DDR1 Promotes Experimental Colitis In Mice By Activating NLRP3 Inflammsome-mediated Pyroptosis

Objective:To investigate the effect of DDR1 on NLRP3 inflammasome-mediated pyroptosis of experimental colitis in mice and its possible mechanisms.Methods:1.SPF wild-type male C57BL/6 mice（6-8 weeks,20±2g）were randomly divided into 4 groups:WT NC group,WT DSS group,WT DSS+low-dose dasatinib group（4 mg/kg/d）and WT DSS+high-dose dasatinib group（8 mg/kg/d）.The WT NC group were administered pure water,while the WT DSS and dasatinib-treatment groups were administered 3.5%DSS for 7 days to induce acute colitis,then the mice were sacrificed on the 8th day after anesthesia,and their colon and spleen were collected:（1）The mental status,hair glossiness,diet and water intake were observed daily,and the body weight,fecal characteristics and blood stool conditions of mice were recorded,then the change rates of body weight and disease activity index（DAI）were calculated;（2）On the 8th day,the colon length,spleen weight and spleen index of the mice were recorded;（3）About 1cm of the bottom colon tissue was taken for HE staining,and the changes of colon structure were observed under the microscope,and then colon histopathological score was performed.（4）q RT-PCR was performed to detect the m RNA expression of NLRP3,caspase-1 and GSDMD in colon tissues of the mice.2.J774A.1 macrophages which express endogenous DDR1 were cultured in vitro:（1）The cytotoxicity of dasatinib on J774A.1 cells was detected by CCK8,and IC₅₀ was calculated;（2）Western blot verified the inhibitory effect of dasatinib on DDR1phosphorylation;（3）Cells were divided into three groups:Control group,LPS group,and Dasatinib+LPS group,and the expressions of NLRP3,pro-caspase-1,cleaved caspase-1,GSDMD,and N-GSDMD were detected by western blot;（4）The m RNA levels of NLRP3,caspase-1 and GSDMD were detected by q RT-PCR;（5）The expressions of IL-1βand IL-6 were detected by q RT-PCR and ELISA.Results:1.Acute colitis models were induced by 3.5%DSS,and the results showed that:（1）Compared with the WT NC group,the WT DSS group experienced much weight loss and had a higher DAI,but both low dose and high dose of dasatinib could alleviated these effects induced by DSS,and the effect of high dose dasatinib was more obvious.（2）Compared with WT DSS group,the colon length of low-dose dasatinib group and high-dose dasatinib group were longer（5.88±0.32 vs 4.95±0.17,p<0.01;6.73±0.19 vs 4.95±0.17,p<0.0001）,as same as the spleen index（5.67±0.90 vs8.35±0.37,p<0.001;3.88±0.69 vs 8.35±0.37,p<0.0001）.（3）Compared with WT DSS group,the colon histopathological scores of low-dose dasatinib group and high-dose dasatinib group were lower（9.33±0.58 vs 12.33±0.58,p<0.05;5.00±1.73 vs12.33±0.58,p<0.0001）.（4）Compared with WT DSS group,the m RNA levels of NLRP3,caspase-1,and GSDMD were decreased in low-dose dasatinib group（NLRP3:29.61±5.02 vs 134.50±7.63,p<0.0001;caspase-1:6.71±1.83 vs 20.71±3.06,p<0.0001;GSDMD:10.88±2.32 vs 33.43±4,76,p<0.0001）,and decreased significantly in WT DSS+high-dose dasatinib group（NLRP3:12.83±3.01 vs 134.50±7.63,p<0.0001;caspase-1:1.48±0.82 vs 20.71±3.06,p<0.0001;GSDMD:3.30±0.59 vs 33.43±4,76,p<0.0001）.2.Cell experiment results:（1）Dasatinib inhibited the activity J774A.1 cells dose-dependently,and the IC₅₀=2.9n M.（2）Dasatinib inhibited the phosphorylation of DDR1 compared with collagenⅠ（ColⅠ）-stimulated J774A.1 cells（0.68±0.07 vs0.90±0.06,p<0.05）.（3）Compared with LPS group,NLRP3,pro-caspase-1,cleaved caspase-1,GSDMD,and N-GSDMD protein expressions decreased in Dasatinib+LPS group（NLRP3:0.90±0.16 vs 0.36±0.04,p<0.01;pro-caspase-1:1.04±0.15 vs0.64±0.02,p<0.01;cleaved caspase-1:1.09±0.04 vs 0.69±0.10,p<0.01;GSDMD:1.06±0.06 vs 0.84±0.01,p<0.05;N-GSDMD:0.47±0.15 vs 0.79±0.10,p<0.05）.（4）NLRP3,caspase-1,and GSDMD m RNA expressions in J774A.1 cells were decreased in Dasatinib+LPS group compared with LPS group（NLRP3:0.98±0.28 vs1.64±0.09,p<0.01;caspase-1:1.57±0.62 vs 2.94±0.65,p<0.05;GSDMD:1.35±0.12 vs2.22±0.57,p<0.05）.（5）q RT-PCR results showed that compared with LPS group,the expression of IL-1βand IL-6 in Dasatinib+LPS group was decreased（373.50±104.50vs 1057±129.30,p<0.0001;38.40±28.22 vs 700.80±112.70,p<0.0001）,ELISA results also showed that dasatinib inhibited the expression of IL-1βand IL-6（16.71±0.57 vs21.43±2.37,p<0.05;208.0±17.11 vs 583.80±33.82,p<0.0001）.Conclusion:（1）Inhibition of DDR1 can significantly relieve experimental colitis induced by DSS in mice;（2）DDR1 promotes colitis by regulating NLRP3inflammasome-mediated pyroptosis of macrophages.