Network pharmacology and experimental validation to explore the mechanism of Changji'an formula against irritable bowel syndrome with predominant diarrhea

Changji'an Formula (CJAF) is a Chinese herbal compound, which is effective against irritable bowel syndrome with predominant diarrhea (IBS-D) in clinic. However, the molecular mechanism has not been well defined. In the current study, the potential targets and signaling pathways of CJAF against IBS-D were predicted using network pharmacology analysis. The pharmacological mechanisms of CJAF against IBS-D and the potential mechanism were validated by using an IBS-D mouse model induced by enema with trinitrobenzene-sulfonic acid (TNBS) plus with restraint stress and further intervened with CJAF. A total of 232 active compounds of CJAF were obtained, a total of 397 potential targets for the active ingredients were retrieved and a total of 219 common targets were obtained as the potential targets of CJAF against IBS-D. GO and KEGG enrichment analyses showed that multiple targets were enriched and could be experimentally validated in a mouse model of IBS-D. The mechanisms were mainly converged on the immune and inflammatory pathways, especially the NF-κB, TNF and IL-17 signaling pathway, which were closely involved in the treatment of CJAF against IBS-D. Animal experiment showed that CJAF alleviated visceral hypersensitivity and diarrhea symptom of IBS-D. CJAF also restored the histological and ultrastructure damage of IBS-D. The result of Western blot showed that CJAF upregulated colonic tight junction proteins of ZO-1, Occludin and Claudin-1. Further results demonstrated that CJAF inhibited the protein expression of NF-κB/NLRP3 inflammasome pathway targets and downregulated proinflammatory mediators of IL-1β, IL-18, TNF-α. In conclusion, CJAF could effectively reduce inflammatory response and alleviate visceral hypersensitivity as well as diarrhea symptom of IBS-D by inhibiting the NF-κB/NLRP3 signaling pathway. This study not only reveals the mechanism of CJAF against IBS-D, but also provides a novel therapeutic strategy for IBS-D.