| Background:Intestinal mechanical barrier dysfunction is the key pathological related to Post infectious irritable bowel syndrome(PI-IBS).Tong-Xie-Yao-Fang is the most frequently used for PI-IBS.At present,there are many studies on the efficacy and mechanism on Tong-Xie-Yao-Fang,lack of research on the effect of Intestinal mechanical barrier dysfunction.Atractylodes macrocephala(AM)and Saposhnikovia divaricate(SD)are the composition of Tong-Xie-Yao-Fang,The taste of AM is sweet and bitter,and it can nourish the spleen.Opposited to AM,the SD,tasted spicy and sweet,can dispel the wind and disperse wet.The synergistic effect of AM and SD is consistent with the pathogenesis of PI-IBS,in which it's mechanism is unknown.Objective:To investigate the effect of AM,SD and compatibility of AM with SD on PI-IBS rats and dyfunctional Caco-2 cell monolayer,gaining the evidence of synergistic protective effects of AM and SD on intestinal barrier.And to vertify the regulation of AM and SD on Caco-2 cell barrier dysfunction induced by PAR-2/CaM/MLCK signaling path way,reveal the underlying molecular mechanism.Methods:Rat model of PI-IBS was established.Through measuring the rat visceral pain pressure threshold and recording the number of tools,the therapeutic effect of AM and SD on PI-IBS rat could be evaluated.The level of IL-6 and IL-8was measured by ELISA.The expression of Mucin-1 protein was analyzed by Western Blot.The level of the tight juction Occludin and Claudin-1 proteins was examined by immunofluorescence.Caco-2 cell monolayer intestinal barrier dyfunction induced by TRYP was established.The transmembrane resistance(TEER)and yellow fluorescence transmission(PFP)rate of intestinal Caco-2 cells were measured to evaluate the effect of compatibility of AM with SD on dyfunctional Caco-2 cells monolayers.The expression of PAR-2 mRNA was analyzed by Q-PCR.The activity of MLCK was examed by ELISA.The level of CAM and ZO-1 protein expression was measured by Western Blot.Results:AM,SD and compatibility of AM with SD could enhanced the rat visceral pain pressure threshold and decrease the number of stools;Furthermore,AM,SD and compatibility of AM with SD could decrease the level of IL-6 and IL-8,increase the expression of Mucin-1 protein,improve the expression of Occludin and Claudin-1 protein in different degrees.AM,SD and compatibility of AM with SD each could increase the transmembrane electrical resistance(TEER)and decrease the percentage of fluorescein permeation(PFP)of Caco-2 cell monolayers;The compatibility of AM with SD can inhibited the expression of PAR-2 mRNA,CAM protein and MLCK activity,increase the level of ZO-1 protein expression.Conclusion:AM,SD and the compatibility of AM with SD has a therapeutic effect on intestinal barrier in different degree,and the compatibility of AM with SD has a synergistic effect.The mechanism may be related to the reduce of IL-6,IL-8 and LDH level,the up-regulation of the expression of Mucin-1 protein,Occludin and Claudin-1 protein,and the regulation of PAR-2/CaM/MLCK signaling path way. |
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