significantly increased(P<0.01); the expression levels of Caspase-3 and Caspase-9 mRNA were significantly increased (P<0.01);the expression level 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 UVB group,the cell proliferation rate of the (1×10^-7, 1×10^-6, and 1×10^-5) mol · L^-1liquiritin+UVB groups were significantly increased (P<0.01);the content of ROS and the total apoptosis rate of cells were significantly decreased (P<0.05, P<0.01); the expression levels of Caspase-3 and Caspase-9 mRNA were significantly decreased(P<0.05, P<0.01);the expression level 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: Liquiritin can promote the expression of anti-apoptotic factor Bcl-2 protein, and decrease the expression of the apoptosis-related factors Caspase-3 and Caspase-9 mRNA and protein, thus inhibiting the apoptosis of HaCaT cells induced by UVB.