Expression and possible role of Smad3 in postnecrotizing enterocolitis stricture

Objective To investigate the expression of Smad3(mothers against decapentaplegic homolog 3)protein in postnecrotizing enterocolitis stricture and its possible mechanism of action.Methods We used immunohistochemistry to detect the expression characteristics of Smad3 and nuclear factor kappa B(NF-κB)proteins in human postnecrotizing enterocolitis stricture.We cultured IEC-6(crypt epithelial cells of rat small intestine)in vitro and inhibited the expression of Smad3 using siRNA technique.Quantitative PCR,western blotting,and ELISA were used to detect the changes in transforming growth factor-β1(TGF-β1),NF-κB,tumor necrosis factor-α(TNF-α),vascular endothelial growth factor(VEGF),and zonula occludens-1(ZO-1)messenger RNA(mRNA)and protein expressions in IEC-6 cells.CCK8 kit and Transwell cellular migration were used to detect cell proliferation and migration.Changes in epithelial-mesenchymal transition(EMT)markers(E-cadherin and vimentin)in IEC-6 cells were detected by immunofluorescence technique.Results The results showed that Smad3 protein and NF-κB protein were overexpressed in narrow intestinal tissues and that Smad3 protein expression was positively correlated with NF-κB protein expression.After inhibiting the expression of Smad3 in IEC-6 cells,the mRNA expressions of NF-κB,TGF-β1,ZO-1,and VEGF decreased,whereas the mRNA expression of TNF-αdid not significantly change.TGF-β1,NF-κB,and TNF-αprotein expressions in IEC-6 cells decreased,whereas ZO-1 and intracellular VEGF protein expressions increased.IEC-6 cell proliferation and migration capacity decreased.There was no significant change in protein expression levels of EMT markers E-cadherin and vimentin and also extracellular VEGF protein expression.Conclusions We suspect that the high expression of Smad3 protein in postnecrotizing enterocolitis stricture may promote the occurrence and development of secondary intestinal stenosis.The mechanism may be related to the regulation of TGF-β1,NF-κB,TNF-α,ZO-1,and VEGF mRNA and protein expression.This may also be related to the ability of Smad3 to promote epithelial cell proliferation and migration.