Haiou Wang,1 Wenkang Huang,1 Xiaoya Pan,1 Meizi Tian,1 Jiahui Chen,1 Xiaotong Liu,1 Qin Li,1 Jianhua Qi,2 Yiping Ye,1 Lijuan Gao1 

1School of Pharmacy, Hangzhou Medical College, Hangzhou, 310013, People’s Republic of China; 2College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China

Correspondence: Lijuan Gao; Yiping Ye, Hangzhou Medical College, 182 Tianmushan Road, Hangzhou, Zhejiang, 310013, People’s Republic of China, Email gaolijuan04141002@126.com; yeyiping2005@163.com

Purpose: To explore the protective effect and underlying mechanism of Quzhou Aurantii Fructus flavonoids (QAFF) on Ulcerative colitis (UC).
Methods: The constituents of QAFF were accurately determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The therapeutic impacts of QAFF were assessed in dextran sulfate sodium (DSS)-induced UC mice, focusing on the changes in body weight, disease activity index (DAI), colon length, histological assessment of colonic tissues, levels of pro-inflammatory cytokines, and expression of tight junction proteins. Western blotting confirmed key regulatory proteins within the differential signaling pathways, guided by transcriptome analysis. Additionally, the influence of QAFF on the gut microbiome was explored through 16S ribosomal RNA (rRNA) sequencing. The alterations in endogenous metabolites were detected by untargeted metabolomics, and their potential correlation with intestinal flora was then examined utilizing Spearman correlation analysis. Subsequently, the regulation of gut microbiome by QAFF was validated by fecal microbiota transplantation (FMT).
Results: Eleven flavonoids, including Naringin and hesperidin, were initially identified from QAFF. In vivo experiments demonstrated that QAFF effectively ameliorated colitis symptoms, reduced IL-6, IL-1β, and TNF-α levels, enhanced intestinal barrier integrity, and downregulated PI3K/AKT pathway activation. Furthermore, QAFF elevated the levels of beneficial bacteria like Lachnospiraceae_NK4A136_group and Alloprevotella and concurrently reduced the pathogenic bacteria such as Escherichia-Shigella, [Eubacterium]_siraeum_group, and Parabacteroides. Metabolomics analysis revealed that 34 endogenous metabolites exhibited significant alterations, predominantly associated with Glycerophospholipid metabolism. These metabolites were significantly correlated with those differential bacteria modulated by QAFF. Lastly, the administration of QAFF via FMT ameliorated the colitis symptoms.
Conclusion: QAFF could ameliorate inflammatory responses and intestinal barrier dysfunction in DSS-induced UC mice probably by modulating the PI3K/AKT signaling pathway and gut microbiome, offering promising evidence for the therapeutic potential of QAFF in UC treatment.

Keywords: Quzhou Aurantii Fructus flavonoids, Ulcerative colitis, inflammation, Gut microbiota, PI3K/AKT, Fecal microbiota transplantation