Tongxieyaofang Decotion Alleviates IBS by Modulating CHRM3 and Gut Barrier

Abstract

Purpose

Through network pharmacology combined with molecular docking and in vivo validation, the study examines the unexplored molecular mechanisms of Tongxieyaofang (TXYF) in the treatment of irritable bowel syndrome (IBS). In particular, the potential pharmacological mechanism of TXYF alleviating IBS by regulating CHRM3 and intestinal barrier has not been studied.

Patients and Methods

LC-MS technique and TCMSP database were used in combination to identify the potential effective components and target sites of TXYF. Potential targets for IBS were obtained from Genecards and OMIM databases. PPI and cytoHub analysis for targets. Molecular docking was used to validate the binding energy of effective components with related targets and for visualization. GO and KEGG analysis were employed to identify target functions and signaling pathways. In the in vivo validation, wrap restraint stress-induced IBS model was employed to verify the change for cytoHub genes and CHRM3 expression. Furthermore, inflammatory changes of colon were observed by HE staining. The changes of Ach were verified by ELISA. IHC and WB validated CHRM3 and GNAQ/PLC/MLCK channel variations. AB-PAS test and WB test confirmed the protection of TXYF on gut barrier. The NF-κB/MLCK pathway was also verified.

Results

In TXYF decoction, LC-MS identified 559 chemical components, with 23 remaining effective components after screening in TCMSP. KEGG analysis indicated that calcium plays a crucial role in TXYF treated for IBS. Molecular docking validated the binding capacity of the effective components Naringenin and Nobiletin with cytoHub-gene and CHRM3. In vivo validation demonstrated that TXYF inhibits the activation of Ach and CHRM3 in IBS, and inhibits for the GNAQ/PLC/MLCK axis. Additionally, TXYF downregulates TNF-α, MMP9, and NF-κB/MLCK, while modulating goblet cell secretion to protect gut barrier.

Conclusion

TXYF inhibits Ach and CHRM3 expression, regulating the relaxation of intestinal smooth muscle via GNAQ/PLC/MLCK. Additionally, TXYF inhibits NF-κB/MLCK activated and goblet cell secretion to protect gut barrier.

Graphical Abstract

Keywords:

• irritable bowel syndrome
• tongxieyaofang decoction
• Ach
• CHRM3
• GNAQ/PCL/MLCK
• gut barrier

Abbreviations

Ach, Acetylcholine; AB-PAS, Periodic Acid Schiff and Alcian Blue Stain; BCL2, B-cell Lymphoma-2; CASP3, Caspase-3; CHRM1, Cholinergic Receptor Muscarinic 1; CHRM3, Cholinergic Receptor Muscarinic 3; ESα, Estrogen Receptor; ELISA, Enzyme Linked Immunosorbent Assay; GO, Gene Ontology; GNAQ (Gq), G Protein Subunit Alpha Q; GNA11 (G11), G Protein Subunit Alpha 11; HE, Hematoxylin and Eosin Staining; IHC, Immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; LC-MS, Liquid Chromatography-Tandem Mass Spectrometry; MLCK, Myosin Light-Chain Kinase; MMP9, Matrix Metalloproteinase-9; OMIM, Online Mendelian Inheritance in Man; PLC, Phospholipase C; PPI, Protein-Protein Interaction Networks; PTGS2, Prostaglandin-Endoperoxide Synthase 2; TNF-α, Tumor Necrosis Factor-α; TTD, Therapeutic Target Database; TCMSP, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.

Data Sharing Statement

All the data involved in this study are available in this published article.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This research was funded by Zhejiang Provincial Natural Science Foundation, China (No. LGD21H290002) and Zhejiang Provincial Natural Science Foundation, China (No. LQ20H030003).