Researches On The Effects And Mechanisms Of Cinnamaldehyde On Ulcerative Colitis

Objective: Ulcerative Colitis(UC)is a chronic inflammatory disease for the characteristics of the inflammation in colorectal mucosa and submucosa,often accompanied by abdominal pain,diarrhea,mucinous pus and bloody stools and other clinical manifestations.Although the pathogenesis of UC remains unclear,intestinal immune dysfunction is considered to be the main cause of the disease.Therefore,exploring the pathogenic mechanism of immune cells in UC and seeking targeted drugs to interfere with immune cells is a new way to treat the disease.Cinnamaldehyde is one of the main active compounds extracted from cinnamon,a natural product of cinnamon.Cinnamaldehyde has superior hypoglycemic,antibacterial and anti-inflammatory activities.However,the effect of Cinnamaldehyde on UC is still unclear.Therefore,this research explored the efficacy and mechanism of cinnamaldehyde on UC based on innate immune cells,macrophages and adaptive immune cells,CD4~+ T lymphocytes.Methods: 1.UC model of mice was established to investigate the efficacy of cinnamaldehyde on UC by evaluating body weight,disease activity index(DAI),histopathological assessment and inflammatory cytokines expression.2.Immunofluorescence was used to investigate the effect of cinnamaldehyde on macrophages in the colon of UC mice;and the macrophages were induced to produce inflammatory response in vitro,so as to verify the effect of cinnamaldehyde on inflammatory cytokines in macrophages.Then,western blotting was used to explore the signaling pathways related to the inhibition of macrophage inflammation by cinnamaldehyde.3.RT-PCR was used to investigate the effects of cinnamaldehyde on NLRP3 inflammasomes,miRNA-21 and miRNA-155 in RAW264.7 cells.Moreover,inhibition or overexpression of miR-21 and miR-155 was used to investigate the effects of cinnamaldehyde on macrophage inflammation.4.Chronic UC model was established in mice to explore the peak time of expression of CD4~+ T cell subsets and determine the optimal administration model.Flow cytometry,immunofluorescence staining and RT-PCR were used to detect the effect of cinnamaldehyde on CD4~+ T cell subsets in UC mice.5.The expression of metabolites in serum of mice was detected by metabonomic to predict the potential target of cinnamaldehyde on UC.Western blotting and RT-PCR were used to detect the expression of target signaling proteins and genes in the colon of UC mice.6.Th17 cells were induced and differentiated in vitro to explore the mechanism of cinnamaldehyde on UC.Antagonizing or knocking out the potential target to verify the key role of target that cinnamaldehyde inhibiting Th17 cells.Results: 1.Cinnamaldehyde can alleviate UC by improving the body weight loss,disease activity index,inhibiting the expression of inflammatory factors,relieving the inflammatory infiltration of the colon,reducing the production of NLRP3 inflammasome,miR-21,miR-155 and the expression of macrophages in the colon of UC mice.2.The expression levels of inflammatory cytokines in RAW264.7 cells,peritoneal macrophages and U937-differentiated macrophage cells were inhibited after cinnamaldehyde administrated in colon of UC mice.Meanwhile,cinnamaldehyde decreased the expression levels of NLRP3 inflammasome,miR-21 and miR-155 in RAW264.7 cells,while inhibition or overexpression of miR-21 or miR-155 had no significant effect of cinnamaldehyde on macrophage inflammatory factors.3.Cinnamaldehyde administration down-regulated the protein expression levels of AKT/m TOR and COX2 in RAW264.7 cells.4.The expression of CD4~+ T lymphocyte subsets in the chronic UC model peaked from 7 to 14 days.In addition,administration of cinnamaldehyde can reduce the expression of Th17 cells and related molecules in spleen,mesenteric lymph nodes and the colon of UC mice.5.The administration of cinnamaldehyde can regulate S1P2 and Rho-GTPase signaling pathways in the colon of UC mice,and the deficiency of S1P2 gene can increase the proportion of Th17 cells in the spleen,and up-regulate the m RNA level of IL-17 A in the colon.6.Cinnamaldehyde could inhibit the differentiation of Th17 cells in vitro,while antagonistic S1P2 expression could reduce the inhibitory effect of cinnamaldehyde on Th17 cell differentiation,and S1P2 gene deletion could block the effect of cinnamaldehyde on Th17 cell differentiation.At the same time,S1P2 gene deletion also leads to the low expression of lnc RNA H19 and MIAT in spleen cells of WT mice.7.Cinnamaldehyde not only inhibited the level of lnc RNA H19,but also up-regulated the level of MIAT in the colon of UC mice.Although cinnamaldehyde can improve the reduction of lnc RNA H19 and MIAT in differentiated Th17 cells,S1P2 gene deletion has no significant effect on the expression of lnc RNA H19 and MIAT in differentiated Th17 cells.Conclusion: Cinnamaldehyde can significantly improve UC,and its mechanism may not only alleviate the inflammatory response of colon macrophages by inhibiting the AKT /m TOR signaling pathway and reducing the expression levels of miR-21 and miRNA-155,but also reduce the expression level of miR-21 and miRNA-155.It is also possible to regulate the S1P2/ Rho-GTPase signaling pathway to inhibit Th17 cell differentiation and coregulate lnc RNA H19 and MIAT to improve the inflammatory response.