The Effect Of GC-C Signaling Pathway On Intestinal Barrier And Inflammation In Ulcerative Colitis

Ulcerative colitis(UC)belongs to a group of chronic idiopathic inflammatory disorders that primarily affects the colon and is a major type of inflammatory bowel disease(IBD).Common clinical symptoms in UC are relapsing abdominal pain,diarrhea and mucous bloody stool.This disease significantly impacts patient quality of life and takes much medical costs due to its repeated remissions,relapses,and high risk of colon cancer.There is an increasing incidence of IBD in China,particularly of UC in recent years.The etiology of IBD is still not well established.Although treatment outcome for IBD has improved with the use of anti-TNFα agents such as infliximab,numbers of patients require colectomy as a result of severe disease that is resistant to current pharmacological therapy.Therefore,a continuous search for more specific etiological factors and the identification of novel pharmacological targets are needed.The structural basis of intestinal mucosal mechanical barrier is tight junction between intestinal epithelial cells(IECs)and the major constituent is tight junction proteins(TJPs).The disruption of mucosal barrier lead to increased intestinal permeability,which contribute the initiation and development of intestinal inflammation.In recent years,several studies have suggested that guanylate cyclase-C(GC-C)signaling pathway regulates epithelial barrier function and intestinal inflammation.GC-C is a transmembrane receptor that is expressed primarily on IECs.The peptides guanylin(Gn)and uroguanylin(Ugn)are the endogenous ligands for GC-C,and are highly expressed in the gastrointestinal epithelium.GC-C is also the receptor for heat-stable enterotoxins(STa)produced by enterotoxigenic Escherichia coli.The binding of these ligands(Gn/Ugn/STa)to GC-C results in the activation of GC-C signaling pathway by the increase of cyclic guanosine monophosphate(cGMP)in cell.This physiological activation of GC-C regulates cellular ion concentration inside and outside as well as intestinal fluid and electrolyte homeostasis.However,the effect of GC-C signaling pathway on the intestinal inflammatory injury is still unclear.In our study,we found that the expression levels of GC-C,Gn and Ugn in colonic mucosa of UC patients were significantly decreased compared with the nornal controls and this decrease was more significant with the increase of disease activity firstly,which suggests that the activity of GC-C signaling is reduced in UC patients and negatively correlated with the clinical severity of UC.The GC-C signaling pathway may be implicated in the genesis and progression of UC.On the basis of the above findings,we used the barrier model of Caco-2 monolayers and tested the crucial mediators of the GC-C signaling pathway(GC-C、Gn、Ugn、cGMP),the TJPs(occludin、claudin-1、ZO-1),and the pro-inflammatory cytokines(IL-8、TNF-α),to explore the role of GC-C signaling pathway in intestinal epithelial barrier and inflammatory injury.We found that IL-1β-treated cells had significantly reduced levels of GC-C、Gn、Ugn、cGMP、claudin-1、ZO-1、cell viability、SOD activity and increased levels of IL-8 and TNF-α、IL-1β-treated cells transfected with Gn overexpression vector had significantly increased levels of cell viability,SOD activity,Gn,GC-C,cGMP,claudin-1 and ZO-1 as well as decreased levels of permeability,IL-8 and TNF-α.Conversely,GC-C-silencing cells had more significantly decreased levels of cell viability,SOD activity,claudin-1 and ZO-1 as well as increased levels of perneability,IL-8 and TNF-a induced by IL-1β.These findings suggest that the dormant GC-C signaling pathway can be restored by the agonists of GC-C.The activation of GC-C signaling pathway can ameliorate the impaired barrier function and inflammatory injury of Caco-2 cells induced by IL-1β.Finally,we performed the experiment in vivo using DSS-induced UC model on mice.In our study,after injection with the lentiviral of Gn overexpression in UC mice,the appearance,feeding and activity of mice were better,weights were increased,the frequency of loose stools and bloody stools was decreased,intestinal permeability and histologic inflammatory score were reduced.The expressions of GC-C、Gn、Ugn,cGMP,claudin-1 and ZO-1 were significantly increased.The levels of IL-8 and TNF-α were decreased in colon tissue and serum.These findings suggest that the application of the lentiviral of Gn overexpression can ameliorate the intestinal inflammatory injury and repair the mucosal barrier in UC mice.Our research showed that the expressions of GC-C,Gn and Ugn in the colonic mucosa of UC patients are negatively correlated with the disease activity of UC,the dormant GC-C signaling pathway can be restored by the agonists of GC-C.The activation of GC-C signaling pathway can ameliorate the impaired barrier function and inflammatory injury of Caco-2 cells induced by IL-1β as well as the intestinal inflammation of DSS-induced mice.To sum up,we revealed that the GC-C signaling pathway played a key role in the initiation and development of UC.These observations suggest that the clinical therapeutic potential of GC-C agonists in UC and provide the important experimental basis of new treatment strategies for IBD.Part I The differential expression of GC-C and its ligands Gn,Ugn in UC patientsObjective:To investigate the differential expression of GC-C and its endogenous ligands,Gn and Ugn,in the colonic mucosa of UC patients with different disease activity,and evaluate the relationship of the GC-C signaling pathway and UC with different severity.Methods:A total of 60 UC patients and 20 normal controls were recruited.The inflamed colonic mucosa were taken from the UC patients and the normal mucosa from the controls.Evaluation of the UC disease activity was performed using a modified Mayo scoring system.The UC patients were divided into mildly active group(18 cases)、moderately active group(23 cases)、severely active group(19 cases).The mRNA and protein expression of GC-C,Gn and Ugn in the colonic mucosa was measured by quantitative real-time PCR(qRT-PCR)and Western blot respectively.To analyze the association of the levels of GC-C,Gn,Ugn and UC as well as its clinical assessments.Results:The results of qRT-PCR showed:GC-C mRNA relative expressions of the Control,Mild,Moderate and Severe were(1.652±0.258),(0.525±0.240),(0.153±0.107)and(0.034±0.020)respectively;Gn mRNA relative expressions were(1.484±0.628),(0.243±0.063),(0.097±0.051)and(0.056±0.014)respectively;Ugn mRNA relative expressions were(1.196±0.609),(0.334±0.058),(0.126±0.066)and(0.082±0.023)respectively.The mRNA relative expressions of GC-C,Gn and Ugn in the colonic mucosa of UC patients were significantly decreased compared with the normal controls.There were statistic differences of the GC-C,Gn and Ugn mRNA relative expressions for the UC groups of mild,moderate and severe.With the increase of disease activity,the mRNA relative expressions of GC-C,Gn and Ugn were all decreased.The results of mRNA were confirmed by the protein relative expressions of GC-C,Gn and Ugn in the colonic mucosa of each group,as assessed by western blot analysis.Conclusion:The transcription and expression of GC-C,Gn and Ugn are downregulated in the colonic mucosa of UC patients and negatively correlated with the disease activity of UC.Part II The effect of GC-C signaling pathway on barrier and inflammatory injury in Caco-2 cellsObjective:To investigate the effect of GC-C signaling pathway on intestinal epithelial barrier and inflammatory injury in the barrier model of Caco-2 monolayers.Methods:Caco-2 cells were grown on Transwell filter to establish the barrier model of monolayers.The integrity of the barrier was determined by measuring the transepithelial electrical resistance(TER)of the cell monolayer.Caco-2 monolayers were stimulated with interleukin-1β(IL-1β)to model the intestinal epithelial inflammatory cells,and the tight junction structure of cells was impaired.GC-C shRNA and Gn overexpression vector were transfected into cells using Lipofectamine,respectively.After different treatment with cells,.the permeability of Caco-2 monolayers was measured by the paracellular passage of the macromolecular substance of fluorescein isothiocyanate(FITC)-dextran(FD-4).The cell viability and SOD activity were evaluated by the CCK-8 and NBT assay respectively.The levels of IL-8 and TNF-a in cell culture medium were measured by ELISA.The expressions of the crucial mediators of GC-C signaling pathway(Gn,Ugn,GC-C and cGMP),TJPs(occludin,claudin-1 and ZO-1)and pro-inflammatory cytokines(IL-8 and TNF-a)were analysed by qRT-PCR and Western blot,respectively.Results:The intestinal barrier model was established characterized by the stable TER above 250 Ω·cm2 at 19 days after the Caco-2 monolayer culture.Cells transfected with Gn overexpression vector for 48h had increased expressions of GC-C and cGMP except the increase of Gn.Expression of Ugn had no significant difference.After transfection with GC-C shRNA for 48h,GC-C-silencing cells also had significantly reduced expressions of Gn、Ugn and cGMP except the reduction of GC-C.After stimulation with IL-1β,IL-1β-treated cells had significantly reduced cell viability,SOD activity and expressions of GC-C、Gn、Ugn、cGMP、claudin-1、ZO-1,as well as increased permeability,levels of IL-8 and TNF-α.Relative to the vector control 1,IL-1β-treated cells transfected with Gn overexpression vector had significantly increased levels of cell viability,SOD activity,claudin-1 and ZO-1 as well as decreased levels of IL-8,TNF-α and permeability.Conversely,cells transfected with GC-C shRNA vector had more significant increased levels of IL-8,TNF-α and permeability as well as decreased levels of cell viability,SOD activity,claudin-1 and ZO-1 induced by IL-1β compared with the vector control 2.But the expression of occludin had no significant difference among each group.Conclusion:The regulation of the ligands Gn,Ugn and their corresponding receptor GC-C is combined,the dormant GC-C signaling pathway can be restored by the agonists of GC-C.The activated GC-C signaling pathway can ameliorate the impaired barrier function and inflammatory injury of Caco-2 cells induced by IL-1β.Part Ⅲ The effect of GC-C signaling pathway on intestinal inflammation in UC miceObjective:To determine the effect of GC-C signaling pathway on intestinal inflammation using DSS-induced UC model on mice.Methods:60 Bal b/c mice were divided into 5 groups(12 mice per group):Control group;DSS+ NS group;DSS+ Mesalamine group;DSS+ Gn group;DSS+Mesalamine+ Gn group.Experimental UC model was established in mice by oral administration of 3%dextran sodium sulfate(DSS)for 1 week.After the establishment of UC model,Mesalamine(30mg/kg)was administered once daily by gavage for 1 week.The lentiviral of Gn overexpression was administered once daily by injection through caudal vein for 1 week.The feeding,activity,body weight and stool of mice were recorded every day since the experiment was conducted.Intestinal permeability of mice was measured by the macromolecular substance of FD-4 after treatment.All mice were killed 2 weeks after the first DSS administration.Serum and colon tissue were collected.Histopathologic score were estimated by the inflammatory injury of HE-staining colon tissue observed with a light microscope.The expressions of the crucial mediators of GC-C signaling pathway(Gn,Ugn,GC-C and cGMP),TJPs(occludin,claudin-1 and ZO-1)and pro-inflammatory cytokines(IL-8 and TNF-α)in colon were detected by immunohistochemical analysis.The levels of IL-8 and TNF-α in serum were measured by ELISA.Results:Compared to the DSS+ NS group,the appearance,feeding and activity of mice in all therapeutic groups were better,weights were increased,and the frequency of loose stools and bloody stools per day was decreased.Among the therapeutic groups,the improvement in DSS+ Mesalamine+ Gn group was the best.The histologic score was significantly higher in DSS+ NS group than Control group.It was lower in all therapeutic groups than DSS+ NS group and the DSS+ Mesalamine+Gn group was the lowest.Compared to the Control group,the expressions of GC-C、Gn、Ugn and cGMP in DSS+ NS group were significantly decreased.They were higher in all therapeutic groups than DSS+ NS group and the DSS+ Gn group was the highest.Compared to the Control group,the intestinal permeability in DSS+ NS group were significantly increased.They were lower in all therapeutic groups than DSS+ NS group and the DSS+ Gn group was the lowest.The expressions of occludin、claudin-1 and ZO-1 were significantly lower in DSS+ NS group than Control group.The expressions of claudin-1 and ZO-1 were significantly increased in all therapeutic groups compared with DSS+ NS group and the DSS+ Mesalamine+Gn group was the highest.Compared to the Control group,the levels of IL-8 and TNF-a of colon and serum in DSS+ NS group were significantly increased.They were lower in all therapeutic groups than DSS+ NS group.The DSS+ Gn group was the lowest.Conclusion:The application of the lentiviral of Gn overexpression separately or combintion with Mesalamine can ameliorate the intestinal inflammatory injury and repair the impaired mucosal barrier function in UC mice,which further support the protective role of GC-C signaling pathway in intestine.