白头翁皂苷D介导AMPK/COX-2通路对食管癌细胞增殖、凋亡的影响

Effects of Pulsatilla saponin D mediated AMPK/COX-2 pathway on the proliferation and apoptosis of esophageal cancer cells in vitro

目的 探讨白头翁皂苷D调控腺苷酸活化蛋白激酶/环氧合酶-2(AMPK/COX-2)信号通路对人食管癌细胞EC9706增殖及凋亡的影响。方法 体外培养EC9706细胞,不同浓度梯度白头翁皂苷D处理后,MTT法检测EC9706细胞增殖,以筛选白头翁皂苷D的作用浓度。将EC9706细胞分为对照组,白头翁皂苷D(PSD)低、中、高剂量组(10、20、40μmol/L白头翁皂苷D),PSD高剂量+AMPK抑制剂(Compound C)组(40μmol/L白头翁皂苷D+10μmol/L Compound C),平板克隆形成实验检测EC9706细胞增殖;流式细胞术检测EC9706细胞凋亡;Western blot法检测EC9706细胞Bax、Bcl-2、p53、磷酸化AMPK(p-AMPK)、磷酸化乙酰辅酶A羧化酶(p-ACC)、COX-2蛋白表达水平。结果 与对照组比较,PSD低、中、高剂量组EC9706细胞克隆形成率、Bcl-2、COX-2蛋白表达水平显著降低,细胞凋亡率、Bax、p53、p-AMPKα、p-ACC蛋白表达水平显著升高,并呈浓度依赖性(P<0.05);与PSD高剂量组比较,PSD高剂量+Compound C组细胞克隆形成率、Bcl-2、COX-2蛋白表达水平显著升高(P<0.05),细胞凋亡率、Bax、p53、p-AMPKα、p-ACC蛋白表达水平显著降低(P<0.05)。结论 白头翁皂苷D通过激活AMPK途径抑制COX-2表达,进而抑制EC9706细胞增殖,促进细胞凋亡。

Objective To investigate the effects of Pulsatilla saponin D(PSD)mediated adenosine 5’-monophosphate-activated protein kinase/cyclooxygenase-2(AMPK/COX-2)signaling pathway on the proliferation and apoptosis of esophageal cancer cells(EC9706)in vitro.Methods EC9706 cells were cultured in vitro and treated with different concentration gradients of PSD,then the proliferation of EC9706 cells was detected by MTT to screen the effect concentration of Pulsatilla saponins D.And the EC9706 cells were divided into control groups,low,medium,and high-dose PSD groups(10,20,40μmol/L,respectively),and high-dose PSD+AMPK inhibitor(compound C)group with 40μmol/L PSD+10μmol/L AMPK inhibitor.The plate clone formation test was used to detect the cell proliferation;flow cytometry was used to detect cell apoptosis;Western Blot was used to detect the expression levels of Bax,Bcl-2,p53,phosphorylated AMPK(p-AMPK),phosphorylated acetyl-CoA carboxylase(p-ACC),and COX-2 in EC9706 cells.Results Compared with those in control group,the EC9706 cell clone formation rate,and the expression levels of Bcl-2 and COX-2 protein were significantly decreased low,medium and high dose PSD groups,however,the apoptosis rate,and the expression levels of Bax,p53,p-AMPKα,p-ACC protein were significantly increased,with a concentration-dependent manner(P<0.05).Compared with those in high-dose PSD group,the cell clone formation rate,and the expression levels of Bcl-2,COX-2 protein were significantly increased in high-dose PSD+compound C group,however,the apoptosis rate,and the expression levels of Bax,p53,p-AMPKα,and p-ACC protein were significantly decreased(P<0.05).Conclusion Pulsatilla saponin D can inhibit the expression levels of COX-2 by activating the AMPK pathway,thereby inhibiting the proliferation and promoting apoptosis of EC9706 cells.