Activation Of GABA_A Receptors In Colon Epithelium Exacerbates Colitis

Background and SignificanceUlcerative Colitis(UC),is one type of inflammatory bowel disease(IBD).This debilitating disease is becoming a worldwide medical concern,with increasing prevalence and incidence in industrialised countries.For the past 30 years,with the development of economy and the following changes in people’s life style,Chinese UC patients also increased dramatically.However,the exact aetiology of UC remains unknown,some scientist believed that genetic predisposition and various environmental and immunological causes would be contributing factors.gamma-aminobutyric acid(GABA)is a major inhibitory neurotransmitter in the adult mammalian brain.In recent years,GABAergic drugs are widely used for anesthesia induction and treating anxietyand alcohol addiction,GABA can also treat some autoimmune disease,such as type 1 diabetes(T1D),experimental autoimmune encephalomyelitis(EAE),collagen-induced arthritis(CIA)and allergic dermatitis.Thus,GABAergic system is believed to have the function of directly suppressing immune and inflammatory responses.Since UC has been characterized by nonspecific inflammation in the colon mucosa.we speculate that GABAergic system could play an important role in the occurrence and development of UC.Our research examined the expression and functions of GABA and GABA A Receptors in colonic nucosa of control and colitis colon.Our results showed that GABA ergic signal activation can aggravate the injury of mucosal barrier and aggravate UC.This finding provides a new perspective and important theoretical basis for the prevention and treatment of UC.At the same time,it has a guiding significance for the application of clinical GABAergic drugs.We also found GABA ergic signal activationcan affect the integrity of colonic epithelial barrier in normal mice.This finding suggested that it should be very carefiul to use GABA supplement food.Methods1.Animal model:Male C57BL/6 mice,8-10 weeks old,were housed under SPF conditions.Mice were divided randomly into 6 groups with 8 mice per group.Acute colitis was induced by administering 2.5%(wt/vol)DSS at a molecular weight of 36,000-50,OOODa(MP Biomedicals,Santa Ana,California,USA)in their drinking water for 7 days ad libitum and euthanized on day 7.The six groups consisted of:1)vehicle-treated controls(Control),2)DSS in drinking water(DSS),3)DSS mice receiving a daily i.p.injection of GABA(Sigma-Aldrich,St.Louis,MO,USA)at 5 mg/kg(DSS+GABA),4)DSS mice receiving a daily i.p.injection of muscimol(Tocris Bioscience,Ellisville,MO,USA)at 2 mg/kg(DSS+muscimol),5)DSS + GABA mice receiving a daily i.p.injection of(+)-bicuculine(Tocris Bioscience,Ellisville,MO,USA)at 5 mg/kg(DSS+GABA+bicuculline)and 6)DSS mice receiving a daily i.p.injection of(+)-bicuculine(DSS+bicuculline).Mice were monitored daily for weight loss,stool consistency,and fecal bleeding.2.Cell model:HT-29 and Human colon carcinoma cell line(Caco-2)was obtained from the Korea Cell Line Bank(Seoul,Korea).Cells were grown at 37 ℃ in DMEM supplemented with 10%FBS,penicillin and streptomycin in a humidified atmosphere of 5%C02.Fully differentiated cell monolayers were then incubated with or without 2%DSS in the absence or presence of GABA(100 μm),muscimol(10 μM)or bicuculline(10 μM)for 48 h.3.Animal model detection:Immunofluorescence staining was used to examine the expression of GAD65/67and GABA A Recepter β2/3 in colon of both DSS-inuced mice model and UC patients;FITC-Drxtran gavage,ussing chamber recording and bacterial culturewere used to analyze the effect of GABA on colonic permeability.Electron microscopy was used to observe the morphology of epithelial tight junction.Immunohistochemistry staining and immunofluorescence were used to analyze the expression of JAM-1,Occludin,MUC-2,ki-67 and cleaved-caspase-3 in both normal and DSS-induced mice colitis colon.AB-PAS was used to analyze the expression of acidic mucin.QPCR were used to analyze the mRNA expression of MUC-2,Notchl,Hesl and Mathl.4.Cell model of detection:Immunofluorescence and Western Blotting were used to analyze the effect of GABAergic signal system to the JAM-1 protein experssion of Caco-2 cells.QPCR were used to analyze the effect of GABAergic signal system to the mRNA expression of MUC-1,MUC-2,MUC-5B and MUC-5AC in HT-29 cells.Results1.GABA A Recepter β2/3 and GAD65/67 were expressed in the colonic epithelium of nomal and UC mouse colon,GAD65/67 mainly expressed in colonic crypt cells,GABA A Recepter β2/3 expressed in both colonic crypt and mature cells of top crypt.GABA A Receptor β2/3 and GAD65/67 were overexpressed in colonic epithelium of UC patient and UC mouse.2.After peritoneal injection of GABA and muscimol,weight loss,colon length,colon damage was examined.the results showed that the DAI score increased and more inflammatory cell invasion into the colon mucosa in UC mice,GABA and Muscimol also increased colon permeability and bacterial invasion.The expression of JAM-1 and occludin,MUC-2,K1-67 decreased seriously in UC mice.the expression of glucogen and MUC-2 protein were reduced;GABA or muscimol administration also increased the number of Cleaved-Caspase-3 positive cell numbers;Peritoneal injection of GABA antagonist inhibited the effects of GABA and muscimol.3.In Cell model,after incubation of GABA or muscimol for 48h,The JAM-1 mRNA and protein expression in Caco-2 cells decreased;In HT-29 cell model,by comparison of control group,exogenous addition of GABA or muscimol decreased the mRNA expression of MUC-1,MUC-2,MUC-5AC,MUC-5B and incubation with Bicuculline block these effects of GABA and muscimol.4.After administration of GABA or muscimol to normol mouse,the colon permeability and bacterial invasion increased.The protein expression of JAM-1 and Occludin increased,GABA or muscimol also reduced the number of glucogen and MUC-2 protein.The positive cells of KI-67 was reduced.Peritoneal injection of bicuculline and or co-administration of GABA and bicuculline increased both the MUC-2 and increased KI-67 positive cell numbers in colon epithelial.5.Compared with the control group,administration of GABA or muscimol decreased the mRNA expression of MUC-2 and Mathl,increased the the mRNA expression of Notch1 and Hesl in normal mouse colon and administration of Bicuculline Reversed these changes.Conclusion1.The GABAergic signaling system were expressed in epithelium in normal colon and upregulated in epithelium in UC mouse and patients’ colon.2.GABA A Receptors activation can interupt the colonic mucosal barrier and aggravate ulcerative colitis.3.GABA A Receptors activation can inhibit the proliferation and induce the apoptosis of epithelial cells thus,lead to the decrement of goblet cells and reduction of tight junctions and which further destroyed the mucosal barrier in ulcerative colitis.4.Notch pathway is partly responsible to the decrement of goblet cell after the GABA or muscimol administration in normal mice.