The Research Of Evodiamine On Targeting The Balance Of Autophagy And NLRP3 To Improve Ulcerative Colitis

Objective:Ulcerative colitis(UC)and Crohn’s disease(CD)are both Inflammatory bowel disease(IBD).Bloody diarrhea is the critical early symptoms of UC,which is easy to relapse.Recurrence increases the risk of colorectal cancer(CRC)and deteriorates the life quality of patients.Treatments of IBD mainly depends on anti-inflammation and immunomodulation,however long-term use of them can lead to serious adverse reactions.Therefore,it is necessary to find novel drugs with effective and fewer adverse effects for treatment of IBD.Previous study has reported that NLRP3 inflammasome is activated in UC,however,the relevant mechanism of UC remains to be clarified.In the present study,we aimed to find novel drugs targeted on NLRP3 inflammasome for UC.Evodia,a kind of Chinese herbal medicine,has an effect on anti-diarrhea.evodiamine is an active component extracted from the fruits of Evodia.The previous data showed that Evodiamine has an anti-inflammatory effect.In this study,we addressed on: 1.To make clear whether evodiamine is able to attenuate damage of experimental UC;2.To illustrate whether over-activated NLRP3 inflammasome in UC is inhibited by evodiamine;3.To elucidate the molecular mechanisms how evodiamine works on the NLRP3 inflammasome to alleviate UC.Methods:Acute colitis in mice was induced by adding DSS to the drinking water at a concentration of 3% for a period of 7 days.During DSS-induced UC,the administrations of different doses of evodiamine(20,40 and 60mg/kg)and positive drug 5-ASA in mice by gavage.The negative control(po.0.5% CMC-Na)and single drug evodiamine groups(60mg/kg)were setted up.All drugs were administered for10 days and DSS drinking water for 7 days and thereafter provided with drinking water for 3 days.Body weight,stool consistency and fecal blood in mice were recorded daily.The mice were sacrificed and colon tissues were collected 10 days after administration.We estimated the improvement of evodiamine on acute UC according to body weight,the length of colons and hematoxyin and esosin staining(HE).We detected the expression levels of interleukin-1β(IL-1β),interleukin-18(IL-18)and myeloperoxidase(MPO)in colon tissues using enzyme-linked immunoassay(ELISA),and the infiltration of inflammatory cells CD11 b in colon tissue using immunofluorescence(IF),investigating the improvement of evodiamine on inflammation.We detected the changes of NLRP3 inflammsome after evodiamine treatment in THP-1 cells stimulated by LPS and ATP.We found that evodiamine affected the autophagy-related proteins P62 and LC3,then we determined the effect of evodiamine on the co-localization of autophagy and inflammasome by immunofluorescence.We detected the effects of evodiamine on NLRP3 inflammasome and inflammatory cytokines by separately intervening in different periods of formation of autophagy,revealing the mechanisms of evodiamine’s anti-inflammatory action.Results:Evodiamine improved the loss of body weight in UC mice,decreased diseased activity index(DAI),increased the length of colons,and repaired damaged colon tissue structures of UC mice in a dose-dependent way.The results of IF and ELISA showed that evodiamine decreased the infiltration of inflammatory cells,the expression level of MPO,the levels of inflammatory cytokines IL-1β and IL-18 in colon tissues,suggested evodiamine has improvement effect on damage of UC.Evodiamine decreased the release of cytokines IL-1β and IL-18,and reduced the protein expression of activated NLRP3 inflammasome in a dose-dependent way in THP-1 cells stimulated by LPS and ATP.Evodiamine also inhibited the oligomerization of ASC,the ASC speck formation,the co-localization of ASC and caspase1.These results demonstrated that evodiamine suppressed the activation of NLRP3 inflammasome by interrupting the NLRP3 inflammasome assembly.The up-regulation of autophagy-related protein P62 and LC3 suggested that evodiamine promoted the autophagy;After inhibiting autophagy,the inhibitory effect of evodiamine on NLRP3 inflammasome was weaken.Conclusion:1.Evodiamine had the protective effect on experimental UC through anti-inflammation;2.Evodiamine suppressed the activation of NLRP3 inflammasome by interrupting the NLRP3 inflammasome assembly and played an anti-inflammatory effect.3.Evodiamine promoted the autophagy,established a balance between autophagy and NLRP3 inflammasome in disease states,thereby playing an improvement effect on UC;4.Evodiamine has a potential effect on UC treatment.