Tongxieyaofang eases IBS through CHRM3 regulation and gut barrier protection

According to the findings of a recent research, Tongxieyaofang (TXYF) reduces acetylcholine (Ach) and cholinergic receptor muscarinic 3 (CHRM3) expression, inhibits the G Protein Subunit Alpha Q/Phospholipase C/ Myosin Light-Chain Kinase (GNAQ/PLC/MLCK) signaling axis, and alleviates irritable bowel syndrome (IBS) diarrhea by dilating the colon. Additionally, it shields the gut barrier by regulating tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB)/MLCK axis, and goblet cell secretion.

Researchers evaluated TXYF's potential targets and pathways for relieving IBS using network pharmacology, providing a more reliable basis for the clinical application of TXYF decoction. The study utilized Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) to determine TXYF's effective components and target sites, obtaining potential targets from Genecards and Online Mendelian Inheritance in Man (OMIM) databases.

Protein-Protein Interaction Networks and cytoHub analysis were used for target identification.  For binding energy validation and visualization, molecular docking was employed. For exploring target functions and signaling pathways, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were utilized. Wrap restraint stress-triggered IBS model was used for in vivo authentication. Inflammatory changes in the colon were observed by hematoxylin and eosin (HE) staining.

Enzyme linked immunosorbent assay (ELISA) confirmed Ach changes, while immunohistochemistry and western blot validated CHRM3 and GNAQ/PLC/MLCK channel variations. The Periodic Acid Schiff and Alcian Blue Stain (AB-PAS) and western blot tests confirmed TXYF's protective effects on the gut barrier, and the NF-κB/MLCK pathway was also validated. Following screening in TCMSP, LC-MS revealed 559 chemical components in the TXYF decoction, leaving 23 efficient components. KEGG analysis revealed that calcium plays a key role in the treatment of IBS with TXYF.

The binding ability of the active ingredients nobiletin and naringenin with cytoHub-gene and CHRM3 was confirmed by molecular docking. In vivo validation showed that TXYF hinders the stimulation of Ach and CHRM3 in IBS and also suppresses the GNAQ/PLC/MLCK axis. Furthermore, TXYF modulates goblet cell secretion to preserve the intestinal barrier while downregulating NF-κB/MLCK, TNF-α, and matrix metalloproteinase-9 (MMP9). These outcomes highlight TXYF's potential as an IBS treatment and support its clinical use.