葛根芩连汤对幽门螺杆菌感染人正常胃黏膜上皮细胞增殖与凋亡的影响及机制研究

Effect of Gegen Qinlian Decoction on the proliferation and apoptosis of Helicobacter pylori-infected human GES-1 cells and the underlying mechanism

目的 观察葛根芩连汤对幽门螺杆菌(Hp)感染人正常胃黏膜上皮(GES-1)细胞增殖与凋亡的影响及相关机制。方法 按照葛根芩连汤干预剂量的不同、是否有Hp感染将人GES-1细胞分为空白对照组、模型组、葛根芩连汤低剂量组(10μmol/L)、葛根芩连汤中剂量组(20μmol/L)、葛根芩连汤高剂量组(40μmol/L)及阳性药对照组(阿莫西林5μmol/L)。培养结束后,采用细胞增殖检测分析法(CCK-8法)检测细胞存活率,酶联免疫吸附法(ELISA)检测上清液肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6水平,流式细胞术检测细胞凋亡率,实时荧光定量聚合酶链式反应(RT-qPCR)检测细胞p38、丝裂原活化蛋白激酶2(MK2)mRNA表达,蛋白免疫印迹(Western bloting)法检测细胞p38、MK2蛋白表达。结果 与空白对照组比较,模型组人GES-1细胞存活率降低(P<0.05);与模型组比较,葛根芩连汤各剂量组与阳性药对照组人GES-1细胞存活率均升高(P<0.05),且葛根芩连汤各剂量组效应呈剂量依赖性(P<0.05);阳性药对照组与葛根芩连汤高剂量组人GES-1细胞存活率比较差异无统计学意义(P>0.05)。与空白对照组比较,模型组TNF-α、IL-1β和IL-6均升高(P<0.05);与模型组比较,葛根芩连汤各剂量组与阳性药对照组TNF-α、IL-1β和IL-6均降低(P<0.05),且葛根芩连汤各剂量组效应呈剂量依赖性(P<0.05);阳性药对照组与葛根芩连汤高剂量组TNF-α、IL-1β和IL-6比较差异均无统计学意义(P>0.05)。与空白对照组比较,模型组人GES-1细胞凋亡率升高(P<0.05);与模型组比较,葛根芩连汤各剂量组和阳性药对照组人GES-1细胞凋亡率降低(P<0.05),且葛根芩连汤各剂量组效应呈剂量依赖性(P<0.05);阳性药对照组和葛根芩连汤高剂量组人GES-1细胞凋亡率比较差异无统计学意义(P>0.05)。与空白对照组比较,模型组人GES-1细胞p38、MK2 mRNA和蛋白表达水平均升高(P<0.05);与模型组比较,葛根芩连汤各剂量组和阳性药对照组人GES-1细胞p38、MK2 mRNA和蛋白表达水平均降低(P<0.05),且葛根芩连汤各剂量组效应呈剂量依赖性(P<0.05);阳性药对照组和葛根芩连汤高剂量组人GES-1细胞p38、MK2 mRNA和蛋白表达比较差异均无统计学意义(P>0.05)。结论 葛根芩连汤可有效提高Hp感染后人GES-1细胞增殖率,同时降低细胞凋亡水平,其机制可能与抑制p38/MK2信号通路、降低细胞炎性反应有关。

Objective To explore the effect of Gegen Qinlian Decoction(GQD)on the proliferation and apoptosis of Helicobacter pylori(Hp)-infected human GES-1 cells and the underlying mechanism.Methods Human GES-1 cells were assigned into the blank control group,the model group,the GQD low-dose,middle-dose and high-dose groups(10μmol/L,20μmol/L,40μmol/L)and the positive control group(amoxicillin 5μmol/L)according to the dose of GQD and the Hp infection.After cell culture and the specific treatment,cell viability was detected by Cell Counting Kit-8(CCK-8)assay.The tumor necrosis factor-α(TNF-α),interleukin-1beta(IL-1β)and IL-6 levels in cell supernatant were measured byenzyme-linked immunosorbent assay(ELISA).Cell apoptosis rate was detected by flow cytometry.The mRNA and protein expressions of p38 Mitogen-activated protein kinase(p38)and mitogen activated protein kinase-activated protein kinase 2(MK2)were determined by real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting,respectively.Results Compared with the blank control group,cell survival rate in the model group was significantly reduced(P<0.05),which in the all GQD groups and the positive control group was significantly higher than that of the model group(P<0.05).Cell survival was dose-dependently elevated with the increasing dose of GQD.TNF-α,IL-1βand IL-6 levels in the model group were significantly higher than those of the blank control group(P<0.05),which in the all GQD groups and the positive control group were significantly lower than those of model group(P<0.05).TNF-α,IL-1βand IL-6 levels were dose-dependently reduced with the increasing dose of GQD.Cell apoptosis rate in the model group was significantly higher than that of the blank control group(P<0.05),which in the all GQD groups and the positive control group was significantly lower than that of the model group(P<0.05).Cell apoptosis rate was dose-dependently reduced with the increasing dose of GQD.The mRNA and protein expressions of p38 and MK2 in the model group were significantly higher than those of the blank control group(P<0.05),which in the all GQD groups and the positive control group were significantly lower than those of the model group(P<0.05).The mRNA and protein expressions of p38 and MK2 were dose-dependently downregulated with the increasing dose of GQD.No significant differences in cell survival rate,TNF-α,IL-1βand IL-6 levels,cell apoptosis rate and mRNA and protein expressions of p38 and MK2 were detected between the positive model group and the GQD high-dose group(all P>0.05).Conclusion GQD can effectively increase the proliferation rate of human GES-1 cells after Hp infection,while reducing the level of apoptosis by inhibiting the p38/MK2 signaling pathway and reducing the cellular inflammatory response.