The Study Of Fibronectin And Collagen In The Pathogenesis Of Hirschsprung Disease

Hirschsprung disease(HSCR)is the most common congenital gut motility disorder,with an incidence of one in 5000 human births,and shows a male to female ratio of 4:1.It is thought to arise from a failure of colonization of the distal gut by enteric nervous system(ENS)precursors during embryonic development.Ultimately,the disease usually results in neonatal bowel obstruction with delayed passage of meconium and bile-stained vomitus,but patients may also present later with intractable constipation or Hirschsprung-associated enterocolitis(HAEC).Although surgical management is available,constipation,fecal incontinence and HAEC are common even after pull through surgery in childhood.The "gene mutation theory" represented by RET gene mutations is one of the pathogenesis of HSCR that is widely recognized.RET gene is the major gene for HSCR,whose mutations are responsible for 40-60%of familial cases and 7-25%of sporadic cases.Other variants involved in the development of ENS account for approximately 30%of patients with sporadic cases.Therefore,the "gene mutation theory" can no longer fully cover the pathogenesis of HSCR,and discussion of the pathogenesis of HSCR is of great significance for searching exploration of other treatment options.There is increasing evidence that the occurrence and development of HSCR involve complex interactions between an unbalanced composition of the extracellular matrix(ECM)and surrounding dysfunctional enteric neural crest cells(ENCCs),which is known as "intestinal microenvironment" theory.The ECM within the cell microenvironment serves not only as a structural skeleton for cells,but also as a source of three-dimensional(3D)biochemical and biophysical cues that trigger and regulate cell behaviors.During the embryonic period,vagal neural crest cells enter the foregut dorsolateral from the neural tube and then migrate in a rostral-caudal direction to colonize the full length of gut.Defects in the migration of neural crest-derived cells in humans result in the complete absence of the ENS in the distal region of the gastrointestinal tract,resulting in an aganglionic segment.The earlier the arrest of migration,the longer the aganglionic segment.This migration process of ENCCs is closely related to the intestinal microenvironment.ECM dysfunction has been widely reported in central nervous system(CNS)diseases that affect cognition.For example,some patients with Alzheimer's disease(AD),Parkinson's disease(PD)or Huntington's disease(HD)also show HSCR.Colorectal aganglionosis in endothelin-3(ET3)and the distal aganglionosis in endothelin receptor B(EDNRB)mutant mice have been attributed to an accumulation of laminin in the aganglionic mesenchyme,leading to premature neuronal differentiation and thus arrested migration and distal aganglionosis.Deletion of ?1 integrin from ENCCs can lead to their impaired migration,abnormal aggregation,and colonic aganglionosis.While the intestinal ECM regulates ENS development,ENCCs in turn affect ECM composition.For example,during the development of ENS,ENCCs can secrete matrix metalloproteases(MMP)and tenascin C to promote their migration.In summary,ENCCs can actively remodeling their surrounding ECM environment during the migration,and these environmental changes have in turn regulated the development of ENCCs.Fibronectin(FN)plays crucial roles in modulating the neural crest cell response and in vascular morphogenesis as a key ECM glycoprotein.Neuroligin-1(NL-1)and neuroligin-2(NL-2)are members of the neuroligin family of postsynaptic adhesion molecules,regulating the formation of excitatory and inhibitory synapses,respectively.We have found a temporal trend in the abnormal expression of FN,neuroligin-1(NL1)and neuroligin-2(NL2)in the diseased colons of HSCR children.However,the real interaction between FN and NLs is not clear.Collagens are the main components of ECM and are the most abundant proteins in mammals,provides support for cell growth,and is responsible for the mechanical resilience of connective tissues.Among them,Collagen I(Col ?)is one of the most abundant ECM proteins,and can form fibers together with collagen ?(Col ?).Collagen ?(Col ?)is a major component of the basal lamina,and plays a key role in maintaining epithelial integrity,gut morphology and intestinal function.Although lots of studies were reported about collagens and ENS in the pathogenesis of HSCR,there is no research involved the expression of Col ?,?and ? in the diseased colons from HSCR children.This project breaks through the limitation of "gene mutation theory" to analyze the pathogenesis of HSCR.Starting from the effect of mutual regulation between ECM and ENCCs on the pathogenesis of HSCR,we hope to further supplement the theory of"intestinal microenvironment" theory.This project is divided into two parts,one is to study the interaction between FN and colonic synapses in HSCR children,the other is to investigate the expression of Col ?,? and ? in the diseased colons in HSCR children,aiming to provide novel theories for the pathogenesis of HSCR.Part one:Fibronectin impairs the function of excitatory/inhibitory synapses in HSCRAim1.To investigate the distribution and functional changes of FN and synapses among ganglionic,transitional and aganglionic segments of the colon tissue of HSCR patients,the regulatory mechanism of FN on excitatory and inhibitory synapses,and clarify the role of FN in the occurrence and development of HSCR.2.To investigate the expression of FN and NLs in the intestinal canal of embryonic rats of different gestational ages,and analyze the correlation between their expression levels and the gestational ages of embryonic mice.3.To investigate the interaction between FN and NLs at the cellular level,providing a new perspective for the occurrence and development of HSCR.Method1.Expression of FN and excitatory/inhibitory synaptic markers in the three parts of colonic ganglionic,transitional and aganglionic segment of HSCR patients.The colon tissues of the ganglionic,transitional and aganglionic segments of HSCR patients were taken to detect the expression levels of mRNA and protein of FN and synapse-related markers by real-time fluorescent quantitative PCR(qRT-PCR)and Western blot(WB).Immunohistochemistry(IHC)methods were used to detect the expression site,protein expression intensity and the proportion of positive staining area of FN and synapse-related markers among the three parts of colon tissue.2.Culture PC12 cells in vitro,knock down FN gene expression,and detect the expression of synapse-related markers.PC 12 cells were transfected with FN-siRNA and negative control NC-siRNA,qRT-PCR,WB and immunofluorescence(IF)methods were used to detect the expression levels of excitatory synaptic markers NL1,PSD-95,vGLUT1,GluA1,GluN1 and inhibitory synaptic markers NL2,SLC32,GABA AR-al,GAD67.3.In vitro culture of embryonic mouse colon tissue in medium containing high concentration of FN to detect the expression of colon synaptic markers.The end colons of the embryos of thirty healthy Wistar rats in embryonic period of 20 days(E20)were placed into completely medium with 20 ?g/mL FN functional domain amino acid fragments(Arginine-glycine-aspartic acid,RGD)for 0,0.5,1,2,4,6,8,and 12 h.WB and IHC staining analysis were used to analyze the protein expression level of excitatory synaptic marker PSD-95 and inhibitory synaptic marker SLC32 of colonic myenteric plexus.4.To investigate the expression of FN and NLs in ENS of embryos at different gestational ages,and analyze their correlation with the gestational age of embryos respectively.The distal colon tissues of embryonic rats at 16 days(E16),18 days(E18)and 20 days(E20)and suckling rats(Ep0)within 24 h after birth were collected,their proteins and mRNA expressions of FN,NL1 and NL2 at the four time periods were analyzed by WB and qRT-PCR.In addition,the correlation between their protein expression levels and the gestational age of embryonic mice were analyzed.5.Culture PC12 cells in vitro,knock down NL1 and NL2 gene expression,and detect the expression of FN.PC 12 cells were transfected with NL1-siRNA,NL2-siRNA,NL1+NL2-siRNA and negative control NC-siRNA,respectively.qRT-PCR and WB methods were used to detect mRNA and protein expression levels of FN under different conditions.Result1.The levels of excitatory/suppressive synaptic marker genes and proteins in ganglionic,transitional and aganglionic segment of HSCR patients were significantly increased,while those of FN were sharply decreased.The results of qRT-PCR and WB showed that the mRNA and protein expression levels of excitatory synapse-related markers(NL1,PSD-95,vGLUT1,GLuN1,GLuAl)and inhibitory synapse-related markers(NL2,SLC32,GABA AR-?1 and GAD67)in the three parts of the ganglionic,transitional and aganglionic segment of HSCR patients gradually increased,while the expression level of FN gradually decreased.The results of IHC staining demonstrated that the immune response intensity of PSD-95 and SLC32 gradually decreased from aganglionic,transition to ganglionic segment,and the FN immune response intensity was the opposite.The proportion of positive staining area indicated that the proportion of synapses gradually decreased from aganglionic,transition to ganglionic segment,while the proportion of FN gradually increased.2.Inhibition of FN expression can promote the expression of synaptic markers in PC12 cells.qRT-PCR,WB and IF results showed that the mRNA and protein levels of NL1,NL2,PSD-95,vGLUT1 and SLC32 in the cells were significantly higher than those in the negative control group after knocking down the expression of FN in PC12 cells.3.FN significantly inhibited the expression level of synaptic markers in colonic myenteric plexus.Thirty E20 embryonic colon tissues were cultured in vitro in complete medium containing 20?g/mL RGD for 0,0.5,1,2,4,6,8 and 12 h,respectively.WB results showed that the protein expression levels of PSD-95 and SLC32 were the lowest at 6 h.Then,the embryonic colon tissues stimulated by RGD for 0 h and 6 h were collected as the control group and experimental group for IHC staining analysis.The results showed that the expression levels of PSD-95 and SLC32 in the colon tissue cultured at 6 h were significantly reduced compared to the colon tissue cultured at 0 h.4.With the development of ENS,the expression of FN in the embryonic colons sharply decreased,while the expression of NLs greatly increased.The expression of FN and NLs was closely related to the gestational age.The results of WB and qRT-PCR showed that the mRNA and protein expression of NL1 and NL2 in the embryonic colons gradually increased and the expression of FN gradually decreased with the development of ENS.Correlation analysis results indicated the expression of FN is negatively correlated with ENS development,while the expression of NL1/NL2 is positively correlated with ENS development.5.FN and NLs are inversely regulated by each other in PC 12 cells.After knocking down the expressions of NL1 and NL2 in PC 12 cells,qRT-PCR and WB results showed that the mRNA and protein levels of FN increased,which combined with the relevant test results of knocking down FN indicated that FN and NLs are mutually reverse regulated.After simultaneously knocking down the expression of NL1 and NL2 in PC 12 cells,there was no obvious difference of the intracellular FN protein expression level compared to those of knocking down NL1 or NL2,which indicated that NL1 and NL2 had no combined inhibitory effect on FNConclusion1.The expression of excitatory/inhibitory synaptic markers gradually increased among aganglionic,transitional and ganglionic segments in HSCR patients,while the expression levels of FN gradually decreased.2.Knocking down the expression of FN can promote the expression of synaptic markers in PC 12 cells.3.Abnormally high expression of FN can significantly reduce the expression of synaptic markers in the colonic myenteric plexus.FN in the medium can significantly inhibit the expression level of synaptic markers of intermuscular plexus in vitro culture of fetal rat colon tissue.4.With the development of ENS,the expression of FN in the embryonic colons sharply decreased,while the expression of NLs significantly increased.The protein expression of FN and NLs were closely related to the gestational ages.5.In PC 12 cells,FN and NLs are mutually reverse regulated,and NL1 and NL2 have no combined inhibitory effect on FN.Part two:The expression patterns of Col ?,? and ? in HSCRAimTo investigate the abnormal expression of collagen ?,? and ? in the diseased colons of children with HSCR.MethodsThe expression of collagen ?,? and ? in HSCR.30 colon specimens of children diagnosed by postoperative pathology were randomly selected as the study objects,and the protein and mRNA expressions of collagen ?(Col ?),Collagen ?(Col ?)and Collagen IV(Col IV)in ganglionic,transitional and aganglionic segments were analyzed by WB and qRT-PCR.IF double staining analysis of the samples visually showed the positional relationship and expression of Col ?,Col ? and Col ? with the intermuscular ganglia.ResultsThe expression of Col ? and ? gradually decreased,while the expression of Col ? gradually increased among ganglionic,transitional and aganglionic segments of HSCR children.WB and qRT-PCR results showed that the protein and mRNA expression levels of Col ? and Col ? were the highest in ganglionic segments,followed by the transitional segments,and the lowest in aganglionic segments,while Col ? presented a completely opposite expression.Double staining by IF showed that Col ? and ? were mainly expressed in and around the intermuscular ganglion,and the intensity of immunoreactivity gradually increased from the narrow segment to the dilated segment.The position of Col ? was the same as that of Col ? and ?,but the intensity of its immune response gradually decreases from the dilated segment to the narrow segment.Conclusion1.Col ?,? and ? abnormalities in the aganglionic bowel are associated with HSCR.2.In muscle strips prepared from colonic tissues of human patients with HSCR,the locations of Col ?,? and ? are both around and within myenteric ganglia.