Role And Mechanism Of 4-octyl Itaconate In Protecting Intestinal Epithelial Barrier In Ulcerative Colitis

Background and aim:Ulcerative colitis(UC)is a chronic non-specific inflammatory bowel disease characterized by abdominal pain,diarrhea and mucopurulent and bloody stool.Its exact pathogenesis is not clear,and it is related to many factors such as genetics,immune disorders,intestinal mucosal barrier destruction,microbial imbalance and so on.In recent years,the incidence of UC has been increasing in China.Clinical drugs such as aminosalicylic acid preparations,glucocorticoids,immunosuppressants and biological agents are used to treat UC.However,the treatment effect is not good,and there are many problems such as drug dependence,resistance,and intolerance.Therefore,it is of great significance to explore safe and effective new drugs for the treatment of UC.Itaconate is one of the most highly induced metabolites in activated pro-inflammatory macrophages.Recent studies have found that it plays an immunomodulatory and antioxidant role in non-immune cells such as keratinocytes.It has shown good therapeutic prospects in disease models such as acute lung injury,sepsis and psoriasis.However,its role and mechanism in colonic epithelial cells and UC remain unclear.The aim of this study is to investigate the effect of 4-octyl itaconate(OI)on the colon mucosal barrier function and its role and mechanism in UC.In addition,this study also compared the therapeutic effects of the combination of OI and Mesalazine,the first-line clinical drug for UC,with those of the two drugs alone,to provide new experimental evidence for the exploration of new drugs for UC.Methods:1.Normal mice were injected intraperitoneally with OI for 6 consecutive days.Histological changes were observed by H&E staining.Western blot was used to detect the expression of tight junction proteins.RT-qPCR was used to detect the expression of inflammatory factors.2.C57BL/6N mice were given 3%Dextran Sulfate Sodium Salt(DSS)for 6 consecutive days to induce UC model.At the same time,25 mg/kg,50 mg/kg and 100 mg/kg OI were injected intraperitoneally.Daily body weight,fecal water content,and fecal occult blood were recorded.Colon length and spleen weight were measured on the sixth day.The changes of body weight and disease activity index(DAI)were recorded every day.Histological scoring was performed using H&E staining.RT-qRCR,Western blot,immunohistochemical staining,immunofluorescence staining and AB-PAS staining were used to detect the degree of colonic inflammation,apoptosis,colonic mucosal injury and oxidative stress related indicators in each group.3.The UC model was induced after intraperitoneal injection of clodronate liposome to deplete systemic macrophages.The daily body weight,fecal water content,and fecal occult blood were recorded.On the day 6,the colon length and spleen weight were measured,and the changes of daily DAI were counted.4.Human normal colonic epithelial cell line NCM460 was treated with 8 mmol/L H2O2 for 24 hours to induce oxidative stress.A co-culture group of H2O2 and 250 μmol/L OI was set up at the same time.Cell viability was detected by CCK-8 assay.Western blot and immunofluorescence staining were used to detect cell apoptosis,tight junction expression and oxidative stress related indicators.5.The UC model was established by drinking 3%DSS for 6 consecutive days,OI,Mesalazine,or a combination of the two drugs were also administered.Body weight,fecal water content,and occult blood were recorded every day.Colon length and spleen weight were measured on the sixth day.Histological scoring was performed using H&E staining.Results:1.Mice received a daily intraperitoneal injection of OI for 6 days.There were no significant differences in the depth,width,and number of colonic crypts between OI treated and control mice.There were no significant changes in the expression of tight junction proteins Claudinl and JAM-1 and inflammatory factors IL-1β,TNF-α,IL-17 and IL-12 in the colon.2.In 3%DSS-induced UC mice model,compared with the DSS group,the colon length was increased,the spleen weight was reduced,and the degree of body weight loss was reduced in the DSS+OI group.The DAI and H&E histological score of colon sections decreased.At the same time,compared with DSS group,the expression of inflammatory factors including IL-1β,IL-6,IL-17 and TNF-α and iNOS in colon tissue of DSS+OI group was significantly decreased,and the number of CD-45 positive cells was also significantly decreased.The apoptosis-related protein Cleaved caspase-3 was decreased.Further study found that the concentration of FITC-Dextran in the serum of DSS+OI group was significantly lower than that of DSS group.The expression of tight junction proteins Claudin1 and ZO-1 and adherens junction E-cadherin was significantly increased in the colon.Mucus secretion and MUC-2 content also increased in the colon.The antioxidant capacity of colon tissue was enhanced,which was manifested as decreased MDA content,increased T-SOD,GSH and GST contents.3.Clodronate liposome was injected intraperitoneally to deplete the macrophages.Acute colitis was then induced with the application of DSS dissolved in drinking water.Compared with DSS+Con-lip group,the number of M1 and M2 macrophages were significantly reduced in both DSS+Clo-lip and DSS+Clo-lip+OI groups.OI still alleviated the colitis induced by DSS such as shortened colons,increased spleen weight and increased DAI score after depletion of macrophages.4.In the H2O2-induced oxidative stress model of colonic epithelial cell NCM460,compared with the H2O2 group,the cell viability of H2O2+OI group was significantly increased.The expression of Cleaved caspase-3 and pro-apoptotic protein Bax was decreased,and the expression of anti-apoptotic protein Bcl-2 was increased.The expression of tight junction including Claudin1,JAM-1 and ZO-1 and adherens junction E-cadherin was significantly increased.The contents of GSH,T-SOD and T-AOC in cells were significantly increased.5.In the DSS-induced UC model,mice were co-treated with OI,Mesalazine or Mesalazine+OI respectively.It was found that OI,Mesalazine or Mesalazine+OI given to UC mice alleviated the shortening of colon length,increase in spleen weight,increase in DAI score,and increase in histological score compared with the DSS group.Compared with the DSS+OI group and DSS+Mesalazine group,the DAI score and colon histological score of UC mice were further reduced after simultaneous Mesalazine+OI treatment.Conclusions:1.OI alleviated DSS induced UC.2.OI inhibited oxidative stress of colon epithelial cells,reduced cell apoptosis,increased the expression of tight junctions,reduced intestinal mucosal permeability and protected intestinal mucosal barrier,thereby alleviating intestinal inflammation.3.OI and Mesalazine had synergistic therapeutic effects on DSS-induced UC.