| Objective:To evaluate the protective effect of inhibition of endoplasmic reticulum stress(ERS)on intestinal epithelial cells of neonatal rats with necrotizing enterocolitis(NEC),and to explore the role of autophagy in this mechanism,so as to provide a part of experimental basis with reference value for further research on the pathogenesis of NEC.Methods:Ten healthy newborn clean-grade SD rats(Sprague-Dawley),male or female,were selected.First,the NEC model of neonatal rats was established by artificial feeding、hypoxia、cold stimulation combined with LPS gastric injection.We isolated the intestinal epithelial cells from the intestinal tissues of newborn rats for cell cultured.The experiment was divided into control group,inhibition group and induction group.The control group was cultured in normal medium without any drugs,the inhibition group was added with 4-phenylbutyric acid(4-PBA;5 mmol/L),and the induction group was added with tunicamycin(Tm;10 μ g/ml)for 24 hours.Subsequently,ELISA was used to determine the expression level of the cellular inflammatory cytokines tumor necrosis factor α(TNF-α)and intestinal fatty acid binding protein(I-FABP)in each group.RT-PCR was used to determine the expression level of glucose regulated protein 78(GRP78)and ORP150 m RNA,which are the markers of endoplasmic reticulum stress in each group;Western blotting was used to determine the expression level of autophagy related proteins LC3II/I and P62 in each group.Results:1.Comparison of the expression of the inflammatory related factor TNF-α and I-FABP in the intestinal epithelial cells of the three groups of newborn rats: compared with those of control group,the expression of TNF-α and I-FABP in the inhibition group was significantly decreased,while the expression of TNF-α and I-FABP in the induction group was significantly increased,the difference was statistically significant(P<0.05).2.Comparison of the expression of the endoplasmic reticulum stress markers GRP78 and ORP150 m RNA in the intestinal epithelial cells of the three groups of newborn rats: compared with those of control group,the expression of GRP78 and ORP150 m RNA in the inhibition group was significantly decreased,while the expression of GRP78 and ORP150 m RNA in the induction group was significantly increased,the difference was statistically significant(P<0.05).3.Comparison of the expression of autophagy-related proteins LC3II/I and P62 in the intestinal epithelial cells of the three groups of newborn rats: compared with the control group,the expression of LC3II/I in the inhibition group was significantly decreased,and the expression of P62 was increased;In the induction group,the expression of LC3II/I was significantly increased,and the expression of P62 was decreased,the difference was statistically significant(P<0.05).Conclusion:1.Compared with the control group,the expression of the inflammatory cytokines(TNF-α 、 I-FABP)and endoplasmic reticulum stress markers(GRP78,ORP150 m RNA)in the inhibition group were obviously reduced,and those of the induction group were significantly increased,suggesting that inhibition of ERS can reduce the inflammatory reaction of intestinal epithelial cells in neonatal rats with NEC,reduce the degree of mucosal damage of intestinal tissue,improve the intestinal barrier function,and thus have a protective effect on NEC.2.Compared with the control group,the expression of LC3II/I in the inhibition group was significantly decreased,the expression of P62 was increased,and the degree of autophagy of cells was significantly reduced.The results in the induction group were contrary,suggesting that autophagy might participate in the inhibition of ERS’ protective effect on NEC,and play a crucial role in the pathogenesis of the disease. |
