内质网自噬在Cajal间质细胞内质网应激损伤中的作用

摘要

目的 探讨内质网自噬在大鼠胃窦Cajal间质细胞(ICCs)内质网应激(ERS)过程中的作用,为改善胃动力障碍提供新靶标.方法 采用酶解法分离大鼠胃窦ICCs并行免疫荧光鉴定,使用衣霉素(TM)诱导ICCs构建ERS模型,三甲基腺嘌呤(3-MA)阻断自噬,雷帕霉素(Rapa)促进自噬.随机将细胞分为4组并分别给予不同的处理,ICCs组正常培养,ERS组给予2μg/mL TM处理ICCs,3-MA组给予2μg/mL TM和4μmol/mL 3-MA联合作用,Rapa组给予2μg/mL TM和2μmol/mL Rapa联合作用,每组作用时间均为6h;通过实时荧光定量PCR法检测ERS相关因子GRP78、自噬相关因子LC3B的mRNA表达;CCK-8法检测各组细胞活性.结果 成功分离并鉴定ICCs.与ICCs组比较,ERS组、3-MA组和Rapa组的GRP78和LC3B表达均升高(P均＜0.05),细胞活性降低(P均＜0.05).与ERS组比较,3-MA组GRP78表达升高(P＜0.05),LC3B表达、ICCs细胞活性降低(P均＜0.05);Rapa组GRP78表达降低(P＜0.05),LC3B表达、ICCs细胞活性升高(P均＜0.05).结论 ICCs的ERS过程伴发内质网自噬,内质网自噬可减轻ICCs的ERS损伤作用.%Objective To explore the role of endoplasmic reticulophagy (ER-phagy) during endoplasmic reticulum stress (ERS) induced by tunicamycin (TM) in rat interstitial cells of Cajal (ICCs),and to provide new targets for improving gastric motility disorders.Methods ICCs were isolated by collagenase digestion and identified by immunofluorescence staining.Tunicamycin (TM) was used to induce ICCs to establish ERS models,the 3-methyladenine (3-MA) was used to block autophagy,while the rapamycin (Rapa) was used to promote autophagy.Then the ICCs were randomly divided into 4 groups and received different interventions,including ICCs group (normal culture),ERS group (the ICCs were stimulated with 2 μg/mL TM),3-MA group (the ICCs were co-stimulated with 2 μg/mL TM and 4 μmol/mL 3-MA),and Rapa group (the ICCs were co-stimulated with 2 μg/mL TM and 2 μmol/mL Rapa);the ICCs in all groups were treated for 6 h.The mRNA expression levels of GRP78 (ERS biomarker) and LC3B (autophagy biomarker) were detected by real-time fluorescent quantitative PCR,and the activity of ICCswas detected by CCK-8 assay.Results We isolated and identifiedICCs successfully.Compared with the ICCs group,the mRNA expression levels of GRP78 and LC3B increased,and the activity of ICCs decreased in the ERS group,3-MA group,and Rapa group (all P ＜ 0.05).Compared with the ERS group,the mRNA expression of GRP78 increased,the mRNA expression of LC3B and the activity of ICCs decreased in the 3-MA group (all P ＜0.05);on the contrary,the mRNA expression of GRP78 decreased,the mRNA expression of LC3B,and the activity of ICCs increased in the Rapa group (all P ＜ 0.05).Conclusion ER-phagy occurs in the process of ERS of ICCs,and ER-phagy can reduce the ERS injury of ICCs.