Oridonin Protects Intestinal Barrier Via Regulating PXR/NF-κB Signaling In Rats With TNBS-induced Post Inflammatory Irritable Bowel Syndrome

Objective:Intestinal mucosal barrier injury play key roles in visceral pain sensitivity in post-inflammatory irritable bowel syndrome(PI-IBS),however,the underlying mechanism is still unclear,and there is also no specific treatment.Our previous study found that oridonin(ORI)can improve intestinal mucosal barrier damage and relieve visceral pain sensitivity in PI-IBS rats.The underlying molecular mechanism of ORI remains to be further studied.Recent studies have shown that PXR/NF-κB negative feedback loop can regulate intestinal mucosal barrier function,but it is not clear whether oridonin can reduce visceral pain sensitivity by regulating intestinal barrier function through this loop.Firstly,the study investigated the efficacy of ORI in reducing the visceral pain sensitivity of PI-IBS;secondly,we explored the beneficial effect of oridonin on the intestinal mucosal barrier function by activating PXR using PXR agonistic/antagonistic in PI-IBS rat model;finally,we clarified the molecular mechanism of oridonin to regulate Caco-2 cell barrier function through the PXR/NF-κB negative feedback loop by using molecular biological methods in cells with overexpress/silence PXR.The research results are of great significance to clarify the molecular mechanism of ORI in the treatment of PI-IBS and to further understand the pathogenesis of PI-IBS.Methods:PI-IBS rat model was induced with 2,4,6-trinitrobenzene sulfonic acid(TNBS).The therapeutic effect of ORI on PI-IBS was determined with the disease activity index(DAI),abdominal contractile reflex test(AWR),colon length and colon histopathological changes as indicators;Using the index of lactulose/mannitol(L/M)ratio,the levels of intestines tight junction proteins as indicators to analyze the protective effect of ORI on the intestinal barrier function in PI-IBS rats and Caco-2 cells;to evaluate the mechanism of ORI in regulating intestinal mucosal barrier function,the m RNA and protein level of PXR/NF-κB signaling pathway and its downstream related factors were determinded in PI-IBS rats and Caco-2 cells after ORI treatment;Finally,the PXR antagonist/antagonist rat and cell models were constructed to explore the potential effect of ORI on PXR.Results:1.ORI pharmacodynamic evaluation: after the induced of TNBS,the PI-IBS group had significantly higher DAI index,lower visceral pain threshold,visual atrophy of colonic edema and increased histopathological score,indicating the successful rat model.And after 14 days ORI treatment,the above conditions were all relieved to different degrees,indicating that ORI has a therapeutic effect on rats.2.The intervention effect of ORI on intestinal barrier of PI-IBS: after 4w TNBS colon perfusion,the urine L/M of the PI-IBS group significantly increased,and the levels of occludin,claudin 1 and zo-1 of the colonic tissue tight junction proteins decreased,indicating that the intestinal barrier function of the PI-IBS group was impaired.After the intervention with ORI,the urine L/M level of the rats decreased,and the tight junction proteins were increased in different degrees.The Caco-2 cell barrier injury model was constructed in vitro,and the expression of tight junction protein S were significantly increased after ORI intervention.It suggested that ORI could improve the intestinal barrier function in PI-IBS rats and caco-2 cells.3.ORI’s role in improving intestinal barrier injury through the PXR/NF-κB signaling pathway: after the treatment of TNBS,PXR gene and protein expression levels in the colon of PI-IBS rats decreased,while the expression levels of p-p65/NF-κB protein increased,and the expression levels of these factors were reversed after ORI intervention.In addition,the levels of CYP3A4 and P-g P,which were the downstream target genes of PXR were decreased,while the expression levels of downstream target genes of NF-κB signaling pathway,NF-κB,i NOS,Cox-2,IL-1,IL-6 and TNF-α were significantly increased.In the Caco-2 cell lines,ORI could also significantly reverse the expression of factors related to the PXR/NF-κB signaling pathway,which was consistent with animal experiments,suggesting that ORI could improve PI-IBS by regulating the PXR/NF-κB signaling pathway.4.The stimulating effect of ORI on PXR: the molecular docking study showed that oridonin has good binding activity with PXR,suggesting that oridonin could activate PXR.Compared with normal control rats,PXR gene and protein expression levels were significantly increased in the colon tissues treated with ORI,which was similar to that of classical agonists.In vitro results showed that ORI could not only increase the expression of PXR in normal Caco-2 cells,but also stimulate the expression of PXR after intervention with PXR inhibitors,suggesting that ORI could be used as a substrate for PXR and had a potential role in stimulating PXR,suggesting that ORI could regulate the PXR/NF-κB signaling pathway to improve intestinal barrier injury.Conclusion:The results of the present study indicated that ORI had a therapeutic effect on PI-IBS rats by increasing the expression of tight junction protein in PI-IBS and LPS-induced Caco-2 cells and repairing the intestinal barrier injury.The molecular mechanism may be related to the regulatory effect of ORI on the PXR/ NF-κB signaling pathway.As a PXR ligand,ORI can be used in the development of PI-IBS therapeutic drugs.