冬凌草甲素缓解脂多糖诱导的Caco-2肠上皮细胞屏障损伤和SIRT1/eIF2α信号通路活性的抑制

Oridonin alleviates LPS-induced barrier dysfunction and SIRT1/eIF2αsignaling pathway activity inhibition in Caco-2 intestinal epithelial cells

目的 探索冬凌草甲素对脂多糖(lipopolysaccharide, LPS)诱导的肠上皮屏障障碍的影响及其生物学机制。方法将Caco-2细胞进行单层培养21 d使其分化后,分为对照组、LPS组(2μg/mL LPS处理24 h)、LPS+低剂量冬凌草甲素组(10μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h)和LPS+高剂量冬凌草甲素组(40μg/mL冬凌草甲素预处理30 min,2μg/mL LPS处理24 h)。采用跨上皮电阻法和FITC-dextran 4通量法评估细胞单层屏障功能;CCK-8法检测细胞活力;乳酸脱氢酶(LDH)法检测LDH释放活性;DCFH-DA探针法检测细胞内活性氧水平;ELISA法检测炎症因子IL-1β和TNF-α分泌水平;免疫荧光法检测细胞的屏障功能相关蛋白闭合蛋白(occludin)和闭锁小带蛋白1(zonula occludens protein 1, ZO-1)的表达分布情况;Western blot法检测SIRT1和p-eIF2α的相对表达水平。结果 与LPS组比较,冬凌草甲素能改善LPS诱导的肠上皮细胞单层屏障障碍,增加细胞活力并降低LDH的释放活性以及细胞中活性氧、IL-1β和TNF-α水平,还能促进闭合蛋白和ZO-1在细胞膜上的表达以及连续分布模式。Western blot检测显示,冬凌草甲素能增加SIRT1的表达,并降低e IF2α的磷酸化。以上作用以高剂量组更佳。结论 冬凌草甲素可缓解LPS导致的肠上皮细胞损伤和单层屏障障碍,SIRT1/eIF2α通路信号激活可能是冬凌草甲素发挥肠上皮细胞保护作用的机制之一。

Objective To explore the effects of oridonin on lipopolysaccharide(LPS)-induced intestinal epithelial barrier disorder and its biological mechanism.Methods Caco-2 cells were cultured in monolayer for 21 days before differentiatation.The differentiated Caco-2 cells were divided into control group,LPS group(treated with 2μg/mL LPS for 24 h),LPS+low-dose oridonin group(pre-treated with 10μg/mL oridonin for 30 min,2μg/mL LPS for 24 h),and LPS+high-dose oridonin group(pre-treated with 40μg/mL oridonin for 30 min,2μg/mL LPS for 24 h).Cell monolayer barrier function was evaluated by transepithelial electrical resistance and FITC-dextran 4 flux assay.Cell viability was detected by CCK-8 method.Lactate dehydrogenase(LDH)releasing ac­tivity was detected by LDH assay.Intracellular reactive oxygen species(ROS)level was detected by DCFH-DA probe.The secretion levels of inflammatory cytokines IL-1βand TNF-αwere detected by ELISA.The expression and distribution of ZO-1 and occludin,the barrier function related proteins,were detected by immunofluorescence method.The relative expression levels of SIRT1 and p-eIF2αwere detected by Western blotting.Results Compared with LPS group,oridonin improved LPS-induced monolayer barrier dysfunc­tion,increased cell viability,and decreased LDH releasing activity,ROS,IL-1βand TNF-αlevels,and promoted the expression and continuous distribution pattern of ZO-1 and occludin on cell membrane.Western blotting results showed that oridonin increased the expression of SIRT1 and decreased the phosphorylation of eIF2α,especially in the high dose group.Conclusion Oridonin can alleviate LPS-induced intestinal epithelial cell injury and monolayer-barrier dysfunction,and the activation of SIRT1/eIF2αsignaling pathway may be one of the mechanisms of oridonin’s protective effect on intestinal epithelial cells.