Melatonin Alleviates The Intestinal Inflammation In DSS-induced Colitis Mice With Circadian Rhythm Disruption Through Inhibiting Redistribution Of Intestinal HMGB1

Objectives: To observe the effect of circadian rhythm disruption?CRD?on the intestinal inflammation,intestinal HMGB1 distribution and TLR4-NF-?B signaling pathway in dextran sulfate sodium?DSS?-induced colitis mice,and to study the effect of melatonin on the intestinal inflammation in these mice and its possible mechanisms.Methods: This study included two parts.?1?Twenty-four C57BL/6 mice were randomly divided into four groups: group CRD circadian rhythm disruption?CRD?,CRD+ Dextrose Sodium Sulfate?DSS?,CRD+DSS group and control group.The mice in group CRD and in group CRD+DSS were raised in single-cage with infrared detection instrument,and were subjected to alternate cycles of light,i.e.12 hours of day-to-night turnover every 7days,lasting for 2 months.The mice in group DSS and those in group CRD+DSS with 2-month CRD were free to drink 2% DSS aqueous solution for 7 days to induce colitis.The control mice received normal light and distilled water.At the end of intervention,mice were anaesthetized and sacrificed.The general condition,colon length and colon histopathology were observed.The expression of HMGB1 and TLR4 in intestinal tissues were detected by immunofluorescence.The level of HMGB1,TNF-?,IL-1? and MPO in the serum and intestinal tissues were respectively detected by ELISA.The expression of HMGB1,TLR4,Myd88 and NF-?B in the intestine were also detected by WB.?2?The C57BL/6 mice with intervention of CRD+DSS were randomly subdivided into 3 groups,that is Group CRD+DSS+ MEL?melatonin?,Group CRD+DSS+MEL+ melatonin receptor antagonism?luzindole,LUZ?,Group CRD+DSS+ HMGB1 inhibitor ethyl pyruvate?EP?.On the 5^th day,all mice were anaesthetized and sacrificed.The general condition,colon length and colon histopathology were observed.The expression of HMGB1 and TLR4 in intestinal tissues were detected by immunofluorescence.The expression of HMGB1,TNF-?,IL-1? and MPO in feces and intestinal tissues were respectively detected by ELISA.WB was used to detect the expression of HMGB1,TLR4,Myd88 and NF-?B in the intestine.Results:?1?Compared with those in group DSS and in Group CRD,the mean body weight was significantly reduced?p<0.05?,the colon length was shortened?p=0.000?,intestinal pathology score increased significantly?p = 0.000?in Group CRD+DSS.The fecal levels of HMGB1,TNF-?,IL-1?,MPO were increased?all p=0.000?,and the colonic concentration of HMGB1,TLR4,Myd88 and NF-?B was also increased respectively?all p=0.000?.Immunofluorescence staining showed that the expression of HMGB1 in cytoplasm and intestinal cells increased significantly.?2?Compared with those in Group CRD+DSS,the extent of weight loss,colon length shortening,and intestinal histopathological scores were significantly reduced in Group CRD+DSS+MEL?p<0.05?.The colonic levels of TNF-?,MPO,HMGB1,TLR4,Myd88 and NF-?B were decreased respectively?p<0.05?.Immunofluorescence staining showed that the expression of HMGB1 in cytoplasm and intestinal cells decreased significantly.The melatonin receptor antagonist luzindole partially reverses the effects of melatonin.After treatment with the HMGB1 inhibitor ethyl pyruvate,the intestinal inflammation improved in Group CRD+DSS,and the expression of HMGB1 in the cytoplasm and intestinal cells were significantly inhibited,which was similar to the melatonin effects.Conclusion: Circadian rhythm disruption may aggravate the intestinal inflammation in colitis mice induced by dextran sulfuric acid by activating the HMGB1-mediated TLR4-NF-?B signaling pathway.Melatonin partially reverses the effect of CRD on intestinal inflammation by inhibiting the redistribution of HMGB1.