Preliminary Study On The Mechanism Of TUDCA Interventing Necrotizing Enterocolitis In Newborn Mice By Inhibiting Apoptosis

Objective: To evaluate the effect of Tauroursodeoxycholic acid(TUDCA),an endoplasmic reticulum stress inhibitor,on necrotizing enterocolitis in neonatal mice,and to explore the protective effect of TUDCA on intestinal epithelial cells and its possible mechanism as well as the effect on Paneth cells in mice.Methods: NEC model of C57BL/6J neonatal mice aged 7-10 days was established by artificial feeding combined with hypoxia and cold stimulation.The experiment was divided into four groups: normal control group(10),NEC model group(14),TUDCA intervention group(10)and NEC model TUDCA intervention group(16).The levels of serum inflammatory factors IL-1beta and IL-6 were detected by ELISA,and the levels of IL-1 beta and IL-6 were detected by real-time fluorescent quantitative PCR.Western blot was used to detect the expression of BiP,p-PERK,p-eIF2? and CHOP in intestinal tissues of mice.The expression of BiP in intestinal tract of mice and human was detected by immunofluorescence and apoptosis in intestinal tract of mice was detected by Tunel.IEC-6 cells were further divided into five groups in vitro: control group,LPS(50 ug/ml)treatment group,LPS and TUDCA(50?M,100?M,200?M)treatment group.LDH release assay was used to detect the toxicity of TUDCA to cells.Flow cytometry and Tunel were used to detect the apoptosis of IEC-6 cells.The expression of BiP was detected by immunofluorescence.Western blot was used to detect the expression of BiP,p-PERK,p-eIF2? and CHOP.Twelve small intestinal specimens from surgical children with NEC and 10 control specimens matching gestational age and surgical age were collected.Paneth cells were counted by immunohistochemical method.Immunohistochemistry was used to detect the expression of antimicrobial peptides(lysozyme,HD-5,secretory phospholipase A2)in Paneth cells.Real-time fluorescence quantitative PCR was used to detect the expression of antimicrobial peptide RNA in Paneth cells.Lysozyme and BiP,HD-5 and Tunel immunofluorescence co-localization staining were detected in intestinal tissues.Immunohistochemistry was used to detect the expression of Lysozyme and Paneth cells count in intestinal tissues of mice;immunofluorescence co-localization staining of Lysozyme and BiP,Lysozyme and Tunel in intestinal tissues;quantitative real-time fluorescence PCR was used to detect the levels of antimicrobial peptides Lysozyme,DEFA3 and DEFA5 mRNA.Results: TUDCA could improve the severity of NEC disease in newborn mice,maintain the weight of NEC mice and improve the survival rate,the difference was statistically significant(P < 0.05).TUDCA can reduce the expression of IL-1? and IL-6 in serum and intestinal wall.TUDCA could inhibit the apoptosis of small intestinal epithelial cells and reduce the expression of endoplasmic reticulum stress markers BiP,p-PERK,p-eIF2 alpha and CHOP(P<0.05).In vitro,TUDCA could inhibit the apoptosis of IEC-6 cells and reduce the expression of endoplasmic reticulum stress markers BiP,p-PERK,p-eIF2? and CHOP in IEC-6 cells(P<0.05).In human clinical specimens,compared with the control group,the lysozyme,HD-5 and secretory phospholipase A2 secreted by Pan's cells in NEC group decreased(P < 0.05).The number of Pan's cells in intestinal specimens of children with NEC was less than that of the control group(P < 0.05).In NEC group,endoplasmic reticulum stress marker BiP and apoptosis increased.In the intestinal specimens of mice,the number of Paneth cells and the level of Lysozyme,DEFA3 and DEFA5 secreted by Paneth cells in NEC group decreased,and the expression of antimicrobial peptides secreted by Paneth cells increased after TUDCA intervention.In NEC group,the expression of endoplasmic reticulum stress marker BiP and apoptosis were increased.After TUDCA intervention,the expression of BiP in Paneth cells was decreased and apoptosis was improved.Conclusion: Intraperitoneal injection of TUDCA can maintain the weight of NEC model newborn mice,improve the severity of the disease,reduce the mortality and inhibit intestinal inflammation.The mechanism may be related to the improvement of endoplasmic reticulum stress and apoptosis in mouse epithelial cells by inhibiting PERK-eIF2 alpha pathway,and the protection of Paneth cells from endoplasmic reticulum stress and apoptosis.