Chang-Kang-Fang alleviates diarrhea predominant irritable bowel syndrome (IBS-D) through inhibiting TLR4/NF-κB/NLRP3 pathway

Abstract

Ethnopharmacological relevance

Chang-Kang-Fang (CKF), originated from traditional Chinese medicine (TCM) formulas, has been utilized to treat diarrhea predominant irritable bowel syndrome (IBS-D) based on clinical experience. However, the underlying mechanism of CKF for treating IBS-D remains unclear and need further clarification.

Aim of the study

The objective of this present investigation was to validate the efficacy of CKF on IBS-D model rats and to uncover its potential mechanism for the treatment of IBS-D.

Materials and methods

We first established the IBS-D rat model through neonatal maternal separation (NMS) in combination with restraint stress (RS) and the administration of senna decoction via gavage. To confirm the therapeutic effect of CKF on treating IBS-D, abdominal withdrawal reflex (AWR) scores, the quantity of fecal pellets, and the fecal water content (FWC) were measured to evaluate the influence of CKF on visceral hypersensitivity and the severity of diarrhea symptom after the intragastric administration of CKF for 14 days. Subsequently, enzyme linked immunosorbent assay (ELISA) was applied to assess the effect of CKF on neuropeptides substance P (SP) and 5-hydroxytryptamine (5-HT), as well as inflammatory cytokines in serum and in intestinal tissues. Further, colonic pathological changes, the amount of colonic mast cells, and the expression level of occludin in rat colon tissues, were investigated by hematoxylin-eosin (HE) staining, toluidine blue staining, and immunohistochemistry, respectively. To explore the underlying mechanisms, alterations in colonic RNA transcriptomics for the normal, model, and CKF treatment groups were assessed using RNA sequencing (RNA-Seq). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays were applied to validate the effect of CKF on predicted pathways in vivo and in vitro. In addition, to elucidate the potential active compounds in CKF, 11 representative components found in CKF were selected, and their anti-inflammation potentials were evaluated using LPS-treated RAW264.7 cell models.

Results

CKF treatment significantly reduced the number of fecal pellets, attenuated visceral hypersensitivity, and decreased 5-HT and SP concentrations in serum and colon tissues, along with a reduction in colonic mast cell counts, correlating with improved symptoms in IBS-D rats. Meanwhile, CKF treatment reduced the colonic inflammatory cell infiltration, lowered the levels of IL-6, TNF-α, and IL-1β in serum and colon tissues, and increased the occludin protein expression in colon tissues to improve inflammatory response and colonic barrier function. RNA-Seq, in conjugation with our previous network pharmacology analysis, indicated that CKF might mitigate the symptoms of IBS-D rats by inhibiting the Toll like receptor 4/Nuclear factor kappa-B/NLR family pyrin domain-containing protein 3 (TLR4/NF-κB/NLRP3) pathway, which was confirmed by WB, IF, and qRT-PCR experiments in vivo and in vitro. Furthermore, coptisine, berberine, hyperoside, epicatechin, and gallic acid present in CKF emerged as potential active components for treating IBS-D, as they demonstrated in vitro anti-inflammatory effects.

Conclusion

Our findings demonstrate that CKF effectively improves the symptoms of IBS-D rats, potentially through the inhibition of the TLR4/NF-κB/NLRP3 pathway. Moreover, this study unveils the potential bioactive components in CKF that could be applied in the treatment of IBS-D.