The Mechanism of Mica on Protecting NSAIDs Induced Small Intestinal Mucosal Barrier Injury via ncRuPAR-PAR2 Pathway

Abstract

Objective: The study was to detect the role of ncRuPAR and Protease activated receptor (PAR)2 in the mechanism of Non-steroidal anti-inflammatory drugs(NSAIDs) induced small intestinal mucosal barrier injury. And to clarify the possible mechanism of mica in treatment NSAIDs induced small intestinal disease. Methods: Caco-2 cells were cultured and divided into 6 groups: control group, diclofenac sodium group, control RNAa group, ncRuPAR RNA activation (RNAa) group, diclofenac sodium + mica group, diclofenac sodium + FSLLRY-NH2 group. 40 μmol/L diclofenac sodium treatment for 24 hours to establish NSAIDs induced small intestinal epithelial cell injury model, 10% mica-containing serum treatment for 24h, 100uM PAR2 blocker FSLLRY-NH2 treatment for 24h. Blank vector or ncRuPAR overexpression vector were transfected into cells for 72h. Then Transwell assay were performed to detect cell permeability, qPCR were used to detect RNA expression of PAR2 and ncRuPAR, and western blot were performed to detect PAR2, occludin and claudin-1 expression. Result: NSAIDs-induced small intestinal epithelial cell injury model was successfully built. Compared with the control group, ncRuPAR and PAR2 were up-regulated, occludin and claudin-1 were down-regulated, cell permeability were increased in diclofenac sodium group (P 0.05). Compared with the control RNAa group, overexpression of ncRuPAR significantly up-regulated the expression of ncRuPAR and PAR2, down-regulated the expression of occludin and claudin-1, and increased cell permeability (P<0.01). Conclusion: When NSAIDs induced small intestinal injury occurs, ncRuPAR activation could up-regulate PAR2, causing the destruction of intestinal epithelial cell tight junction and the increase of intestinal epithelial cells permeability. Mica-containing serum has a similar effect as PAR2 blocker, which could antagonize the above-mentioned damage process and play a role in protecting the mechanical barrier function of the intestinal mucosa. Funding Statement: This research was supported by funding from Zhejiang Provincial Natural Science Foundation of China under Grant No.LY18H030001； the Medicine and Health Science and Technology Plan Projects in Zhejiang province(2017KY413), Traditional Chinese Medicine Science and Technology Plan of Zhejiang Province(2017ZA089, 2016ZB071, 2015ZZ012, 2014ZA030); National Natural Science Foundation of China (81573760);Medical Health Platform Plan Projects of Zhejiang Province (2015RCA020); Zhejiang Provincial Natural Science Foundation of China (LY16H030010). Declaration of Interests: The authors state: There is no conflict of interest. Ethics Approval Statement: Not required.