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.

Background: Changji’an Formula (CJAF) is an effective Chinese herbal prescription to treat irritable bowel syndrome with predominant diarrhea (IBS-D), which is derived from two famous classical prescription: Sijunzi decoction and Tong-Xie-Yao-Fang. However, the molecular mechanism has not been well defined. This study aims to illustrate the molecular mechanism of CJAF in the treatment of IBS-D. Methods: Chemical components of CJAF were determined by ultrahigh performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) and further verified by reference standards. IBS-D model was induced in C57BL/6J mice by a single edema with colonic infusion of 0.1 mL trinitrobenzene sulfonic acid (TNBS, 50 mg/mL) combined with 7 days of restraint stress, 2 h/d. The treatment group was given rifaximin (100 mg/kg) and high, moderate and low doses of CJAF by gavage for 7 days, respectively (n = 7). After administration, the main symptoms of IBS-D were tested, and behavioral tests were conducted using sucrose preference test and open field test. The colonic tissues of mice were obtained. Gene and protein expression of mast cell tryptase, nerve growth factor (NGF), tyrosine kinase receptor A (TrkA), phosphorylated TrkA, growth associated protein 43(GAP43) and neuro-specific enolase (NSE) were determined by RT-PCR, western blot and immunohistochemistry. Results: 36 compounds were identified by UPLC-Q-Orbitrap HRMS, and the determined components can be categorized into 7 chemical types, including 16 flavonoids, 7 triterpenoids, 4 alkaloids, 3 sesquiterpenoids, 2 monoterpene glycosides, 2 organic acids, 1 phenylpropanoids and 1 tannin. Animal experiment showed that the abdominal pain and diarrhea symptoms of IBS-D mice were alleviated by CJAF. The sucrose preference, total translocation distance and average velocity of movement in the open field test was upregulated. The mRNA and protein expression of mast cell marker tryptase, as well as NGF, phosphorylated TrkA, GAP43 and NSE in IBS-D mice colonic tissues were down-regulated by CJAF. Conclusions: CJAF could effectively alleviate abdominal pain and diarrhea symptoms of IBS-D by inhibiting the activation of colonic mast cells and the resultant activation of NGF/TrkA signal pathway. Therefore, CJAF affords a potential candidate for the treatment of IBS-D. Graphical