Studies On Molecular Mechanisms For Regulation Of NLRP3 Inflammasome And Ulcerative Colitis By Circadian Clock Protein REV-ERB?

Objective:Ulcerative colitis?UC?is a kind of inflammatory bowel disease,which is chronic and often recurrent,seriously affecting patients'normal life.UC patients are often accompanied by symptoms such as insomnia and circadian rhythm disruption,suggesting close relationship between UC and circadian clock.However,whether UC is regulated by REV-ERB?and its mechanism is still unknown.This thesis will analyze the correlation between the circadian clock and UC,and clarify the regulatory effects and mechanisms for regulation of UC by circadian clock protein REV-ERB?.The specific research objectives are as follows:?1?to establish a mouse UC model,to determine gene expression profiling in colon and to investigate changes of clock gene expression;?2?to establish physiological and genetic mouse models with disrupted circadian clock and evaluate their susceptibility to UC;?3?to determine Rev-erb?in mice with UC or circadian rhythm disruption,and to investigate susceptibility of Rev-erb?knockout mice to UC;?4?to clarify the regulatory effects of REV-ERB?to UC,and to elucidate the regulatory mechanism;?5?to explore preventive and therapeutic effects on UC by using REV-ERB?agonist.Methods:1.Wild-type mice were used as experimental subjects.Acute UC model was induced by using dextran sodium sulfate?DSS?.The expressions of colonic genes for UC mice and normal mice at six time points were determined by RNA sequencing.Cycling genes in normal and UC mice were identified.We analysed changes of the clock genes in mouse colon by using real-time quantitive PCR?q PCR?.2.Mouse model with physiological circadian clock disruption was established with a jet lag schedule.After vadiated by wheel-running experiment,UC was induced by using DSS.Through performing comprehensive characterization of mouse UC,including body weight,disease activity index?DAI?,colon length,H&E staining score and myeloperoxidase?MPO?activity analysis,susceptibility of mice to UC with physiological circadian clock disruption was explored.In addition,the expression of Rev-erb?in mouse colon was determined by q PCR.3.Bmal1 knockout mouse model?genetic circadian clock disruption?was estabolished by using CRISPR/Cas9 technique.After validated by wheel-running test,PCR genotyping,q PCR and Western blotting,UC was induced by using DSS.UC susceptibility of mice with genetic circadian clock disruption was evaluated by the inflammation index values?i.e.,weight loss,DAI,histopathological score,colon length,and MPO?.In addition,the expression of Rev-erb?in mouse colon was determined by q PCR.4.Rev-erb?knockout mouse was estabolished by using CRISPR/Cas9 technique.After validated by PCR genotyping and Western blotting,UC was induced by using DSS.The susceptibility of Rev-erb?knockout mice to UC was evaluated by the inflammation index values?i.e.,weight loss,DAI,histopathological score,colon length,and MPO?.In addition,the expression of Rev-erb?in mouse colon was determined by q PCR.In addition,the effects of Rev-erb?knockout on NLRP3 inflammasome were examined by ELISA and Western Blotting.5.By using q PCR,the cycling patterns of Nlrp3 and related genes in liver and colon were determined.In addition,effects of Rev-erb?knockout on colonic Nlrp3 were evaluated.The transcriptional regulation mechanisms of REV-ERB?on NLRP3 inflammasome were demonstrated by q PCR and Western Blottomg,combined with biological methods such as dual luciferase reporter assay,electrophoretic mobility shift assay?EMSA?and chromatin immunoprecipitation?Ch IP?.6.By using wild-type,Nlrp3 knockout and Rev-erb?knockout mice as experimental subjects,UC was indued by using DSS.The small molecule agonist SR9009 was used to activate REV-ERB?to explore the preventive and therapeutic effects for UC.Results:1.Clock genes are dysregulated in mice with experimental colitis.RNA sequencing suggested that DSS-induced UC led to persistent and genome-wide gene deregulation in mouse colon.Oscillations of core clock genes were diminished and most of core clock genes?i.e.,Rev-erb??were blunted in mice with UC.Dysregulation of clock genes?Rev-erb?,Rev-erb?,Clock,Bmal1,Npas2,Per2,Cry1,Ror?and Dbp?were further confirmed by q PCR analyses.2.Disruption of circadian clock exacerbates experimental colitis.Jet-lagged mice were established and validated by wheel-running test.Compared with normal mice,jet-lagged mice showed aggravated weight loss,elevated DAI and MPO values,increased histopathological scores,and shorter colons.These data indicated physiologic disruption of circadian clock sensitized mice to UC.Furthermore,Rev-erb?was reduced in jet-lagged mice.Bmal1 knockout mice were established and validated.Bmal1 knockout mice were much more sensitive to DSS-induced colitis as evidenced by the relative indexes?i.e.,weight loss,DAI,histopathological score,colon length,and MPO?.These data indicated genetic disruption of circadian clock sensitized mice to UC.Furthermore,Rev-erb?was reduced in Bmal1 knockout mice.3.Rev-erb?ablation sensitizes mice to UC and NLRP3 inflammasome activation.Rev-erb?knockout mice were eatablished and validated.Rev-erb?knockout mice were much more sensitive to DSS-induced colitis as evidenced by relative indexes?i.e.,weight loss,DAI,histopathological score,colon length,and MPO?.In addition,REV-ERB?ablation caused increased levels of NLRP3 proteins and IL-1?/IL-18 proteins in colon.4.Identification of Nlrp3 as a clock-controlled gene.Hepatic and colonic Nlrp3 displayed robust diurnal fluctuations.This oscillation was in antiphase to Rev-erb?.The rhythmicity in Nlrp3 expression was dampened in Rev-erb?knockout mice.These data suggested that Nlrp3 is a clock controlled gene and a potential direct target of REV-ERB?.5.Rev-erb?inactivates NLRP3 inflammasome at the priming stage.SR9009 prior to LPS treatment suppressed NLRP3 and IL-1?m RNAs and proteins in PMs,whereas SR9009 post LPS treatment exerted no effects.Stimulation with LPS/ATP for a short period of time?30min?caused activation of caspase-1 in PMs and SR9009 treatment exerted no effects on caspase-1 activation.These data indicated that REV-ERB?inactivated NLRP3 inflammasome at the priming stage rather than assembling step.6.REV-ERB?represses Nlrp3 transcriptionREV-ERB?activation attenuated LPS-induced NLRP3 expression in Raw264.7 cells and PMs,indicating a repressive action of REV-ERB?on NLRP3 expression.Luciferase reporter,Ch IP and EMSA assays showed that REV-ERB?suppressed Nlrp3 transcriptional activity through binding to Rev RE?-1139 to-1129bp?of Nlrp3 promoter.Moreover,mutation of Rev RE?-1139/-1129 bp?failed to abrogate the transcriptional repression effects of SR9009.Only a mutation of both Rev RE and?B sites completely abolished the effects of SR9009.Luciferase reporter,EMSA,and immunofluorescence assays indicated REV-ERB?activation repressed NF-?B signaling.Further,REV-ERB?suppressed p65transcription through binding to Rev RE?-484/-474 bp?on p65 promoter.7.REV-ERB?activation alleviates UC Compared with vehicle-treated mice,SR9009-treated mice displayed reduced body weight loss,lower DAI score,higher survival rate,longer colons,decreased histological score and suppressed MPO activity.The protective effects of SR9009 on DSS challenge were lost in Nlrp3^-/-mice and Rev-erb?^-/-mice.Collectively,REV-ERB?activation protected mice from UC via suppression of NF-?B and inactivation of NLRP3 inflammasome.Conclusion:1.DSS induced UC causes persistent and genome-wide gene deregulation.Oscillations of clock genes are diminished.2.Both physiologic and genetic circadian clock disruption sensitize mice to UC3.REV-ERB?plays an important role in regulation of NLRP3 inflammasome and colitis development.4.REV-ERB?blocks the priming of NLRP3 inflammasome through repression of Nlrp3transcription.5.REV-ERB?activation by SR9009 attenuates DSS-induced colitis in wild-type mice,and represents a promising approach for prevention and management of colitis.6.The discovery of regulation of UC by biological clock provides a scientific mechanism for the circadian rhythm changes of Chinese medicine diseases,and establishs a foundation for time-based treatment.