Preliminary Study On The Molecular Mechanism Of Abnormal Contraction In The Aganglionic Gut Of Hirschsprung’s Disease

Hirschsprung’s disease（HSCR）,a common cause of neonatal intestinal obstruction,is a challenging condition for surgeons to treat,and remains an enigma,albeit a partially solved one to scientists.Pathological changes is aganglionosis in the myenteric plexus and submucosal plexus,meanwhile the parasympathetic nerve fibers increased and thick,which caused by abnormal embryonic development of the enteric nervous system.The cause of abnormal embryonic development of ENS dysplasia is very complex,many factors involved,but the specific mechanism is not clear at present.The surgical treatment is the mainstream,also had used Gut Neural Crest Stem Cells（GNCSCs）transplanted to mice,at present.In Clinical feature we foundthat the aganglionosis gut was spasm,stiffness in Hirschsprung’s disease,which caused the gut functional obstruction,which in turns results in severe chronic constipation and abdominal distention.Intestinal smooth muscle contraction maybe lead to luminal pressure change of the aganglionosis gut in Hirschsprung’s disease,except the basic pathological changes,but we don’t know the mechanism.Studies had revealed that 20 kDa myosin light chain（MLC20）and its regulatory proteins myosin light chain phosphatase target subunit 1（MYPT1）,myosin light chain phosphatase catalytic subunit（PP1c）and PKC-related the phosphorylation of protein-17kDa（CPI-17）are associated with intestinal smooth muscle contraction.This study aimed to assess the expression and phosphorylation levels of MLC20,MYPT1,PP1c and CPI-17 in the normal ganglionic（N）,oligoganglionic（O）and aganglionic（A）intestinal tract tissue samples from HSCR patients.In the present study,a retrospective study was performed to discusses the HSCR gut smooth muscle abnormal changes,from the clinical point of view.At the same time,we collected the intestinal tract tissue samples from HSCR patients that pathologically diagnosed.Immunohistochemistry was performed to observed the expression of MLC20and MYPT1 in N,O and A intestinal smooth muscle tissue from HSCR patients.Moreover,Western-blot was also performed to observed the expression of MLC20 and MYPT1 in the N,O and A intestinal smooth muscle tissue from HSCR patients.Based on the of research ofMLC20 and MYPT1,we used Immunohistochemistry and Western-blot performed to observed the expression of PP1c and CPI-17 in the N,O and A intestinal smooth muscle tissue from HSCR patients.Western-blot was also performed to observed the expression of MLC20、MYPT1、PP1c and CPI-17 in the control group and the A group of HSCR.Correlation analysis in the Westem-blot gray level data of the phosphorylated and nophosphorylated of MLC20,MYPT1,PP1c and CPI-17 in three groups of HSCR,as well as.First of all,we found MYPT1 and p-MYPT1 downregulated at the stenotic segment in HSCR patients,then,we indicated p-CPI-17 was upregulated,and PP1c was downregulated.Changes of p-MYPT1 and p-CPI17 levels might caused p-MLC20upregulation and these abnormal changes might directly participate in abnormal intestinal smooth muscle contraction in HSCR patients,which will thus provide a basis for further understanding of the spasm,stiffness aganglionosis gut.Part one:Clinical research of the gut smooth muscle change in Hirschsprung’s diseaseObjective A retrospective study was performed to discusses the HSCR gut smooth muscle abnormal changes,from the clinical point of view.Methods Forty-three neonatal patients（22 males and 9 females;21 and 10 cases of short and long-segment types,respectively）pathologically diagnosed with HSCR.To analysis and summary the imaging characteristics,anorectal pressure measurement,digital rectal examination,operation data and postoperative pathological.Results Abdominal vertical slice:38 cases of low ileus,5 cases of unobvious low ileus.Barium enema radiography,the neonatal period has the typical imaging features,short groupof 29 cases,14 cases of long group.Anorectal pressure measurement:43 cases were not elicited rectal inhibitory reflex,19 cases had abnormal reflection.Rectal resting pressure:HSCR group:7.481±0.299 cmH₂O,control group:3.169±0.269 cmH₂O（p=0.043,P<0.05）;short7.086±0.373 cmH₂O;long 7.672±0.499 cmH₂O（P=0.365,P>0.05）.Digital rectal examination:the anus closemouthed and the rectal constriction was obvious.In intraoperative descriptionclearly distinguished normal ganglionic,oligoganglionic and aganglionic segment,aganglionic segment was spasm,abnormal peristalsis and stiffness.Myenteric plexus and submucosal plexus did not see the ganglion cells,hyperplasia of nerve plexus,nerve fiber hyperplasia and the gut smooth muscle enlargement,thickening,disorder in Postoperative pathological HE of aganglionic segment of HSCR.Conclusions Abnormal changes in smooth muscle of aganglionic segment in HSCR from the postoperative pathological.Part two:MYPT1 and MLC20 expression changes and its significance in Hirschsprung’s diseaseObjective Retrospective study found HSCR gut smooth muscle with abnormal changed,we speculated that the abnormal changes had abnormal protein expression in regulation of contraction.but we don’t known wether had changed in regulate proteins.It is known that 20 kDa myosin light chain（MLC20）and its regulatory proteins MYPT1 are associated with intestinal smooth muscle construction.This study aimed to assess the expression and phosphorylation levels of MYPT1 and MLC20 proteins in intestinal tract tissue samples from HSCR patients,aim to preliminary explanat the abnormal contraction from molecular level.Methods Thirty-one pediatric patients（22males and 9 females;21 and 10 cases of common and long-segment types,respectively）pathologically diagnosed with HSCR were divided into normal ganglionic（N）,oligoganglionic（O）and aganglionic（A）groups.Western blot and immunohistochemistry（S-P method）were used to assess protein amounts of MYPT1 and MLC20 at the stenotic segment of smooth muscles in HSCR patients.Immunohistochemistry was used to detect these proteins in intestinal smooth muscles.Western blot was used to detect the MYPT1and MLC20 exspresion between control group and A group of HSCR.Used correlation analysis study the index of Western blot-gray data between the three groups.Results Western blot showed higher p-MLC20（P=0.0345,P<0.05）levels and lower MLC20（P=0.0413,P<0.05）amounts in HSCR group A compared with group N;meanwhile,MYPT1（P=0.0297,P<0.05）and p-MYPT1（P=0.0185,P<0.05）levels were reduced.Immunohistochemistry showed MLC20（P=0.0592,P>0.05）no statistical differences in HSCR group A compared with group N;meanwhile,MYPT1（P=0.0312,P<0.05）levels were reduced.Western blot analyzed the comparison results between the control group with group A of HSCR and group A and N in HSCR.We had revealed the line of correlation analysis between three groups:p-MLC20 and p-MYPT1 showed had negative correlation between group A of HSCR（p=0.047,correlation index﹣0.428）.Conclusions p-MLC20 was upregulated,with MYPT1 and p-MYPT1 downregulated at the stenotic segment in HSCR.Changes of p-MYPT1 levels might caused p-MLC20 upregulation,and these abnormal changes might directly participate in abnormal intestinal smooth muscle contraction in HSCR.Part three:Preliminary study on the regulatory abnormal contraction mechanism of CPI-17 and PP1c cooperation with MYPT1 in the aganglionic gut of Hirschsprung’s diseaseObjective We had found that MYPT1 and MLC20 levels were changed in the stenotic segment of HSCR,we don’t known other regulate proteins if participate in smooth muscle contraction.PP1c and CPI-17 was also involved in the regulatory pathways of smooth muscle.This study aimed to assess the expression levels of PP1c and CPI-17 proteins in intestinal tract tissue samples from HSCR patients,in order to recearch if they cooperate with MYPT1 regulate the abnormal contraction mechanism in the aganglionic gut of Hirschsprung’s disease.Methods Thirty-one pediatric patients（22males and 9 females;21 and 10 cases of common and long-segment types,respectively）pathologically diagnosed with HSCR were divided into N,O and A groups.Western blot and immunohistochemistry（S-P method）were used to assess protein amounts of CPI-17and PP1c at the stenotic segment of smooth muscles in HSCR patients.Immunohistochemistry was used to detect these proteins in intestinal smooth muscles.Western blot was used to detect the CPI-17 and PP1c exspresion between control group and A group of HSCR.Used correlation analysis to study MYPT1,MLC20,PP1c and CPI-17 index of Western blot-gray data between the three groups.Results Western blot showed higher levels p-CPI-17（P=0.0254,P<0.05）and lower CPI-17（P=0.0489,P<0.05）amounts in HSCR group A compared with group N;meanwhile,PP1c（P=0.871,P>0.05）levels were no statistical differences.Immunohistochemistry showed higher levels CPI-17（P=0.0329,P<0.05）in HSCR group A compared with group N;meanwhile,PP1c（P=0.0338,P<0.05）levels were reduced.We had revealed the line of correlation analysis between the three groups:p-MLC20 and p-CPI-17showed hadpositive correlation between group A of HSCR(P=0.0489,correlation index 0.425,but p-MLC20and PP1c didn’t have correlation（P=0.253,P>0.05）.Conclusions p-CPI-17 was upregulated,with PP1c downregulated at the stenotic segment in HSCR patients.Changes of p-CPI-17 and PP1c levels ultimately caused p-MLC20 upregulation,cooperate with p-MYPT1 lead to p-MLC20 changed,and these abnormal changes might directly participate in abnormal intestinal smooth muscle contraction in HSCR patients.