益心解毒方抑制H9C2心肌细胞凋亡的作用机制

Mechanism of Yixin Jiedu Fang in inhibiting apoptosis of H9C2 cells

目的研究益心解毒方对H9C2心肌细胞凋亡的抑制作用及可能机制。方法实验分为正常组、模型组、益心解毒方组和槲皮素组,采用缺氧缺糖/复氧复糖诱导H9C2心肌细胞凋亡,研究益心解毒方的保护作用。运用CCK-8法检测细胞存活率,镜下观察细胞的形态改变,Fluo-3AM荧光染色法检测细胞内钙超载,Hoechst 33342荧光染色法检测细胞凋亡的情况,实时荧光定量PCR检测Fas和Fas L的基因表达,Western Blot测定p-Akt和caspase-8蛋白的表达。结果与正常组比较,模型组细胞存活率显著下降,细胞皱缩变形,细胞发生钙超载,凋亡细胞增多,Fas和Fas L的mRNA表达增加,caspase-8蛋白表达升高,p-Akt表达降低,均有统计学差异(P<0.05)。与模型组比较,益心解毒方组细胞存活率增加,细胞形态有改善,细胞钙超载减轻,凋亡细胞的数量减少,pAkt蛋白的表达上调,Fas L mRNA和caspase-8蛋白的表达下调,均有统计学差异(P<0.05)。结论益心解毒方能抑制H9C2心肌细胞凋亡,其作用机制可能与减轻钙超载和调控外源性凋亡途径相关。

Objective To study the effect and possible mechanism of Yixin Jiedu Fang Supplementing Heart and kemoving Toticity Decoction in inhibiting apoptosis of H9C2 myocardial cells. Methods H9C2 cells were divided into normal group, model group, Yixin Jiedu Fang group and quercetin group. The apoptosis of H9C2 myocardial cells was induced by using hypoxia-hypoglycemia/reoxygenation- reglycemia method, and protective effect of Yixin Jiedu Fang was studied. The survival rate of H9C2 cells was detected by using CCK-8 assay. The morphological changes of H9C2 cells were observed with microscope. Intracellular calcium overload was detected by using Fluo-3AM method and apoptosis of H9C2 cells was detected by using Hoechst 33342 method. The genetic expressions of Fas and FasL were detected by using real-time fluorescence quantitative polymerase chain reaction （RT-PCR）, and expressions of p-Akt protein and caspase-8 protein were detected by using Western blotting method. Results Compared with normal group, the survival rate of H9C2 cells decreased, cell shrinkage and deformation, intracellular calcium overload and more apoptosis cells were observed, expressions of Fas mRNA and FasL mRNA increased, expression of caspase-8 protein increased, and expression of p-Akt protein decreased in model group （ all P 〈0.05 ）. Compared with model group, the survival rate of H9C2 cells increased, cellular morphology were improved, intracellular calcium overload was relieved, expression of p-Akt protein was up-regulated, and expressions of FasL mRNA and caspase-8 protein were down-regulated in Yixin Jiedu Fang group （ all P 〈 0.05 ）. Conclusion Yixin Jiedu Fang can inhibit the apoptosis of H9C2 cells, and the mechanism maybe related to the relief of intracellular calcium overload and control of exogenous apoptotic pathway.