目的 探讨抑制内质网应激(endoplasmic reticulum stress,ERS)诱导的自噬对新生大鼠坏死性小肠结肠炎(necrotiz-ing enterocolitis,NEC)的影响.方法 首先建立新生大鼠NEC模型,然后从中分离提取出肠上皮细胞,分为对照组、抑制组、诱导组.对照组正常培养,抑制组加4-苯基丁酸,诱导组加衣霉素处理24h.采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测各组细胞炎性细胞因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、肠脂酸结合蛋白(intestinal fatty acid binding protein,I-FABP)的表达水平;实时荧光定量聚合酶链反应(real-time quantitative polyme rase chain reaction,RT-qPCR)检测各组细胞 ERS 标志物葡萄糖调节蛋白 78(glucose regulated protein 78,GRP78)、氧调节蛋白 150(oxygen-regulated pro-tein 150,ORP150)的mRNA表达水平;Western blot法检测各组细胞自噬相关蛋白LC3 Ⅱ/Ⅰ、p62的表达水平.结果 与对照组比较,抑制组p62表达明显增高,TNF-α、I-FABP、GRP78、ORP150、LC3 Ⅱ/Ⅰ表达显著降低,而诱导组p62表达明显降低,TNF-α、Ⅰ-FABP、GRP78、ORP150、LC3Ⅱ/Ⅰ表达显著增高,差异均有统计学意义(P<0.05).结论 抑制ERS诱导的自噬激活可减轻NEC新生大鼠肠黏膜损伤和炎性反应,改善肠道屏障功能.
Objective To investigate the effect of inhibiting autophagy induced by endoplasmic reticulum stress(ERS)on necrotiz-ing enterocolitis(NEC)in neonatal rats.Methods First,the NEC model of neonatal rats was established.Then,the intestinal epitheli-al cells were isolated and divided into three groups:control group,inhibition group and induction group.The control group was cultured normally,the inhibition group was added with 4-phenylbutyric acid,and the induction group was added with tunicamycin for 24hours.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of the cellular inflammatory cytokines tumor necrosis factor-α(TNF-α)and intestinal fatty acid binding protein(I-FABP)in each group.Real-time quantitative polymerase chain reac-tion(RT-qPCR)was used to detect the mRNA expression level of the markers of ERS glucose regulated protein 78(GRP78)and oxy-gen-regulated protein 150(ORP150).Western blot was used to detect the expression of autophagy related proteins LC3 Ⅱ/Ⅰ and p62.Results Compared with the control group,the expression of p62 in the inhibition group increased significantly,the expression of TNF-α,I-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ in the inhibition group was significantly decreased,while the expression of p62 in the induc-tion group was significantly decreased,the expressions of TNF-α,Ⅰ-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ were significantly increased,and the differences were statistically significant(P<0.05).Conclusion Inhibition of ERS induced autophagy activation can alleviate intestinal mucosal injury and inflammatory response in neonatal rats with NEC and improve intestinal barrier function.