Abstract
To investigate the expression of Smad3 (mothers against decapentaplegic homolog 3) protein in postnecrotizing enterocolitis stricture and its possible mechanism of action. 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. 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-α did not significantly change. TGF-β1, NF-κB, and TNF-α protein 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. 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.
Highlights
What is already known about this subject? ► The most common complication after conservative treatment in patients with necrotizing enterocolitis (NEC) is intestinal stenosis. ► The mechanism of intestinal stenosis may be related to intestinal tissue ischemia, inflammation, fibrous hyperplasia, and so on. ► Activation of transforming growth factor-β1 (TGF-β1)/Smad[3] signaling pathway is an important mechanism in promoting intestinal fibrosis. ► Smad[3] is the most direct effector protein in intestinal fibrosis
What are the new findings? ► We found that intestinal fibrosis was the primary manifestation of intestinal stenosis secondary to NEC. ► Smad[3] protein is overexpressed in intestinal stenosis secondary to NEC. ► There was a synergistic relationship between Smad[3] protein expression and collagen fiber expression in intestinal stenosis secondary to NEC. ► Smad[3] protein can regulate the expression of TGF-β1, nuclear factor kappa B, tumor necrosis factor-α, zonula occludens-1, and vascular endothelial growth factor in intestinal epithelial cells. ► Smad[3] protein can promote intestinal epithelial cell proliferation and migration
We examined the effects of Smad[3] on the proliferation and migration of intestinal epithelial cells, on the expression of tight junction protein zonula occludens-1 (ZO-1), and on epithelial–mesenchymal transition (EMT) to explore the mechanism of Smad[3] affecting postnecrotizing enterocolitis stricture
Summary
Correspondence to Neonatal necrotizing enterocolitis (NEC) is Key messagesWhat is already known about this subject? ► The most common complication after conservative treatment in patients with necrotizing enterocolitis (NEC) is intestinal stenosis. ► The mechanism of intestinal stenosis may be related to intestinal tissue ischemia, inflammation, fibrous hyperplasia, and so on. ► Activation of TGF-β1/Smad[3] (transforming growth factor-β1/mothers against decapentaplegic homolog 3) signaling pathway is an important mechanism in promoting intestinal fibrosis. ► Smad[3] is the most direct effector protein in intestinal fibrosis.What are the new findings? ► We found that intestinal fibrosis was the primary manifestation of intestinal stenosis secondary to NEC. ► Smad[3] protein is overexpressed in intestinal stenosis secondary to NEC. ► There was a synergistic relationship between Smad[3] protein expression and collagen fiber expression in intestinal stenosis secondary to NEC. ► Smad[3] protein can regulate the expression of TGF-β1, nuclear factor kappa B, tumor necrosis factor-α, zonula occludens-1 , and vascular endothelial growth factor in intestinal epithelial cells. ► Smad[3] protein can promote intestinal epithelial cell proliferation and migration.How might it impact on clinical practice in the foreseeable future? ► Exploring Smad[3] inhibitors may help prevent intestinal fibrosis and may reduce the length of the narrow bowel.main cause of death in premature infants. ► Smad[3] protein can promote intestinal epithelial cell proliferation and migration. ► We found that intestinal fibrosis was the primary manifestation of intestinal stenosis secondary to NEC. ► Smad[3] protein is overexpressed in intestinal stenosis secondary to NEC. ► There was a synergistic relationship between Smad[3] protein expression and collagen fiber expression in intestinal stenosis secondary to NEC. How might it impact on clinical practice in the foreseeable future?
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