The Role Of β-arrestin2 In Neonatal Necrotizing Enterocolitis And Its Molecular Mechanism

OBJECTIVE: Necrotizing enterocolitis(NEC)is one of the most common gastrointestinal emergencies in premature infants,and is associated with significant morbidity and mortality.At present,the pathogenesis of NEC is still unclear.β-arrestin2 is an important G protein-coupled receptor regulatory protein which expresses in intestinal epithelial cells and is involved in the regulation of cell apoptosis.In this study,we will explore the role of β-arrestin2 in NEC and the potential mechanisms.Methods: The intestinal tissue samples of NEC patients and the control group with intestinal atresia were collected to detect the expression of β-arrestin2.As NEC always occur in the premature,the expression of β-arrestin2 in intestinal tissues of mice with different gestational age was compared,and the expression of β-arrestin2 in intestinal tissues of premature infants and term infants was also detected.The NEC mouse model was constructed to detect the expression ofβ-arrestin2 in the intestine of mice in the NEC group and the control breast-fed group.NEC modeling was simultaneously performed in β-arrestin2 knockout and wild type mice.The body weight changes,survival curves,intestinal inflammation scores were compared and the expression of Cleaved-Caspase3,CHOP and Bi P were detected of four groups of mice(KO+Ctrl(Knockout Control Group),KO+NEC(Knockout NEC Group),WT+Ctrl(Wild Type Control Group),WT+NEC(Wild Type NEC Group)).The expression levels of CHOP in the intestine of mice with different gestational age,term infants and premature infants were detected.IEC-6 cells were used as the research object to detect the effect of LPS stimulation on the expression of β-arrestin2.β-arrestin2 si RNA and plasmids were transfected respectively to construct an IEC-6 cell line in which β-arrestin2 was overexpressed or silenced stably.After stimulation with LPS or ER stress positive control Thapsigargin,the effect of β-arrestin2 on the expression of Cleaved-Caspase3,CHOP and Bi P was examined.The β-arrestin2 and Bi P plasmids were simultaneously transfected within IEC-6 to detect differences in expression of Cleaved-Caspase3 and CHOP.The effect of HA15(a Bi P inhibitor)alone or in combination with β-arrestin2 overexpression on the expression of Cleaved-Caspase3,CHOP and Bi P was examined.Co-IP was used to detect the interaction between β-arrestin2 and Bi P,and the effect of Salmeterol on the interaction between the two proteins was observed.Fluorescence double labeling was used to detect the localization relationship of two proteins.The effect of β-arrestin2 on Bi P ubiquitination and BIK release were examined.The difference in BIK expression between preterm and fullterm intestinal tissue was compared.Results: Compared with the control group,β-arrestin2 was significantly increased in the intestinal tract of children with NEC and NEC mice.With the increase of gestational age,the expression ofβ-arrestin2 in the intestine of mice decreased,and the expression of β-arrestin2 in the intestinal tissues of the fullterm children was also significantly lower than that in preterm infants.Compared with the WT+NEC group,the mice in the KO+NEC group had a slower weight loss,a higher survival rate,a lighter intestinal inflammatory disease,and a lower NEC score.In addition,the expression of Cleaved-Caspase3 and CHOP was decreased in the KO+NEC group compared with the WT+NEC group,while the expression level of Bi P was increased.The expression of CHOP in the intestinal tissues of premature baby and premature mice were also higher than that in the fullterm.LPS stimulation of IEC-6 significantly increased β-arrestin2 expression.Overexpression of β-arrestin2 increased the levels of Cleaved-Caspase3 and CHOP induced by LPS or Thapsigargin and decreased the expression of Bi P,while the effect of β-arrestin2 silencing was just the opposite.Bi P overexpression can attenuate the increased expression of Cleaved-Caspase3 and CHOP caused by overexpression of β-arrestin2.HA15 promoted apoptosis and was not affected by β-arrestin2 expression.Co-IP experiments revealed an interaction between β-arrestin2 and Bi P,which could be enhanced by Salmeterol,which further promoted apoptosis.β-arrestin2 and Bi P were co-localized in the cytoplasm of cells.Bi P was regulated by β-arrestin2 ubiquitination,and its ubiquitination level increased with the increase of β-arrestin2 transfection.β-arrestin2 significantly increased the release of BIK only when overexpressed,and it did not induce BIK release when at normal level or silenced.Consistent with β-arrestin2 and CHOP expression trends,the expression level of BIK in premature intestinal tissue was significantly higher than that in fullterm intestinal tissue.Conclusion: β-arrestin2 is expressed in NEC intestinal epithelial cells and reveals a potential effect of promoting ER Stress and the development of NEC.β-arrestin2 may regulate the ubiquitination and degradation of Bi P by binding to Bi P and then promoting the release of BIK to induce intestinal epithelial cell apoptosis and NEC.