Protective Effect And Mechanism Of Itaconate On DSS-induced Ulcerative Colitis Mice

Background:Inflammatory bowel disease（IBD）is a multifactorial inflammatory disease that includes Crohn’s disease（CD）and ulcerative colitis（UC）.UC is a kind of chronic inflammatory bowel disease that occurs in the mucosa and submucosa of colon and rectum,it’s prone to attack repeatedly and likely to cause many complications,such as intestinal perforation,peritonitis,or colorectal cancer.Itaconate is one of macrophage metabolites,it is generated by decarboxylation of cis-aconitate in activated macrophages under the stimulation of lipopolysaccharide（LPS）and other factors.Studies have shown that itaconate contributes to the regulation of cellular metabolism,oxidative stress,and it has significant anti-inflammatory effect.But the role and mechanism of itaconate on ulcerative colitis（UC）are not clear.In this research,we used the dextran sulphate sodium（DSS）-induced colitis models on mice,and investigated the possible impact and mechanism of itaconate on UC.On the other hand,immune-responsive gene 1（Irg1）is a key one regulating the expression of itaconate,so our research focused on whether Irg1 deficiency will affect the structure and function of intestinal epithelium in mice,to further explore the effect role of Irg1 in UC.Methods:Frist,C57BL/6 male mice were orally received 3%and 5%DSS for 9 days,then C57BL/6 male mice were orally received 3.5%DSS for 9 days.Data were collected by recording mice bodyweight,characteristics of feces,hematochezia,and the general motion daily.At the end of the experiment,mice were sacrificed,the colon lengths were compared,and histopathological examination was confirmed by hematoxylin and eosin（HE）staining.According to our previous results,we determined that 3.5%DSS was used for 7 days and tap water for 2 days to induce UC mice model in C57BL/6 mice.For treatment,DSS-induced mice were injected intraperitoneally with 4-octyl itaconate（4-OI）once a day.The body weight,hematochezia,fecal characteristics,colon lengths of mice were assessed.Four hours before execution,the mice were given fluorescein-isothiocyanate-dextran（FITC-dextran）by gavage.After the mice were sacrificed,the blood was collected and centrifuged immediately.Colon tissues were harvested and the length was measured.Serum was used for assaying fluorescence intensity of FITC to reflect intestinal epithelial permeability.The tight junction protein claudin-1 and occludin of colon tissue were analyzed by Western Blot to further observe the alteration of intestinal epithelial barrier function.Part of the colon tissue were embedded in paraffin for HE staining.Intestinal mucus and cell apoptosis were assessed by periodic acid-Schiff（PAS）and TUNEL staining.The expression of Nuclear Factor-E2-related factor 2 and NADPH:quinone oxidoreductase 1were detected by Western Blot to analyze the possible mechanism of itaconate on DSS-induced mice.Finally,to compare the differences of intestinal structure between Irg1knockout mice and the wild type mice,HE staining was used for observing the changes in intestinal structure.For the purpose of the discrepancy between the intestinal epithelial cell apoptosis and proliferation,TUNEL and immunohistochemistry staining of proliferating cell nuclear antigen（PCNA）were applied.The secretion of intestinal mucus was examined by PAS and immunohistochemistry staining of mucin2.Results:1.The DSS-induced mice presented significant symptoms and histopathological changes of UC characterized by weight loss,diarrhea,and gradually aggravated bloody stool.Histopathological changes showed shorten colon,thicken intestinal submucosa,abnormal crypt structure,and infiltration of inflammatory cells in UC mice.2.Compared with 3%DSS,the 5%DSS treated mice displayed worse symptoms of colitis,the weight loss,diarrhea,and bloody stool were more serious,even death under the 5%DSS treatment.Compared with 5%DSS,3%DSS treated mice presented less weight loss,and the appearance time of change in fecal characteristics and hematochezia was relatively late.By contrast,the weight loss and fecal characteristics change of 3.5%DSS treated mice were in a moderate time,no mice death during the experiment.UC induced with 3.5%DSS was more coincident with this experiment.3.3.5%DSS-induced mice were treated with 4-OI,the situation of weight loss,diarrhea,and hematochezia were improved,the extent of shortening colon decreased.Histologically showed that there were no edema and thickening intestinal submucosa,complete intestinal lumen structure and crypt structure,a few inflammatory cells infiltration.4.4-OI treatment alleviate the increased intestinal epithelial barrier permeability induced by DSS.The expression of tight junction protein claudin-1 and occludin was up-regulated,and the FITC levels were decreased.Strong evidence manifested 4-OI alleviated the damage of intestinal epithelial barrier in DSS-induced mice.5.4-OI treatment attenuated the apoptosis of intestinal epithelial cells and mucosal injury induced by DSS,decreased the number of TUNEL positive cells and increased the content of intestinal mucus.6.4-OI treatment enhanced the expression of Nrf2 and NQO1 proteins in colon of UC mice,which can alleviate oxidative stress injury induced by DSS.7.The deficiency of Irg1 affected the structure of intestinal epithelium,Irg1^-/-mice presented deepen crypts,increased cell apoptosis,disturbed the balance between cell apoptosis and proliferation,reduced secretion of intestinal mucus and mucin2 content.Conclusion:This research investigated the colitis symptoms,intestinal epithelial barrier function,and intestinal mucosal injury in DSS-induced mice,and the evaluation confirmed that 4-OI can play a protective role in DSS-induced mice,this effect may be related to the activation of the Nrf2antioxidant pathway.On the other hand,Irg1^-/-mice with the intestinal barrier dysfunction and damaged intestinal mucosal barrier indicated that the deficiency of Irg1 may impact intestinal epithelium structure and function.Taken together,these findings suggested an important role for Irg1 in modulating the structure and function of intestinal epithelium under the basic circumstances.In DSS-induced colitis mice,itaconate plays an antioxidant effect by activating the Nrf2 signal,so as to alleviate the progression of the disease,which provides a new idea for the treatment of oxidative stress in UC.