Bidirectional Regulation Of Circadian Disturbance And Inflammation In Inflammatory Bowel Disease

Ulcerative colitis(UC)and Crohn’s disease(CD)are the two major forms of inflammatory bowel disease(IBD),which characterized by chronic inflammatory pathologies of the intestine,periods of symptomatic crisis and remission that cause a substantial worsening of life quality.It has been confirmed that IBD patients suffered from circadian rhythm abnormalities of sleep disturbance and inflammation in the GI tract.Circadian rhythm exists in many physiological and behavioral activities in mammals and is controlled by the central pacemaker in the brain’s suprachiasmatic nucleus(SCN)and subsidiary oscillators in nearly all body cells.It has revealed that up to 10% of gene expressions in mice and human are rhythmical,including cell growth,DNA damage-repair,and metabolic processes or immune function.As a result,disruption of the circadian system can lead to pathological conditions such as metabolic syndrome,cancer and immune disorders.As IBD patients suffer from circadian rhythm abnormalities,chronic inflammation and pathogenic production of TNF-α,IL-6,all of which are affected by the molecular clock,we hypothesized that the circadian timekeeping was perturbed in IBD.Methods:1.51 ulcerative colitis(UC)patients,39 Crohn’s disease(CD)patients and 42 healthy controls were recruited.The expressions of circadian genes in recruited patientswere measured by qRT-PCR.The correlations between circadian gene expressions and ESR,CRP and Mayo score or SES-CD were made.2.Dextran sodium sulfate(DSS)was used to induce the experimental colitis.Fifty mice were randomly divided into two groups,DSS groups and control groups.Each group were randomly divided into five groups by Zeitgeber time(ZT)(n =5 in each).Mice in DSS group werefed with 3%(wt /vol)DSS dissolved in drinking water for 7 days.Control mice received the same water without DSS.The colon wascollected at ZT0,ZT6,ZT12,ZT18 or ZT24.Peritoneal macrophages were isolatedcultured.After clock resetting,the peritoneal macrophages were treated with LPS(1 μg/ mL).The expression levels of circadian genes in mice model and peritoneal macrophageswere measured by qRT-PCR.3.A light/dark shift model was used to evaluate the influence of circadian disturbances on DSS induced colitis.28C57BL/6 mice were randomly divided into 4groups(n=7).The phase shift+DSS model was made by a delayed time(six hours)to turn off the lights every one week,and during the 8th week,3%(wt /vol)DSS was used for one week for inducing colitis.Mice in non-phase shift group were group-housed under normal 12:12-hour light-dark cycle.Mice in non-phase shift group and phase shift group received the same water without DSS.4.We detected the protein level of Bmal1 in mucosal samples of UC,CD and the non inflamed tissues of the individuals based on the gross appearance determined by the endoscopy operator.Western blot and immunofluorescence were used to detect the expression level and location of Bmal1.Mice were divided into 4 groups,Wild type group(wt),Bmal1-/-group(ko),Wild type group+DSS group(wt+DSS),and Bmal1-/-+DSS group(ko+DSS),n=7.Mice in colitis group received 3%(wt /vol)DSSdissolved in drinking water for 7 days.The other groups received the same water without DSS.Results1.In both intestinal biopsies and peripheral blood mononuclear cells(PBMC),the expressions of circadian genes were almost all reduced in IBD compared with healthy controls(p < 0.05).Circadian genes of biopsy tissues showed a negative correlation with Mayo Score or SES-CD in IBD.2.In DSS induced colitis and LPS stimulated peritoneal macrophages,the circadian gene had altered a lot.It seemed that there was a broad lowering of basal expression of circadian gene expression in DSS induced colitis.After LPS stimulation,Bmal1,Clock,Per1 and Cry1 significantly decreased within 2h(p<0.05).3.Inflammation in phase shift mice were more serious compared with thenon-phase shift mice after being exposure to DSS,and phase shift exacerbated DSS induced colitis with increased inflammatory cytokines and chemokines.And the expression level of p-STAT3 and p-P65 were higher in phase shift +DSS mice compared with non-phase shift+ DSS mice(p<0.05).4.Bmal1 located in nuclear area and inflamed regions of UC patients showed a significant down-regulation of Bmal1 level.Inflammation in ko+DSS mice were more serious compared with the wt+DSS mice after being exposure to DSS,and Baml1 knock out exacerbated DSS induced colitis with increased inflammatory cytokines and chemokines(p<0.05).Conclusion:1.Disturbed circadian genesinvolved in the pathogenesis of IBD.2.Inflammation and circadian genes produced a complex bidirectional regulation that was relevant to IBD.3.Circadian clock appeared to be a potential target of IBD therapy.