Active components Alignment of Gegen Qinlian Decoction for inflammatory bowel disease: exploring the possible role of the SphK1/S1P/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gegenqinlian Decoction (GQD) has been used as a folk remedy for gastrointestinal diseases in China over thousands of years, and demonstrates significant efficacy against inflammatory bowel disease (IBD). We previously showed that the active components alignment of GQD inhibits nuclear factor kappa B (NF-κB) p65 signal transduction and plays a key role in preventing and treating IBD. With growing insights into the gut microbiota and its metabolites, the molecular mechanisms underlying therapeutic effects of GQD against IBD require further elucidation. This is particularly relevant given the previously reported relationship between the host NF-κB pathway and the gut microbiota.

AIM OF THE STUDY: To explore the therapeutic mechanisms of GQD and its aligned active components against IBD, with a focus on microbiota-involved regulation of the host sphingosine kinase 1 (SphK1) /sphingosine-1-phosphate (S1P) / NF-κB signaling pathway, using both 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced rat colitis and dextran sulfate sodium salt (DSS)-induced mouse colitis models and cultured cells.

MATERIAL AND METHODS: In this research, the therapeutic effect of GQD on TNBS-induced colitis rats was evaluated through the measurement of body weight, colorectal length, disease activity index (DAI), colon length and hematoxylin-eosin staining. 16S rRNA gene sequencing and untargeted metabolomic analyses were performed to evaluate the restoration of gut microbial and metabolic homeostasis by GQD. Network pharmacology and molecular docking were then utilized to explore the potential relationships between specific targets and the active components alignment (ACA) of GQD on IBD treatment. To further validate the in vivo effects of the ACA, a dextran sulfate sodium salt (DSS)-induced mouse colitis model was established, and body weight, DAI, colorectum length, histopathology, S1P levels, and SphK1/S1PR1 expression were assessed. Experimental validation in LPS-stimulated RAW264.7 macrophages and NCM460 colonic epithelial cells was subsequently conducted to determine whether this active components alignment suppressed the SphK1/S1P/NF-κB pathway via molecular and cellular assays, including cell metabolomics, ELISA, Western blot, and immunofluorescence staining.

RESULTS: GQD significantly relieved symptoms, colonic inflammation, and intestinal damage in TNBS-induced colitis rats. It partially reversed gut microbiota dysbiosis, modulated sphingolipid metabolic pathways, and downregulated the expression of sphingolipid-related Sphk1 and S1PR1 proteins, as well as S1P levels, in colon tissues (p < 0.05). In the DSS-induced mouse colitis model, the ACA of GQD significantly alleviated body weight loss, DAI, colorectum shortening, and histopathological damage, and reduced S1P levels and SphK1/S1PR1 expression in colon tissues (p < 0.05 or p < 0.01). In vitro, the ACA of GQD attenuated LPS-induced inflammatory responses in RAW264.7 macrophages and NCM460 colonic epithelial cells, as reflected by significant reductions in the levels of NO, ROS, and pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β, and S1P production (p < 0.05 or p < 0.01). Mechanistic dissection revealed that this ACA of GQD suppressed SphK1 expression and S1P production, inhibiting IκBα degradation and NF-κB p65 nuclear translocation, as directly evidenced by immunofluorescence staining, thus repressing the transcription of downstream pro-inflammatory genes and alleviating intestinal inflammation.

CONCLUSION: GQD restored gut microecological homeostasis and rectified sphingolipid metabolic disturbances. Its active components alignment afforded anti-inflammatory and colitis-protective effects, partially via modulation of the SphK1/S1P/NF-κB signaling pathway. The consistent therapeutic and mechanistic effects observed in both TNBS-induced rat colitis and DSS-induced mouse colitis support that the eight-component alignment largely recapitulates the key effects of the complete GQD formulation. These findings provide novel mechanistic insights and a scientific rationale for the clinical application of GQD in the management of IBD.