SIRT1抑制高良姜素诱导的HepG2细胞凋亡的研究

The study of the inhibition effects of SIRT1 on the apoptosis induced by Galangin in HepG2 cells

目的高良姜素是一种具有抗肿瘤活性的天然黄酮类化合物,SIRT1是参与许多生理过程的Sirtuin家族蛋白重要成员之一。文中旨在探讨SIRT1在高良姜素诱导Hep G2细胞凋亡过程中的作用。方法实验设置DMSO溶剂对照组、EX-527组(50μmol/L EX-527处理细胞2h)、高良姜素组(高良姜素处理细胞24h)和EX-527+高良姜素组(先用50μmol/L EX-527处理细胞2 h再用高良姜素处理细胞24 h)。用Hochest33342染色法和流式细胞术、Western blot分析细胞凋亡的变化。RNA干扰和转染外源基因调控SIRT1的表达后,130μmol/L高良姜素处理细胞24 h,并设置未感染腺病毒组、载体对照组(感染空载体腺病毒)、SIRT1敲降组(感染敲降SIRT1腺病毒)、未转染质粒组、空载体对照组(转染空载体质粒组)、SIRT1上调组(转染过表达SIRT1质粒),用流式细胞术和Western blot检测细胞凋亡。结果与DMSO溶剂对照组细胞凋亡率、PARP1剪切带与GAPDH灰度比值[(2.49±0.22)%,0.06±0.00]比较,高良姜素组[(11.62±0.55)%,0.89±0.01]、EX-527+高良姜素组[(25.75±0.61)%,1.15±0.06]均升高(P<0.01);其中EX-527+高良姜素组较高良姜素组亦明显升高(P<0.01)。Western blot结果表明,SIRT1敲降组中SIRT1与GAPDH灰度比值(0.06±0.01)较载体对照组(1.11±0.05)和未感染腺病毒组(1.10±0.04)明显减小(P<0.01)。与载体对照组和未感染腺病毒组比较,SIRT1敲降组细胞凋亡率、PARP1剪切带与GAPDH灰度比值均增加(P<0.01)。SIRT1上调组中SIRT1与GAPDH灰度比值(1.63±0.04)较载体对照组(0.89±0.02)和未转染质粒组(0.87±0.03)相比明显增大(P<0.01)。与未转染质粒组和空载体对照组比较,SIRT1上调组细胞凋亡率和SIRT1剪切带与GAPDH灰度比值降低(P<0.01)。结论 SIRT1抑制高良姜素诱导的Hep G2细胞凋亡。

Objective Galangin is a natural flavonoid with antineoplastic activity. SIRT1 is an important member of Sirtuin family which parcitipate in many physiological process. The aim of this study was to investigate the effect of SIRT1 on HepG2 cell apoptosis induced by galangin. Methods HepG2 cells were pre-treated with SIRT1 inhibitor EX-527 for 2 hours, and then galangin for 24 hours. DMSO solvent control group, EX-527 treatment group, galangin treatment group and EX-527 and galangin co-treatment group were established. Hoechst 33342 staining, flow cytometry and western blot were performed to detect the apoptosis of HepG2 cells. After regulating the expression of SIRT1 in HepG2 cells with RNA interference and transfection of exogenous genes, these cells were treated with galangin for 24 hours. Negative control group, vector control group, SIRT1 knock down group, blank control group, blank vector group,and SIRT1 upregulation group were established. Western blot and Flow cytometry were performed to detect the apoptosis of HepG2 cells. Results The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of galangin group [（ 11.62 ± 0.55） % ,0.89±0.01]and EX-527＋ galangin group[ （25.75±0.61） % ,1.15±0.06] were all increaset（P〈0.01 ） ,when these were compared with DMSO solvent control group[ （2.49±0.22） % ,0.06±0.00] ; and those in EX-527＋ galangin group were also markedly increased compared with galangin group（ P〈0.01）. The result of western bolt was that the gray level ratio of PARP1 and GAPDH of SIRT1 knocked down group（ 0.06±0.01） was markedly decreased compared with vector control group（ 1.11 ±0.05） and without adenovi-rus infection group （ 1.10±0.04） （P〈0.01）. The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 knocked down group were markedly increased compared with vector control group and without adenovirus infection group（P〈 0.01）. The gray level ratio of PARP1 and GAPDH of SIRT1 up-regulated group （1.63±0.04） was markedly increased compared with blank control group （0.89±0.02） and without plasmid transfection group （0.87±0.03） （P〈0.01） . The apoptosis rate and the gray level ratio of shear band of PARP1 and GAPDH that of SIRT1 up-regulated group were markedly decreased compared with blank control group and without plasmid transfection group （P〈0.01）. Conclusion SIRT1 inhibited galangin-induced apoptosis in HepG2 cells.