Mechanism Of Visceral Hypersensitivity Caused By Circadian Rhythm Disorder And The Effect Of Rifaximin Intervention

Object:Most physiological processes in the human body show biological rhythms,such as sleep-wake cycle,body temperature,hormone secretion,metabolism,immune response and so on.Circadian rhythm is a common kind of biological rhythm.Clinical studies have found that the prevalence of IBS is higher in shift workers,which suggests that circadian rhythm disorder may involve in the occurrence and development of IBS,while the pathophysiological changes of IBS often involve changes in visceral sensitivity.Therefore,the purpose of this study is to explore the mechanism of visceral hypersensitivity caused by circadian rhythm disorder.As an intestinal selective antibiotic,the role of rifaximin in the treatment of IBS has attracted much attention in recent years.This study also explored the intervention effect of rifaximin on this pathological process.Methods:(1)8-week-old C57BL6 J male mice were randomly divided into Non-Phase shift group(NN)and Phase shift group(PN).Each group was divided into five subgroups according to the zeitgeber time which expressed of ZT0、ZT6、ZT12、ZT18、and ZT24.Visceral sensitivity was evaluated by CRD-AWR score and response of colonic smooth muscle strips to acetylcholine.intestinal histopathological score was evaluated by HE staining,transepithelial electrical resistance(TEER)and FD4 permeability were measured by Ussing chamber technique,intestinal mucosal tight junction protein was detected by Western blot,(2)The mice were given rifaximin intragastrically in the last week of the model of circadian rhythm disorder.The mice were divided into Non-Phase shift group(NN),Phase shift group(PN)and Phase shift + Rifaximin group(PR).The expression of intestinal inflammatory cytokines was detected by PCR,The transepithelial electrical resistance(TEER)and FD4 permeability of mice in each group were detected by Ussing Chamber technique.response of colonic smooth muscle strips to acetylcholine reflects visceral sensitivity.(3)The expression of tight junction proteins in intestinal mucosa of IBS patients treated with rifaximin was detected by immunofluorescence technique.Human colon cancer epithelial cell line Caco2 was stimulated by LPS in vitro,and then treated with 100 μ g / m L rifaximin.immunofluorescence and PCR technique were used to detect the expression of tight junction proteins.Results:(1)Phase shift mice showed visceral hypersensitivity: 1)the AWR score of phase shift mice increased.2)the pain threshold of phase shift mice decreased.3)the response of colonic smooth muscle strips of phase shift mice to acetylcholine increased in vitro.(2)The expression of colonic inflammatory factors and mucosal permeability increased in phase shift mice: 1)the expression of intestinal IL-17α,IL-1β and IL-6 in PN group was higher than that in NN group(P<0.05).2)the expression of Claudin1 and Occludin in PN group was lower than that in NN group(P<0.05).3)TEER in PN group was lower than that in NN group,and the permeability of FD4 was increased in PN group(P<0.05).Correlation analysis showed that pain threshold was positively correlated with TEER and negatively correlated with FD4 permeability as well as IL-17α,IL-1β and IL-6.(3)Disruption of circadian rhythm affects the expression of genes for the colon clock:before ZT12,the expression of Bmal1 and Clock gene in PN group was lower than that in NN group,and the expression of Pers gene was higher than that in NN group.After ZT6 time point,the expression of Crys gene in PN group was higher than that in NN group.(4)Rifaximin improved visceral hypersensitivity and intestinal inflammation caused by circadian rhythm disturbance: Compared with the PN group,the response of colonic smooth muscle strips to Ach was decreased in the PR group;The expressions of IL-17α,IL-1β and IL-6 in PR group were significantly decreased(P < 0.05).(5)Rifaximin improved intestinal barrier function: compared with PN group,TEER increased and FD4 permeability decreased in PR group(P<0.05).Compared with IBS patients,the expression of Occludin and ZO-1 in intestinal mucosa was relatively increased after treatment with rifaximin.(6)The cell experiment in vitro showed that rifaximin could increase the expression of tight junction protein in intestinal epithelial cells: After Caco2 cells were stimulated by LPS,TEER decreased,and the results of immunofluorescence and PCR showed that the expression of tight junction proteins decreased.while TEER and the expression of tight junction proteins increased after treated with rifaximin.Conclusion:Circadian rhythm disorder can produce visceral hypersensitivity similar to IBS pathophysiology.Moreover,increased visceral sensitivity is related to intestinal low-grade inflammation and increased intestinal mucosal permeability.Rifaximin can improve visceral hypersensitivity caused by circadian rhythm disorder which may be related to its anti-inflammatory and restoration of intestinal epithelial barrier function.