Component characterization of Zanthoxylum nitidum extract and its anti-colitis effect through regulating PPARγ/NF-κB/iNOS signaling pathway and gut microbiota.

Ulcerative colitis (UC) is a chronic, recurrent inflammatory disease of the gastrointestinal tract, for which there are currently no effective therapeutical targets or curative agents. Zanthoxylum nitidum (Roxb.) DC., known as "liangmianzhen" in China, is a woody liana plant belonging to the Rutaceae family. Its dried root is widely used as a traditional Chinese herbal medicine that possesses anti-inflammatory, antibacterial, analgesic, and gastrointestinal protective properties. However, its protective effect against UC is still unclassified. This study aims to investigate the active ingredients, therapeutic efficacy, and probable mechanism of Z. nitidum root in experimental UC. An ethanol extract of Z. nitidum root (ZNE) was prepared, and its chemical composition was characterized. A murine UC model was established via affording 3 % dextran sulfate sodium (DSS), a well-established method to induce colitis resembling human UC. Disease condition was monitored to assess the efficacy of ZNE. ELISA, qRT-PCR, immunohistochemistry, Western blotting and molecular docking were used to elucidate the potential mechanism of ZNE against UC. Additionally, bacterial 16S rRNA gene sequencing was used to assess changes in gut microbiota. Component characterization indicated that the main components of ZNE are alkaloids, including magnoflorine, diosmin, hesperidin, sanguinarine, nitidine chloride, chelerythrine, skimmianine, and L-sesamin. Animal experiment displayed that ZNE treatment prominently attenuated the manifestations of DSS-elicited colitis, reduced DAI scores and histological damage, which exhibited more effectiveness than positive drug 5-ASA. Furthermore, ZNE markedly decreased pro-inflammatory cytokine levels while enhancing anti-inflammatory factors. Mechanistically, ZNE remarkably regulated the PPARγ/NF-κB/iNOS signaling pathway by irritating PPARγ, inhibiting IκBα phosphorylation, and preventing NF-κB p65 nuclear translocation, thereby suppressing iNOS expression. However, the role of ZNE in alleviating UC and regulating PPARγ/NF-κB/iNOS signaling pathways was partially abolished by treatment with PPAR-γ antagonist GW9662, indicating that ZNE activated PPARγ to exert its activities. Molecular docking analysis revealed that the above eight alkaloids from ZNE demonstrated significant binding affinity to PPARγ by interacting with critical amino acid residues in its active site. Additionally, ZNE alleviates UC by restoring gut microbiota balance, specifically by promoting the proportion of beneficial bacteria such as Bacteroidetes, Porphyromonadaceae, Prevotellaceae and Akkermansia_muciniphila, while reducing the Firmicutes/Bacteroidetes ratio and the abundance of Proteobacteria, Enterobacteriaceae, Bacteroides, Bacteroides_acidifaciens and Escherichia. Correlation analysis results suggested that these altered bacteria are closely related to the colitis-related parameters. In conclusion, this study is the first to demonstrate that ZNE exerts significant protective effects against UC, and the main active ingredients of ZNE are alkaloids. Its anti-colitis mechanism involves modulation of the PPARγ/NF-κB/iNOS signal pathway and restoration of gut microbiota homeostasis.