| Background:Ulcerative colitis(UC)is a type of gastrointestinal disease characterized by bloody stools,abdominal pain and weight loss.Patients are often accompanied by mental symptoms such as anxiety and depression.However,its etiology has still been unclear.Elevated interleukin-13(IL-13)has been found in the circulatory system of UC patients,indicating that type 2 immunity is an important factor involved in the pathogenesis of UC.There are various drug options for the clinical treatment of UC,however,some of them have obvious adverse effect and some others(e.g.monoclonal antibodies)are not suitable for all patients.So it is still the general trend to find safe and effective treatment methods against UC.Melatonin(N-acetyl-5-methoxytryptamine,MT)was thought to be a hormone secreted only by the pineal gland in the early days,but subsequent studies found that melatonin is enriched in the gastrointestinal tract.The concentration of melatonin is 100-400 times that of melatonin in the circulatory system.Moreover,instead of being regulated by circadian rhythms,intestinal melatonin is regulated by dietary rhythms.The hepatic clearance of intestinal melatonin is higher than 99.9%which leads to a local physiological effect of it.In UC patients,the concentration of melatonin in the circulating system decreases,and the function of EC changes at the same time.Since intestinal melatonin and circulating melatonin are relatively independent,the intestinal melatonin,which is located at the site of UC,is more affected and credible during UC.Therefore,it is of clinical significance to explore the correlation between the content of intestinal melatonin and UC.Due to the complex etiology of UC,experimental colitis mice with a clear etiology is the main method to explore the mechanisms of UC.The inducers of experimental colitis are dextran sulfate sodium(DSS),trinitrobenzenesulfonic acid(TNBS)and oxazolone.Different from DSS and TNBS,oxazolone colitis is mainly induced by type 2 immunity,corresponding to the phenomenon of elevated type 2 immunity in clinical UC patients.The effect of melatonin on oxazolone-induced experimental colitis remains unknown.In recent years,the important role of type 2 immunity in the process of UC has gradually attracted by researchers,at the same time,drugs targeting type 2 immunity have entered clinical trials.Among these three models,oxazolone-induced colitis is the only one mediated by type 2 immunity,which corresponds to the elevation of type 2 cytokines in UC patients.However,the effect of melatonin on oxazolone-induced experimental colitis and its mechanism of action are still unknown.Part I:Effect on the role of melatonin in ulcerative colitis.Objectives:The main purpose of this part was to explore the relationship between the concentration of intestinal melatonin and the severity of UC,and to explore the effect of melatonin on oxazolone-induced colitis in mice.MethodsClinical research:According to the diagnostic criteria of UC,11 UC patients and 17 healthy controls were included.Mucosal biopsy was performed from ulcerated lesions of the descending colon to sigmoid colon from UC patients under colonoscopy,and the corresponding of healthy controls were randomly collected.The obtained tissues were detected by Enzyme-Linked immunoabsorbent assay(ELISA).and the severity of colitis was assessed by pathological score,then the correlation between the level of melatonin expression and the severity of colitis was analyzed.Quantitative real-time polymerase chain reaction(qPCR)was used to detect the expression of AANAT and HIOMT,which are key enzymes for melatonin synthesis in intestinal tissue.Basic research:Oxazolone was used to induce type 2 immune-mediated experimental colitis in mice and melatonin was performed.Western Blot,qPCR and flow cytometry were used to evaluate the severity of colitis in mice of control group(without melatonin intervention)and experimental group(melatonin intervention).Results:ELISA was used to explore the concentration of intestinal melatonin of both healthy controls and UC patients,showed that the concentration of intestinal melatonin of UC patients was significantly reduced(P<0.0001).By evaluating the relationship between intestinal melatonin content and intestinal pathological score,it was found that with the increase of pathological severity,the tissue concentration of melatonin gradually decreased,and there was a significant negative correlation between the two groups(r=-0.7481,P=0.0013).Subsequently,qPCR was used to detect the key enzyme of melatonin synthesis and it was found that the mRNA expression levels of AANAT and HIOMT,key enzymes of melatonin synthesis,were significantly lower than those of healthy controls(P<0.001).Melatonin could mitigate the oxazolone-induced experimental colitis,as shown in weight loss(P<0.001),colon length(P<0.01),pathological score(P<0.001),neutrophil infiltration(P<0.001)TNFα and IL-1β expression(P<0.05),and the expression of tight junction proteins ZO-1 and Occludin(P<0.001).Conclusions:(1)Melatonin in colon tissue was significantly negatively correlated with the severity in UC patients.(2)The synthesis function of melatonin in UC patients was significantly decreased.(3)Melatonin treatment can significantly alleviate type 2 immune-mediated experimental colitis in mice.Part Ⅱ:The mechanism of melatonin on oxazolone-induced colitis in miceObjectives:In this part,we will base on melatonin and oxazolone-induced experimental colitis,and discuss in detail in the mechanism section,focusing on and exploring the immune and microbial mechanisms.While filling the gaps in the field,it is expected to guide clinical medication and provide more optional treatment options for UC patients.Methods:Immune mechanisms:determine the immune components in mice that respond to oxazolone and use techniques such as flow cytometry and qPCR to detect melatonin treatment(experimental group)and no treatment(control group)in colitic mice induced by oxazolone.Discover the difference between experimental group and control group of this component type 2 immune effector molecules in mice with experimental colitis;and sort these cells,stimulate them with melatonin in vitro,and detect their functional changes.Microbial mechanism:The gut microbiota in the experimental group and the control group were communicated in the same cage,and various indicators related to the severity of inflammation after the oxazolone-induced colitis were evaluated in the mice;Fecal microbial sequencing and other methods were used to analyze the effects of melatonin on the gut microbiota,and specific microbial changes were analyzed at the genus level.Fecal microbiota transplantation was used to detect whether the mouse microbiota affected by melatonin could relieve oxazolone-induced experimental colitis independently.Results:In oxazolidon-induced experimental colitis in mice,type 2 innate lymphocyte(ILC2)is the dominant inflammatory cell group.Melatonin significantly inhibited the inflammatory cytokine IL-13 produced by ILC2 in mice with colitis(P<0.01),and the mRNA expressions of IL-5 and IL-13 were significantly decreased(P<0.05)as well.In vitro,melatonin inhibits IL-5 and IL-13 production of ILC2 in intestinal lamina propria lymphocytes in a time-and dose-dependent manner.When mice were kept in same cages,the effect of melatonin on relieving colitis disappeared,and there were no significant differences in body weight,colon length,intestinal pathology,immune cell infiltration,inflammatory factor secretion and tight junction protein expression(P>0.05).It was found that melatonin could significantly change the α-diversity and β-diversity of intestinal microbiota as well as the composition of intestinal flora at the genus level in mice.Finally,it was confirmed that the intestinal microbiota interfered with melatonin could alleviate oxazolone-induced experimental colitis by fecal microbiota transplantation.Conclusions:(1)Melatonin inhibits the secretion of type 2 cytokines from ILC2 cells.(2)Melatonin modulates the gut microbiota in mice,increasing beneficial bacteria and reducing opportunistic pathogens.(3)The alleviation of oxazolone-induced experimental colitis by melatonin is dependent on the gut microbiota.(4)Transplantation of melatonin-treated microbiota alleviated oxazolone-induced colitis. |
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