摘要
目的:探讨紫花牡荆素(casticin)促进肝星状细胞(hepatic stellate cell,HSC)-T6凋亡的作用机制.方法:用终浓度为0、0.5、1.0、2.0μmol/L的Casticin作用于HSC-T6,于12、24、48 h后采用MTT法检测HSC-T6增殖抑制率;24 h后收集各组细胞,流式细胞术检测细胞周期及凋亡率;DNA琼脂糖凝胶电泳法检测HSC-T6凋亡;RTPCR法检测促凋亡基因FAS/FASL mRNA及抑凋亡基因Bcl-2 mRNA的表达;免疫组织化学法检测凋亡蛋白Caspase3的表达.结果:MTT法检测显示,Casticin对HSC-T6有增殖抑制作用,并存在浓度和时间依赖关系;流式细胞仪检测HSC-T6凋亡,发现2.0μmol/L的Casticin作用48 h后,HSC-T6凋亡率达到最大值55.70%±5.56%,各药物组凋亡率与空白对照组比较,差异有统计学意义(P<0.05).DNA琼脂糖凝胶电泳法检测HSC-T6凋亡,显示有特征性的DNA梯度带形成.RT-PCR法检测促凋亡基因FAS/FASL mRNA表达明显上调,而抑凋亡基因Bcl-2 mRNA的表达下调.免疫组织化学法检测凋亡蛋白Caspase3的表达明显增加,当Casticin浓度2.0μmol/L作用48 h,其表达率71.33%±2.68%,与空白对照组比较,差异有统计学意义(P<0.05).结论:Casticin诱导HSC-T6细胞凋亡的机制可能是通过上调Fas/FasL基因表达,下调Bcl-2,促使线粒体通透性增加,再激活Caspase3蛋白诱发细胞凋亡.
AIM： To investigate the effect of casticin on apoptosis of hepatic stellate cells（HSCs） in vitro and the possible mechanisms involved. METHODS： Rat HSC-T6 cells were cultured in high-glucose DMEM and then treated with different concentrations of casticin（0, 0.5, 1.0, and 2.0 μmol/L） for 12, 24 and 48 h. HSC-T6 apoptosis was identified by flow cytometry（FCM） and agarose gel electrophoresis. The mRNA expression of apoptosis-related genes Fas/FasL and Bcl-2 was examined by RT-PCR. The expression of Caspase3 was studied by immunocytochemical staining assay（SABC）. RESULTS： Casticin treatment significantly increased the apoptosis of HSC-T6 in a dose- and time-dependent manner compared with the control group（P〈0.01）. The highest apoptosis rate was observed in HSC-T6 cells treated with 2.0 μmol/L of casticin for 48 h（55.70% ± 5.56%）. An oligonucleosomal DNA ladder was demonstrated by SABC, indicating DNA break in HSC-T6cells. The expression of Fas/FasL mRNA was increased, while expression of Bcl-2 mRNA was reduced. After HSC-T6 cells were treated with casticin at concentrations of 0.5, 1.0, and 2.0 μmol/L for 48 h, the positive rates of Caspase3 protein expression were 12.78% ± 0.74%, 41.00% ± 1.51% and 71.33% ± 2.68%, respectively. Casticin treatment significantly increased Caspase3 protein expression in a dose- and time-dependent manner compared with the control group（P〈0.001）. CONCLUSION： Casticin induced apoptosis of HSC-T6 cells may involve mitochondrial pathways and Bcl-2 family proteins. Casticin might be a potential Chinese medical component for inhibiting liver fibrosis.
出处
《世界华人消化杂志》
CAS
北大核心
2014年第7期907-914,共8页
World Chinese Journal of Digestology
