Study On The Mechanism Of Intestinal Glial Cells Regulating Intestinal Mucus Barrier Through MUC2

BackgroundUlcerative colitis(UC)is one of the most common types of inflammatory bowel disease(IBD),and its main clinical manifestations are abdominal pain,diarrhea,mucus,pus,and blood stool.In recent years,the incidence of UC has increased year by year,however its pathogenesis is still not fully understood.Increased intestinal mucus barrier permeability and bacterial migration may lead to the occurrence and progression of UC inflammation,and the decrease of muc-skeleton protein MUC2 may predate and contribute to the inflammation activation.Exploring the underlying mechanisms of UC midgut mucus barrier dysfunction may prevent relapse and prolong remission in patients with UC.Enteric glial cells(EGCs)can interact with various non-neuronal cells in the intestinal wall and are important local regulators of various intestinal functions.S-nitrosoglutathione(GSNO)secreted by EGCs can regulate intestinal barrier function and inflammatory response.GSNO expression level in the body by the secretion of EGCs nitroso glutathione reductase(S-nitrosoglutathionereductase,GSNOR)regulation,can be GSNOR degradation.GSNO is an endogenous NO donor,and NO can regulate the synthesis and secretion of MUC2 by gobletcells(GCs).Can EGCs affect GCs synthesis and secretion of MUC2 by regulating the generation of GSNO and cause intestinal mucus barrier abnormalities?In patients with UC,LPS can cross the intestinal barrier and be recognized by EGCs surface TLR4.In the central nervous system,astrocytes after TLR4 activation induce nitroso glutathione reductase(Snitrosoglutathionereductase,GSNOR)generation,and GSNO degradation increases.TLR4 is a major receptor for lipopolysaccharide(LPS),which can cross the intestinal barrier to contact EGCs in UC.Based on the TLR4 pathway of central glial cells,we speculated that intestinal LPS may activate the TLR4 signal on EGCs,initiate the synthesis of GSNOR,and increase the degradation of GSNO,which result in the reduction of the synthesis and secretion of MUC2 in goblet cells,the destruction of intestinal mucus barrier structure and the increase of permeability,and ultimately lead to the progression of inflammation in UC.This study included two parts to verify the above hypothesis.The first part was to probe the correlation between EGCs in the colonic mucosa of patients with active UC and the synthesis and secretion of MUC2 by goblet cells,and the second part was to determine the effect of LPS on EGCs and the signaling pathway through in vitro cell experiments,and then to discuss the influence of EGCs on the synthesis and secretion of MUC2 by goblet cells and the related mechanism.Aim1.To investigate the correlation between intestinal mucosal EGCs phenotype changes and goblet cell function changes in patients with active UC.2.To determine the effect of LPS on EGCs and its signaling pathway through in vitro cell experiments,and the effect of EGCs on MUC2 and its related mechanism through in vitro co-culture experiments.MethodsPart Ⅰ:Clinical studyIn this study,patients with active UC and normal controls were enrolled.The diagnostic criteria for patients with active UC were the consensus opinion of the inflammatory enterology group of the gastroenterology branch of the Chinese medical association on the diagnosis and treatment of inflammatory bowel disease(Beijing 2018).The disease activity was evaluated using the modified Mayo score,which is more suitable for scientific research,and the histological score was assessed using the Geboes index.Normal control group was those with colon polyp found by colonoscopy,but with normal colon mucosal histology and hematology results.Under colonoscopy,3 pieces of colon mucosa at the lesion of UC patient and rectal sigmoid junction of control patient were obtained.One paraffin section was stained with AB-PAS to observe the quantity and secretion of GCs.1 piece was used for immunohistochemical staining of MUC2 and GFAP,and the last one piece was used for protein extraction and detection of MUC2 protein expression level.The correlation between the expression level of GFAP and the changes of MUC2 was analyzed using GraphPadPrism9.0 software according to the immunohistochemical scoring method.Part two:Basic experiment1.After the EGCs was stimulated by LPS of different concentrations for 24h,westernblots assay was used to detect the expressions of GFAP,TLR4 and GSNOR,the immunofluorescence was used to detect the locations of GFAP and GSNOR,.2.The expression of TLR4 in EGCs was down-regulated by siRNA transfection,and the influence of TLR4 changes on GSNOR was detected by western blot and qRT-PCR.3.A EGCs-GCs co-culture model was established and western blot assay was used to detect the effect of LPs-activated EGCs on MUC2 expression,and NO content in the supernatant of cells was detected by NO kit;as well as the effect of down-regulating TLR4 in EGCs and inhibiting GSNOR in EGCs on MUC2 expression.4.After 24h co-culture of EGC and GCs,western blot assay was used to detect the expression of MUC2,and qRT-PCR assay was used to detect the expression of GSNOR,iNOS and GFAP in EGCs in basement chamber.ResultsPart I:The clinical studyThere were no significant differences in gender(48.39%vs 44%),age(47.50±13.06 vs 44.46±10.01)and body mass index(23.82±3.49 vs 22.71±3.89)between active UC patients and controls(P>0.05)..Interpatient score of active UC Mayo score[mean(SD)]was 6.61(3.43),Mayo endoscopy score[mean(SD)]was 2.03(0.71),and histology score[mean(SD)]was 4.10(0.23).AB-PAS staining showed colon mucosa damage,intestinal recess atrophy and distortion,reduced mucus secretion and number of cup cells in active UC patients(P<0.001).Westernblot analysis showed that the expression of MUC2 protein in the intestinal mucosa of UC patients was less than the control group(P<0.01).The formation of glial networks of EGCs in intestinal mucosa was observed in immunohistochemical staining,and the content of MUC2 in goblet cells was reduced.Quantitative analysis suggested that the expression level of GFAP in colon mucosa of active UC patients was significantly higher than that of control group(P<0.001),and the expression level of MUC2 was lower than that of control group(P<0.01).The high expression of GFAP was negatively correlated with the downregulation of MUC2(R2=0.1789,P<0.05).Part Two:Basic experiment1.LPS up-regulated the expression of GFAP,TLR4 and GSNOR proteins in glial cells,.mRNA and protein expression of GSNOR were decreased by down-regulating TLR4 in EGCs.2.LPS-activated EGCs inhibited the expression of MUC2 protein in co-culture system.However,the expression of MUC2 protein decreased after down-regulating TLR4 in EGCs or inhibiting GSNOR in EGCs.3.A EGCs-GCs co-culture model was established and western blot assay was used to detect the effect of LPs-activated EGCs on MUC2 expression,and decreased the content of NO in the supernatant of LPS stimulated EGCs cells;as well as the effect of down-regulating TLR4 in EGCs and inhibiting GSNOR in EGCs on MUC2 expression.4.EGCs co-cultured with GCs significantly up-regulated the expression of MUC2,iNOS mRNA in basal compartment EGCs significantly up-regulated,and mRNA expressions of GSNOR and GFAP decreased.ConclusionThe abnormal synthesis and secretion of MUC2 by goblet cells in active UC patients is related to the activation of intestinal glial cells.Lps-activated EGCs inhibit the synthesis and secretion of MUC2 by goblet cells,leading to the structural destruction and permeability increase of intestinal mucus barrier,which is related to the increased degradation of glia-derived GSNO caused by the activation of TLR4 signal on EGCs surface.In addition,glia-derived iNOS is found to be involved in the direct communication between EGCs and goblet cells.MeaningIn this study,the hypothesis was verified by clinical specimens and cell experiments in vitro,that is,intestinal LPS activates TLR4 signal on the surface of intestinal glial cells,initiates the synthesis of GSNOR,and induces the increase of GSNO degradation,which leads to the decrease of MUC2 synthesis and secretion of goblet cells,the destruction of intestinal mucus barrier structure and the increase of intestinal mucus barrier permeability,and finally leads to the occurrence and progress of inflammation in UC.It expands the role of EGCs in intestinal barrier and to some extent provides a new idea for exploring the mechanism of intestinal mucus barrier damage caused by the decrease of MUC2 in inflammatory intestine associated with UC.