Research On Mechanisms Of TRPA1 Modulators In Improving Ulcerative Colitis Of Mice Induced By Dextran Sulphate Sodium

Ulcerative colitis（UC）,a chronic and recurrent inflammatory disease,mainly involves the mucosa and submucosa of the colon and rectum.The main clinical features include diarrhea,abdominal pain,and mucus blood stool.The pathogenesis of UC is not fully elucidated,and the currently used drugs have limited efficacy,obviously side effects and drug resistance.Therefore,novel and effective drugs are urgently required.Studies show that the disease is mainly caused by abnormal immune responses mediated by CD4⁺T cells.In patients with inflammatory bowel disease,there are a large number of CD4⁺T cells infiltration and pro-inflammatory cytokines in the inflamed colon.Therefore,it is crucial to regulate the function of CD4⁺T cells for treatment of inflammatory bowel disease.TRPA1 is a non-selective calcium channel that is functionally expressed in CD4⁺T cells.Previous studies revealed that TRPA1 was correlated with neuro-inflammation and related pain.However,the role of TRPA1 in CD4⁺T cells is not well-elucidated in the pathology of ulcerative colitis.We investigated the effects of TRPA1 desensitizer allyl isothiocyanate（AITC）and inhibitors such as A967079 and HC030031 on colitis in mice and explored the underlying mechanisms from the perspective of CD4⁺T cells in the present study.Objective:The present study aimed to investigate the effects of TRPA1 modulators on UC in mice and the underlying mechanisms from the perspective of CD4⁺T cells.Methods:Colitis in mice was induced by dextran sulphate sodium（DSS）.Real-time PCR and Western Blot were used to determine the mRNA and protein levels of cytokines secreted by CD4⁺T cells in mouse colon and mesenteric lymph nodes.CCK-8 assay was used to examine the activity of TRPA1 modulators on the proliferation of spleen CD4⁺T cells.Enzyme linked immunosorbent assay（ELISA）was employed to examine the activity of TRPA1 modulators on secretion of cytokines by spleen CD4⁺T cells.We further examined the activity of TRPA1 modulators on the proliferation and secretion of cytokines of spleen lymphocyte cells from wild-type and Trpa1 knock-out mice by CCK-8 assay and ELISA.Whole cell patch-clamp technique was used to record the membrane current induced by AITC in HEK-293 cells transfected with TRPA1.Results:TRPA1 desensitizer AITC（10 mg/kg）and antagonist A967079（10 mg/kg）obviously ameliorated the weight loss and colon contraction in mice with colitis.Compared with the DSS group,loss of epithelial cells,destruction of crypts,and infiltration of inflammatory cells were obviously improved in the TRPA1modulator-treated group,suggesting the efficacy of AITC and A967079 in treatment of colitis induced by DSS in mice.We found that the used TRPA1 modulators obviously decreased the mRNA and protein levels of IFN-γ,IL-4,and IL-17A,while increased the levels of TGF-βin the colon tissues and mesenteric lymph nodes of mice with colitis.As the cytokines IFN-γ,IL-4,IL-17A,and TGF-βwere not merely secreted by CD4⁺T cells in colon tissues,we next isolated CD4⁺T cells from the spleens of mice with colitis using immunomagnetic beads and investigated the activities of TRPA1 desensitizer AITC and antagonists including A967079 and HC030031 on the proliferation and secretion of cytokines of CD4⁺T cells in vitro.We found that AITC（3,10,30μmol/L）,A967079（10,30,100μmol/L）and HC030031（10,30,100μmol/L）suppressed the proliferation of CD4⁺T cells after activation.These modulators significantly inhibited the secretion of IFN-γ,IL-4,and IL-17A,while promoted the production of TGF-βin activated CD4⁺T cells.Given the possible multiple targets of small chemical molecules,we investigated the role of TRPA1 in the function of CD4⁺T cells by examining the activities of AITC（3,10,30μmol/L）,A967079（10,30,100μmol/L）and HC030031（10,30,100μmol/L）on the proliferation and secretion of cytokines of Trpa1 knock-out mouse splenic lymphocytes in vitro.We found that the efficacy of these tool agents were obviously weakened in suppressing the proliferation and cytokine section of splenic lymphocytes isolated from Trpa1^-/-mice compared with that from wild type mice,suggesting the critical role of TRPA1 in function of CD4⁺T cells.We finally examined the activity of AITC in activating TRPA1 and recorded the membrane current using the whole-cell patch clamp technique.It was found that membrane current obviously increased after exposure to AITC and decreased over time,suggesting desensitivity of AITC on TRPA1.Conclusions:1.TRPA1 desensitizer AITC and antagonist A967079 obviously ameliorated the colitis of mice induced by DSS.2.TRPA1 desensitizer AITC and antagonist A967079 reversed the changes of IFN-γ, IL-4,IL-17A,and TGF-βin colon tissues and mesenteric lymph nodes of mice with colitis.3.TRPA1 desensitizer AITC and antagonists A967079 and HC030031 suppressed the section of IFN-γ,IL-4,and IL-17A and promoted the production of TGF-βin CD4⁺T cells in vitro.4.The effect of TRPA1 modulators on ulcerative colitis is mainly produced by the TRPA1 channel acting on CD4⁺T cell.