Study On The Effect Of Saposhnikovia Divaricata On Intestinal Barrier Of PI-IBS And Its Mechanism

Background:Intestinal barrier dysfunction plays a significant role in the mechanisms leading to Post infectious irritable bowel syndrome(PI-IBS).Tong-Xie-Yao-Fang(TXYF) is an important prescription in the treatment of IBS, which contains Saposhnikovia divaricata(SD).And ancient medical books has some related exposition such as ―wind resistance like barrier‖.Objective:To prove the protective effect of SD on intestinal mechanical barrier in PI-IBS rats,illustrate the scientific connotation ―wind resistance like barrier‖; And to observe the role of SD on Caco-2 cell monolayer barrier model,further the study of ―wind resistance like barrier‖, vertify the regulation of SD on Caco-2 cell barrier dysfunction induced by PAR-2 / MLCK / ZO-1 signaling pathway, reveal the molecular mechanism of the defend of SD on Caco-2 cell barrier.Methods:1、Rat model of PI-IBS were established, through measuring the rat visceral pain pressure threshold and the number of tools within 24 hours, the therapeutic effect of high, medium and low dose of SD on rat of PI-PBS could be evaluated, and the intestinal epithelial cells in rats were observed by TEM.2、Caco-2 cell monolayer model were established, in order to assess the therapeutic effect of high, medium and low dose of SD on Caco-2 cell barrier, barrier function was analyzed by measurement of transepithelial electrical resistance(TEER) and percentage of fluorescein permeation(PFP),and the change of TEER and PFP in TRYP-induced intestinal barrier dysfunction Caco-2 cellswhich has been treated apically with the high, medium and low dose of SD were dectected; the expression of PAR-2 and MLCK m RNA was analyzed by RT-PCR, and the level of ZO-1 protein expression was measured by western blot; the distribution of the tight juction(TJ) proteins was examined by immunofluorescence.Results:1、SD can enhance the rat visceral pain pressure threshold and decrease the number of stools within 24 hours 14 days later;Furthermore, SD can improve intestinal barrier dyfunction of PI-IBS rats in different degrees;2、The safty dose of SD on Caco-2 cell is in the range of 0-250g/ml and 30g/ml, 60g/ml, 120g/ml dose are feasible; Additionally, SD elevate the TEER and the PFP of Caco-2 cell monolayers, SD also increase the TEER and the PFP of TRYP-induced intestinal barrier dysfunction cells; SD inhibitthe expression of PAR-2 m RNA and MLCK m RNA and increase the level of ZO-1 protein expression as well as ameliorate the distribution of the TJ proteins in TRYP-induced intestinal barrier dysfunction Caco-2 cells.Conclusion:SD can enhance the intestinal mechanical barrier of rat that support the exposition of ―wind resistance like barrier‖ and the underlying mechanism is to mediate PAR-2 / MLCK / ZO-1 signaling pathway.