The Mechanisms And Protective Roles Of SDR5-Fc In Experimental Colitis

BackgroundStudies have shown that ulcerative colitis(UC)is caused by multiple factors that are characterized by immune disorders and the activation of inflammatory cells.Therefore,the activation of macrophages plays an important role in the development of the disease.Activation of the NLRP3 inflammasome is one of the major forms of macrophage activation,and it is also the major source of IL-1? inflammatory cytokine production.Under long-term inflammatory conditions,colonic epithelial cells are damaged and the rate of apoptosis is significantly increased,further aggravating the progression of the disease.For the disease,inhibition of inflammatory responses and reduction of colonic epithelial damage are the primary methods.TNF-related apoptosis-inducing ligand(TRAIL)is a type 2 transmembrane protein of the tumor necrosis factor superfamily,also known as Apo2 L,combination with its receptor DR5(TRAIL-R2),can trigger the programmed death of cells.Moreover,DR5 and TRAIL can activate NF-?B,MAPK pathway.It can also induce the production of pro-inflammatory cytokines,such as IL-1? and TNF?.This suggests that the TRAIL-DR5 pathway may regulate macrophage activation through the NLRP3 inflammasome.In this study we discussed whether TRAIL regulates macrophage activation through the NLRP3 inflammasome and participates in inflammation.At the same time,we use sDR5-Fc fusion protein to block TRAIL-DR5 pathways to observe whether it has an improvement on mice with acute ulcerative colitis.ObjectsTo study the effect and mechanism of sDR5-Fc on ulcerative colitis in mice.MethodsAcute ulcerative colitis model was established with 3% DSS.Animals were randomly divided into six groups: Normal,DSS,hIgG,rhTNFR-Fc,sDR5-Fc 10 mg/kg and sDR5-Fc 20 mg/kg.sDR5-Fc was injected every two days and the disease activity index(DAI)which mainly included weight loss,diarrhea,and bleeding was measured daily.The mice were sacrificed on the sixth day of modeling,Mouse blood was centrifuged to measure serum IL-1? levels by ELISA;mesenteric lymph nodes were taken and macrophage changes were detected by flow cytometry;Then we got the whole colons and measure length.Collecting the distal colon in triplicate,Western blot was used to detect the expression of NLRP3 inflammasome and related inflammatory factor.RT-PCR was used to detect the gene expression of NLRP3 inflammasome and related inflammatory factor.The last part of colon was put in 4% paraformaldehyde,we made a co-location with NLRP3 and macrophage by serial section cutting technique.HE was designed to validate the damage of colon tissue and detect the side effects of sDR5-Fc on heart,liver,spleen,lung and kidney in mice.IHC was used to test change of macrophages in colon tissue.In NCM460 cells,we used different concentrations of LPS(100 ng/mL,200 ng/mL,400 ng/mL)alone or 100 ng/mL LPS and different concentrations of TRAIL(100 ng/mL,200 ng/mL,400 ng/mL,800 ng/mL)co-stimulated NCM460 cells,then we detected cell damage and apoptosis using caspase 3/7 activity detection kit and flow cytometry;After collecting cell protein,we used Western blot to detect DR5,Bax and Bak;Flow cytometry was used to detect cellular ROS release.We used 100 ng/mL LPS and different concentrations of TRAIL(50 ng/mL,100 ng/mL,200 ng/mL,400 ng/mL),sDR5-Fc(100 ng/mL,200 ng/mL,400 ng/mL)to costimulate THP-1.Subsequently,expression of NLRP3,pro-caspase-1,casp-p20,p-p65 and DR5 were detected by Western blot.Results(1)In vivo,our results showed that mice in the acute ulcerative colitis model group had body weight loss,increased DAI scores and shorter colon length;In addition,the colon tissue structure was damaged,inflammatory cell infiltration was increased,and the number of intestinal crypt was significantly decreased.However,in sDR5-Fc group,The above symptoms of mice were significantly improved.At the same time,sDR5-Fc can reduce macrophage infiltration in colon tissue,upregulate M2 macrophages and downregulate M1 macrophages in mesenteric lymph nodes.The results of immunohistochemistry on NLRP3 and macrophage localization indicated that infiltrating macrophages in the tissue were the main cells which expressed NLRP3.Whereas the genes and protein levels of NLRP3,Caspase1,and IL-1? in the colon tissue of sDR5-Fc group mice significantly decreased compared with IgG group.In addition,RT-PCR results showed that the gene levels of MCP-1 and TNF? in sDR5-Fc group were significantly lower than those in IgG group.(2)In vitro,our results showed that sDR5-Fc inhibited TRAIL-induced the activation of NLRP3 inflammasome in THP-1 cell.In addition,sDR5-Fc inhibited TRAIL-induced apoptosis of NCM460 cells.(3)HE showed that there was no difference in the histological structure of heart,liver,spleen,lung and kidney between treatment group and the normal group.ConclusionssDR5-Fc inhibited the activation of macrophage,NLRP3 inflammasome and reduced the damage of the colonic epithelium by binding to TRAIL,thus,it improved the disease progression of acute ulcerative colitis.