The Role And Its Mechanism Of ENS-SIP Syncytium In Colonic Dysmotility Of Hirschsprung’s Disease

Normal gastrointestinal(GI)tract motility is regulated by enteric nervous system(ENS)and interstitial cells in smooth muscle layer.The interstitial cells of GI tract include interstitial cells of Cajal(ICCs)and platelet-derived growth factor receptor alpha positive(PDGFRα~+)cells,which form gap junctions with smooth muscle cells respectively,and futher form a functional syncytium similar to motor unit together,SIP syncytium.Neurotransmitters released by ENS mainly act on ICCs and PDGFRα~+cells in SIP syncytium,and then affect smooth muscle movement through gap junction.The aim of this study was to observe the distribution and effect of ICCs and PDGFRα~+cells in ENS and SIP syncytium in colonic smooth muscle of children with Hirschsprung’s disease(HSCR)and animal models,and to explore the mechanism of colonic dysmotility in HSCR.Thirty-one isolated colon specimens of HSCR children admitted to Xinhua Hospital from April 2017 to October 2018 were collected.The contraction of smooth muscle in narrow segment,dilated segment and proximal margin was recorded by contractile experiments,and treated with various ion channel agonists and antagonists.Real-time fluorescence quantitative PCR(q RT-PCR)and Western blot analysis were used to detect the expression of c-Kit,PDGFRA,ANO1 and SK3 in different segments.Then we established the partial intestinal obstruction(PCO)mice model.Western blot and fluorescence immunohistochemical staining were used to observe the expression of c-Kit,PDGFRα,ANO1 and SK3.The colonic dysmotility in PCO mice was verified by colonic migrating motor complexes(CMMCs)and spontaneous smooth muscle contraction.Firstly,the response of smooth muscle to electric field stimulation(EFS)in the narrow/dilation and proximal colon was observed in HSCR colon.The results showed that there was an inhibitory response followed by excitatory response in the dilated and proximal smooth muscle segments,while the contractile excitability of the narrow smooth muscle segments was significantly increased.Secondly,the effects of TTX,NOS inhibitors,ANO1 channel inhibitors,SK3 channel agonists and antagonists on the contraction of smooth muscle in 3 segments of the HSCR colon were observed.The results showed that the above drugs had the weakest effect in the narrow segment.Finally,the distribution of ICCs and PDGFRα~+cells in SIP syncytium of HSCR colon was detected by molecular biology.The results showed that the protein and m RNA expression of c-Kit,ANO1,PDGFRα,SK3 in the narrow segment was relative lower.In PCO mice,the contraction of colonic smooth muscle and CMMCs were abnormal.The effects of PCO on NOS inhibitors and ANO1 channel inhibitors were significantly weakened,whereas the responses to SK3 channel agonists and antagonists were significantly enhanced.Molecular biology experiments showed that ICCs and ANO1 channels in SIP syncytium were significantly downregulated and PDGFRα~+cells and SK3 channels were significantly upregulated.The above results suggest that:The excitability of cholinergic nerve increased significantly in the narrow segment of colon of HSCR children,while the inhibitory nitric oxide nerve response almost disappeared completely.The function of ICCs-ANO1-SMCs and PDGFRa-SK3-SMCs axes in the narrow segment were significantly decreased.In PCO mice,the function of ICCs and ANO1 channels was downregulated while that of PDGFRα~+cells SK3 channels was upregulated.Although SIP syncytium changed differently in HSCR patients or PCO mice,the relative balance of ICCs-ANO1-SMCs and PDGFRα~+cells-SK3-SMCs pathways plays an important role in the regulation of colonic motility.