Oridonin Attenuates TNBS-Induced Post-Inflammatory Irritable Bowel Syndrome via PXR/NF-κB Signaling
Abstract
To investigate the beneficial effects of oridonin, a diterpenoid compound isolated from Rabdosia rubescens, on the inflammatory response in a TNBS-induced post-inflammatory irritable bowel syndrome (PI-IBS) model and to explore the underlying mechanism, we utilized a PI-IBS rat model and Caco-2 cell lines. We found that intestinal barrier function, reflected by the lactulose/mannitol (L/M) ratio and tight junction protein levels, was significantly improved by oridonin treatment. Additionally, oridonin reduced inflammation by inhibiting the phosphorylation of NF-κBp65 and decreasing the levels of its downstream genes, including iNOS, COX-2, IL-1β, and IL-6. Molecular docking studies demonstrated a strong binding affinity between oridonin and PXR. In Caco-2 cells, oridonin markedly inhibited LPS-induced NF-κB activation in a PXR-dependent manner while inducing PXR and its target genes, CYP3A4 and P-gp. This was associated with decreased NF-κB expression and the restoration of the intestinal barrier. These findings indicate that the therapeutic effects of oridonin on experimental PI-IBS, achieved through the repair of intestinal barrier function, may be closely associated with the regulatory role of the PXR/NF-κB signaling pathway. Oridonin may serve as a PXR ligand for the development of therapies for PI-IBS.
Introduction
Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder affecting 10–20% of the general population. Up to one-third of IBS patients develop post-infectious or post-inflammatory IBS (PI-IBS) following acute gastrointestinal infection or inflammation. The condition significantly impacts patients’ quality of life and places a substantial burden on healthcare systems. PI-IBS is considered a multifactorial disease with an unclear precise mechanism, although recent evidence suggests that impaired intestinal mucosal barrier function may play a significant role in its pathogenesis. Preservation of barrier function offers potential for developing new therapeutic options for treating the disease.
The intestinal barrier prevents the entry of pathogenic microorganisms into the body. When this barrier is compromised, harmful intraluminal substances can penetrate, triggering chronic low-grade inflammation, which has been observed in multiple inflammatory bowel diseases, including PI-IBS. Elevated levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 have been reported in the serum and mucosa of PI-IBS patients. These cytokines disrupt tight junctions by modulating critical cell signaling pathways, including the NF-κB pathway, which is a central regulator of the inflammatory response and is implicated in the pathology of IBD and PI-IBS.
Pregnane X receptor (PXR) is a ligand-activated nuclear receptor abundantly expressed in the liver and intestine. It can be activated by various chemicals, including rifampicin and herbal compounds, through binding to its ligand-binding pocket. Initially recognized for regulating drug-metabolizing enzymes and transporters, PXR has recently been shown to have therapeutic potential in IBD by reducing the expression of NF-κB and its target genes, thereby decreasing intestinal inflammation. Therefore, the PXR/NF-κB signaling pathway may play a critical role in the mechanism of PI-IBS and represent a potential therapeutic target.
Oridonin, a diterpenoid compound from Rabdosia rubescens, has demonstrated various pharmacological effects, including anti-inflammatory, antibacterial, and antitumor properties. Previous studies have shown that oridonin can inhibit NF-κB activation and has therapeutic effects on PI-IBS. However, it remains unclear whether the therapeutic effects of oridonin on PI-IBS are related to its anti-inflammatory activity and whether this mechanism involves the PXR/NF-κB signaling pathway. This study aims to investigate the protective effects of oridonin in PI-IBS by repairing intestinal barrier function and elucidating the role of the PXR/NF-κB signaling pathway in this process.
Materials and Methods
Chemicals and Reagents
Oridonin, rifampicin, ketoconazole, lactulose, mannitol, and LPS were obtained from commercial suppliers. Cell culture materials, including DMEM and fetal bovine serum, were prepared for experiments on Caco-2 cells.
Animals and Model Preparation
Male Sprague-Dawley rats were housed under controlled conditions before experiments. A PI-IBS model was induced using TNBS enema under anesthesia following fasting. After 28 days, an abdominal withdrawal reflex (AWR) test was conducted to confirm visceral hyperalgesia, and a disease activity index (DAI) test assessed the degree of intestinal inflammatory activity.
Experimental Design
Rats were divided into normal, PI-IBS, and oridonin-treated groups to investigate the therapeutic effects of oridonin. After 14 days of oridonin administration, AWR tests were conducted, followed by intestinal permeability tests using the lactulose/mannitol ratio, colon length measurement, and histopathological evaluation. Additional experiments were conducted to observe the effect of oridonin on PXR expression, including groups treated with rifampicin and ketoconazole.
Abdominal Withdrawal Reflex
AWR scores were recorded to evaluate visceral sensitivity by inserting a latex balloon into the colon of anesthetized rats and measuring the response to incremental colorectal distension.
Disease Activity Index
DAI was calculated based on body weight, stool consistency, and intestinal bleeding to assess the degree of inflammation in the intestine.
In Vivo Intestinal Permeability Assay
Urine samples were collected after administration of a lactulose/mannitol solution to measure the L/M ratio, reflecting intestinal permeability using high-performance liquid chromatography.
Molecular Docking
Autodock software was used to perform molecular docking studies between oridonin and the ligand-binding domain of PXR to evaluate potential binding interactions.
Cell Culture and Drug Treatment
Caco-2 cells were cultured and treated with oridonin and LPS to investigate their effect on NF-κB activation and PXR signaling. Cell viability assays determined appropriate dosing for oridonin treatment.
Western Blotting
Protein samples from tissues and cells were analyzed by western blotting to evaluate the expression of NF-κB, PXR, and tight junction proteins.
Real-Time PCR
mRNA levels of target genes were measured using quantitative real-time PCR, normalized to β-actin levels, to assess gene expression changes associated with treatments.
Statistical Analysis
Data were analyzed using one-way ANOVA and Student’s t-tests, with p-values <0.05 considered statistically significant.
Results
Oridonin Attenuated PI-IBS Symptoms in TNBS-Induced PI-IBS Rats
Oridonin treatment reduced DAI scores, improved colon length, and increased AWR thresholds, indicating a reduction in visceral hypersensitivity and inflammation in PI-IBS rats.
Oridonin Ameliorated Intestinal Permeability and Gut Barrier Function in PI-IBS Rats
Oridonin significantly decreased the L/M ratio in PI-IBS rats and upregulated tight junction protein expression, indicating improved intestinal barrier integrity.
Oridonin Suppressed Inflammatory Action by Regulating NF-κB Signaling Pathway in PI-IBS Rats
Oridonin reduced the mRNA expression of inflammatory cytokines and NF-κB while inhibiting NF-κB p65 phosphorylation, demonstrating anti-inflammatory effects via NF-κB pathway suppression.
The Effect of Oridonin on PXR Signaling
Oridonin treatment increased the expression of PXR and its target genes, CYP3A4 and P-gp, in PI-IBS rats and normal rats, indicating activation of PXR signaling.
Molecular Docking of PXR to the Ligand-Binding Domain
Docking studies confirmed that oridonin can bind to the ligand-binding domain of PXR, supporting its role as a potential PXR agonist.
Oridonin Improved Gut Barrier Function in Caco-2 Cells by Regulating PXR/NF-κB Signaling Pathway
Oridonin treatment reversed LPS-induced NF-κB activation while increasing PXR and CYP3A4 expression in Caco-2 cells, supporting its role in enhancing gut barrier function via the PXR/NF-κB signaling pathway.
Discussion
Oridonin was found to alleviate PI-IBS symptoms by restoring intestinal barrier integrity and reducing inflammation through the regulation of the PXR/NF-κB signaling pathway. These findings indicate that oridonin, as a PXR agonist, could serve as a potential therapeutic agent for treating PI-IBS by targeting impaired intestinal barrier function and chronic inflammation.