Expression Of NIX-mediated Mitophagy In Neonatal Necrotizing Enterocolitis And Its Possible Mechanism

Part ⅠObjective:To detect the expression level of NIX mediated mitochondrial autophagy in children with NEC.Methods:We collected intestinal tissue samples excised during surgery from children in the control group(n=8)and NEC group(n=6)and used fluorescence real-time quantitative PCR(qRT PCR)to detect the mRNA expression level of the BCL2/Adenovirus E1B 19kDa interacting protein 3-like(NIX/BNIP3L),hypoxia inducible factor-1α(HIF-1α),and autophagic markers(MAP1LC3,LC3)in intestinal tissue samples from children with NEC.To detect the protein expression level of NIX,HIF-1α and LC3 in intestinal tissue samples from children with NEC by Western blot(WB)and immunohistochemical methods.Results:The experimental results suggest that compared with the control group,the mRNA and protein expression levels of NIX,HIF-la and LC3B in intestinal tissue of children with NEC decreased significantly,with a statistically significant difference(P<0.05).Part ⅡObjective:To detect the expression level of NIX mediated mitochondrial autophagy in mouse with NEC.Methods:We used 60 5-day-old SPF grade C57BL/6 mice as experimental animals and randomly divided them into two groups:the control group and the NEC modeling group.According to the NEC pathological scoring standard,a double blind method was used to score the damage of the NEC model mice.A score of ≥2 indicates that the model was successful.Intestinal tissue samples were collected and used fluorescence real-time quantitative PCR(qRT PCR)to detect the mRNA expression level of the BCL2/Adenovirus E1B 19kDa interacting protein 3-like(NIX/BNIP3L),hypoxia inducible factor-1α(HIF-1α),and autophagic markers(MAP1LC3,LC3)in intestinal tissue samples from mouse with NEC.To detect the protein expression level of NIX,HIF-1α and LC3 in intestinal tissue samples from mouse with NEC by Western blot(WB).Results:The experimental results show that compared with the control group mice,the general situation of NEC group mice is poor.Further testing and analysis of the obtained mouse intestinal tissue samples shows that compared with the control group mice,the mRNA levels of NIX,HIF-1α and LC3B in NEC murine intestinal tissue were significantly decreased,with a statistically significant difference(P<0.05),indicating that the experimental results in the mouse model were consistent with those of children with NEC.Part ⅢObjective:In vitro experiments were conducted to investigate the possible mechanism of NIX mediated mitochondrial autophagy in influencing the progression of NEC disease.Methods:In order to further study the possible mechanism of NIX in NEC diseases,we constructed a NIX overexpression lentivirus,transfected the NIX overexpression lentivirus into human intestinal epithelial cells(FHs 74 Int),and then verified the functional changes of intestinal epithelial cells.The expression level of NIX was detected by PCR and WB;Immunofluorescence was used to observe the expression and localization of NIX;WB was used to detect the expression level of autophagy related proteins,CCK-8(Cell Counting Kit-8)was used to detect the level of cell proliferation,and flow cytometry was used to detect changes in apoptosis.The above experiments were used to verify the effect of overexpression of NIX on the function of small intestinal epithelial cells.Results:The experimental results of human small intestinal epithelial cells(FHs 74 Int)showed that the expression of NIX and Caspase-3 was localized in the cytoplasm,and was transfected into small intestinal epithelial cells overexpressed with NIX lentivirus,and the protein expression of NIX was significantly increased.After overexpression of NIX,the expression of autophagy receptor protein P62 significantly decreased,the expression of autophagy effector protein 1 significantly increased,and the expression ratio of LC3II/LC3I significantly increased,indicating autophagy activation;There was no significant change in apoptosis related protein Caspase-9 and an increase in Caspase-3 cleavage,indicating significant activation of apoptosis;The level of cell proliferation decreased significantly from the second day of the experiment;The significantly increased rates of early and late apoptosis and total apoptosis suggest that NIX can inhibit the proliferation level of human intestinal epithelial cells,promote autophagy and apoptosis.