芝麻素对幽门螺杆菌诱导人胃黏膜上皮细胞损伤的影响及机制

Effects and mechanism of sesamin on human gastric mucosal epithelial cell injury induced by Helicobacter pylori

目的探讨芝麻素(sesamin,SES)是否通过调控微小RNA(microRNA,miR)-204的表达影响幽门螺杆菌(Helicobacter pylori,Hp)诱导的人胃黏膜上皮细胞损伤。方法将人胃黏膜上皮细胞分为对照组、Hp组、Hp+SES低剂量(SES-L)组、Hp+SES中剂量(SES-M)组、Hp+SES高剂量(SES-H)组、Hp+miR-204模拟物(miR-204)组、Hp+miR-204模拟物阴性对照(miR-204 negative control,miR-NC)组、Hp+SES-H+miR-204抑制剂阴性对照(anti-miR-NC)组及Hp+SES+miR-204抑制剂(anti-miR-204)组。采用细胞计数试剂盒8法检测细胞生长抑制率,流式细胞术检测细胞凋亡,蛋白质印迹法检测活化的半胱氨酸天冬氨酸蛋白酶(cleaved-caspase)3、cleaved-caspase9蛋白表达,检测乳酸脱氢酶(lactate dehydrofenase,LDH)活性及白介素(interleukin,IL)-1β、IL-8水平,逆转录定量聚合酶链反应检测miR-204表达。结果与Hp组比较,Hp+SES-L组、Hp+SES-M组和Hp+SES-H组人胃黏膜上皮细胞的生长抑制率、凋亡率、cleaved-caspase3蛋白及cleaved-caspase9蛋白的表达量均降低,并呈浓度依赖性(P<0.05);与Hp组比较,Hp+SES-L组、Hp+SES-M组和Hp+SES-H组人胃黏膜上皮细胞的LDH活性、IL-1β及IL-8水平均降低,并呈浓度依赖性(P<0.05);miR-204过表达后,Hp+miR-204组人胃黏膜上皮细胞的miR-204表达量较Hp+miR-NC组增加,而生长抑制率、凋亡率、cleaved-caspase3蛋白、cleaved-caspase9蛋白的表达量、LDH活性、IL-1β以及IL-8水平均较Hp+miR-NC组降低(P<0.05);干扰miR-204表达后,Hp+SES+anti-miR-204组人胃黏膜上皮细胞的miR-204表达量低于Hp+SES+anti-miR-NC组,而生长抑制率、凋亡率、cleaved-caspase3蛋白、cleaved-caspase9蛋白的表达量、LDH活性、IL-1β以及IL-8水平均高于Hp+SES+anti-miR-NC组(P<0.05)。结论SES可通过上调miR-204表达,增加Hp诱导的人胃黏膜上皮细胞活力,并减少凋亡和炎症的发生。

Objective To investigate whether sesamin(SES)affects the human gastric mucosal epithelial cell injury induced by Helicobacter pylori(Hp)by regulating the expression of miR-204.Methods Human gastric mucosal epithelial cells were divided into control group,Hp group,Hp+SES low-dose(SES-L)group,Hp+SES medium dose(SES-M)group,Hp+SES high dose(SES-H)group,Hp+miR-204 simulant negative control(miR-NC)group,Hp+miR-204 simulant(miR-204)group,Hp+SES-H+miR-204 inhibitor negative control(anti miR-NC)group,and Hp+SES+miR-204 inhibitor(anti miR-204)group.Cell growth inhibition rate was detected by cell counting kit 8,cell apoptosis was detected by flow cytometry,the protein expression of cleaved caspase 3 and 9 were detected by western blot,lactate dehydrogenase(LDH)activity and interleukin-1β(IL-1β)and IL-8 level were detected by the kit,and reverse transcription quantitative polymerase chain reaction was used to detect miR-204 expression.Results Compared with Hp group,the Hp+SES-L group,Hp+SES-M group and Hp+SES-H group had significant reductions in the growth inhibition rate,cell apoptosis rate,and the protein expression of cleaved-caspase 3 and 9 in gastric mucosal epithelial cells in a concentration-dependent manner(P<0.05).Compared with Hp group,the LDH activity,the levels of IL-1βand IL-8 in human gastric mucosal epithelial cells were decreased in a concentration-dependent manner in Hp+SES-L group,Hp+SES-M group and Hp+SES-H group(P<0.05).After miR-204 overexpression,the expression of miR-204 in human gastric mucosal epithelial cells in Hp+miR-204 group was higher than that in Hp+miR-NC group,but the growth inhibition rate,apoptosis rate,the protein expression of cleaved caspase-3,cleaved caspase-9,LDH activity,and IL-1βand IL-8 levels were decreased compared with Hp+miR-NC group(P<0.05).After interfering with the expression of miR-204,the expression of miR-204 in human gastric mucosal epithelial cells in Hp+SES+anti miR-204 group was lower than that in Hp+SES+anti miR-NC group,while the growth inhibition rate,apoptosis rate,the protein expression of cleaved-caspase3,cleaved-caspase 9,LDH activity,IL-1βand IL-8 were higher than those in Hp+SES+anti miR-NC group(P<0.05).Conclusion SES can increase Hp induced human gastric mucosal epithelial cells vitality and reduce apoptosis and inflammation by up regulating the expression of miR-204.