牡荆素鼠李糖苷通过EGFR抑制酒精引起的胃上皮细胞焦亡

Pectin rhamnoside inhibits alcohol-induced gastric epithelial cell pyroptosis through EGFR

目的探讨牡荆素鼠李糖苷(Vitexin-2″-o-rhamnoside,RHV)对酒精所致胃上皮细胞(human normal gastric epithelial cells,GES-1)焦亡是否有改善作用及其作用机制。方法GES-1细胞分别用不同浓度(0,200,400,600,800,1000 mmol/L)酒精干预,检测细胞存活率、焦亡相关蛋白的表达、乳酸脱氢酶(LDH)的释放量,筛选引起细胞焦亡的最佳干预浓度。GES-1细胞分别用800 mmol/L酒精干预0,2,4,6,8 h,CCK-8检测细胞存活率,筛选最佳干预时间。GES-1细胞分别用不同浓度RHV(5,15,30,60μmol/L)预处理,检测酒精引起的细胞存活率及焦亡相关蛋白的变化,筛选RHV的最佳干预浓度。采用网络药理学分析及分子对接预测RHV的可能作用靶点。GES-1细胞分别用酒精及联合RHV预处理干预,检测磷酸化表皮生长因子受体(p-EGFR)的表达。GES-1细胞分别用不同浓度EGFR抑制剂(0,1,2,4,8,10 nmol/L)及激活剂(0,5,10,20μmol/L)处理,检测p-EGFR蛋白表达,观察核心靶点改变是否影响细胞焦亡。GES-1细胞分别用不同浓度RHV(0,15,30,60μmol/L)预处理,再用EGFR激活剂干预,检测p-EGFR的表达。Western blot和免疫荧光检测焦亡相关蛋白的表达,乳酸脱氢酶(LDH)试剂盒检测LDH的释放量。结果酒精处理后GES-1细胞存活率呈剂量和时间依赖性降低(P<0.05),LDH释放量及cleaved caspase-1,GSDMD-N,NLRP3,IL-1β蛋白表达剂量依赖性增多(P<0.05),考虑模型的稳定性,后续实验选择800 mmol/L酒精干预4 h。RHV预处理后,酒精诱导的GES-1细胞存活率剂量依赖性升高(P<0.01),NLRP3,COX-2,cleaved caspase-1,IL-18蛋白表达剂量依赖性减少(P<0.05),RHV最佳干预浓度为60μmol/L。酒精性胃炎及牡荆素共同作用的核心靶点可能为IL-6,EGFR,IL-1β等,相关通路集中于氧化还原、代谢、受体活化及EGFR相关通路,且RHV与EGFR可能有较好结合性。与酒精单独处理比较,联合RHV预处理后p-EGFR水平升高(P<0.05)。EGFR抑制剂使酒精诱导的LDH释放量及p-EGFR,GSDMD-N,IL-1β蛋白表达剂量依赖性减少(P<0.05),而EGFR激活剂使p-EGFR和GSDMD-N表达增多(P<0.05)。30,60μmol/L RHV预处理使EGFR激活剂诱导的p-EGFR蛋白水平降低(P<0.05)。结论RHV可通过抑制EGFR磷酸化改善酒精诱导的GES-1细胞焦亡。

Objective To clarify the improvement effect of Vitexin-2″-o-rhamnoside(RHV)on alcohol-induced gastric epithelial cell pyroptosis and its mechanism.Methods GES-1 cells were intervened with different concentrations of alcohol(0,200,400,600,800,1000 mmol/L),and then the cell viability,the expressions of pyroptosis-related proteins and the lactate dehydrogenase(LDH)release were detected to screen the optimal intervention concentration for inducing the cell apoptosis.GES-1 cells were intervened with 800 mmol/L alcohol for 0,2,4,6,8 h respectively,and the cell viability was detected using CCK-8 to screen the optimal intervention time.GES-1 cells were pretreated with different concentrations of RHV(5,15,30,60μmol/L)and then treated with 800 mmol/L alcohol,and the cell viability and the expressions of pyroptosis-related proteins were detected to screen the optimal intervention concentration of RHV.Network pharmacology analysis and molecular docking were employed to predict the possible action targets of RHV.GES-1 cells were intervened with alcohol or combined with RHV pretreatment,and the expression of phosphorylated epidermal growth factor receptor(p-EGFR)was detected.GES-1 cells were treated with different concentrations of EGFR inhibitor(0,1,2,4,8,10 nmol/L)and activator(0,5,10,20μmol/L),and the expression of p-EGFR protein was detected to observe whether the changes of core targets affect the cell pyroptosis.GES-1 cells were pretreated with different concentrations of RHV(0,15,30,60μmol/L)and then intervented with EGFR activator,and the expression of p-EGFR was detected.Western blot and immunofluorescence were used to detect the expressions of pyroptosis-related proteins,and the lactate dehydrogenase(LDH)assay kit was used to measure LDH release.Results After alcohol treatment,the survival rate of GES-1 cells was decreased in a dose-and time-dependent manner(P<0.05),while LDH release,and the protein expressions of cleaved caspase-1,GSDMD-N,NLRP3,and IL-1βwere dose-dependently increased(P<0.05).In view of the stability of the model,800 mmol/L alcohol intervention for 4 h was chsoen for the subsequent experiments.After RHV pretreatment,the alcohol-induced GES-1 cell survival rate was dose-dependently increased(P<0.01),while the protein expressions of NLRP3,COX-2,cleaved caspase-1,and IL-18 were decreased dose-dependently(P<0.05).The optimal intervention concentration of RHV was 60μmol/L.The core targets of the combined action of alcoholic gastritis and resveratrol may be IL-6,EGFR,IL-1β,etc.,the related pathways focused on oxidation-reduction,metabolism,receptor activation,and EGFR-related pathways,and RHV may have good binding affinity with EGFR.Compared with alcohol treatment alone,the level of p-EGFR was increased after combined with RHV pretreatment(P<0.05).EGFR inhibitor dose-dependently reduced the levels of LDH release and p-EGFR,GSDMD-N,and IL-1βprotein expression induced by alcohol(P<0.05),while EGFR activator increased the expressions of p-EGFR and GSDMD-N(P<0.05).Pretreatment with 30,60μmol/L RHV reduced the protein level of p-EGFR induced by EGFR activator(P<0.05).Conclusion RHV can improve the alcohol-induced pyroptosis of GES-1 cells by inhibiting EGFR phosphorylation.