化瘀祛痰方药含药血清调控PI3K/AKT通路对肝癌细胞自噬及上皮间质转化的调控作用研究

Effect of Serum Containing Huayu Qutan Recipe(化瘀祛痰方)Regulating PI3K/AKT Pathway on Autophagy and Epithelial Mesenchymal Transition of Hepatoma Cells

目的探讨化瘀祛痰方药含药血清调控PI3K/Akt通路对肝癌细胞自噬及上皮间质转化的调控作用。方法20只SD大鼠随机分成两组:空白组、化痰祛瘀方组,化瘀祛痰组给予16 g/kg灌胃,空白组给予等量生理盐水灌胃。各组大鼠连续灌胃10 d,1次/d。将HepG2细胞分为4组:空白对照组,转化生长因子β1(transforming growth factorβ1,TGF-β1)处理组,TGF-β1+化痰祛瘀含药血清组,TGF-β1+阿霉素组。收集上述各组细胞,CCK-8法分析化瘀祛痰方对肝癌细胞增殖的抑制作用,MDC染色法检测HepG2细胞自噬,Western Blot、qPCR检测各组细胞自噬、上皮间质转化相关蛋白和基因Beclin-1、LC3、E-cadherin和Vimentin的影响以及PI3K/Akt通路蛋白的表达。结果化瘀祛痰含药血清能抑制肝癌HepG2细胞的增殖,MDC染色法显示化瘀祛痰方促进自噬小泡的形成。Western Blot和qPCR结果显示化瘀祛痰方上调Beclin-1、LC3 II水平促进自噬(P<0.01),下调E-cadherin水平(P<0.01),同时上调Vimentin水平(P<0.01),抑制HepG2细胞上皮间质转化,p-PI3K、p-Akt蛋白水平均显著下调(P<0.01)。结论化瘀祛痰方可能通过抑制PI3K-Akt通路活化,诱导HepG2细胞自噬,抑制细胞上皮间质转化。

Objective To investigate the effect of the regulation of PI3K/Akt pathway by serum containing Huayu Qutan Recipe(化瘀祛痰方)on autophagy and epithelial mesenchymal transformation of liver cancer cells.Methods Twenty SD rats were ran-domly divided into two groups:blank group and Huatan Quyu Recipe group.The Huayu Qutan Recipe group was given Huayu Qutan Recipe(16 g/kg)by gavage and the blank group was given the same amount of normal saline by gavage.Rats in each group were given the administration for 10 days,once a day.HepG2 cells were divided into four groups:blank control group,TGF-β1 treatment group,TGF-β1+serum containing Huatan Quyu Recipe group,TGF-β1+adriamycin group.The cells of the above groups were collected.The inhibitory effect of Huayu Qutan Recipe on the proliferation of liver cancer cells was analyzed by CCK-8 method.The autophagy of HepG2 cells was detected by MDC staining.The effects of autophagy,epithelial mesenchy-mal transformation related proteins and the levels of genes Beclin-1,LC3,E-cadherin and vimentin and the expressions of PI3K/Akt pathway proteins were detected by Western Blot and qPCR.Results The serum containing Huayu Qutan Recipe can in-hibit the proliferation of HepG2 cells and promote the formation of autophagic vesicles.Western Blot and qPCR results showed that Huayu Qutan Recipe up-regulated the levels of Beclin-1 and LC3 II(P<0.01),down-regulated E-cadherin levels(P<0.01)and simultaneously up-regulated Vimentin levels(P<0.01).The levels of p-PI3K and p-Akt protein were sig-nificantly down-regulated(P<0.01)and the differences were statistically significant.Conclusion Huayu Qutan Recipe may in-duce HepG2 cell autophagy and inhibit cell epithelial-mesenchymal transition by PI3K-Akt pathway activation.