Uncovering key anti-inflammatory constituents and mechanism of Wuwei Xiaodu Decoction by a combined strategy of phytochemistry, AIDD, network pharmacology, and in vitro and in vivo assay.

BACKGROUND: Wuwei Xiaodu decoction (WXD), a renowned prescription in traditional Chinese medicine, is widely used to treat various skin diseases, including atopic dermatitis. However, the bioactive constituents and anti-inflammatory mechanisms remain unclear. PURPOSE: The study was designed to investigate the most important bioactive components and the anti-inflammatory molecular mechanisms of WXD that have not been fully elucidated. MATERIALS AND METHODS: The mixed herbs of WXD were extracted by different solvents, including ethyl-acetate (EA) and petroleum-ether (PE). HPLC/LC-MS and GC-MS were performed to identify the chemical composition of WXD-EA and WXD-PE, respectively. The RAW264.7 cell line was used to assess the inflammatory responses, while CCK8 assay was employed to assess the cytotoxicity. Nitric oxide (NO) levels were quantified using the Griess assay. The protein expression levels were evaluated by western blot analysis. Nuclear translocation of p-STAT3 were analyzed by immunofluorescence and western blot. The targets and pathways were predicted by open-source databases. 1-chloro-2,4-dinitrobenzene (DNCB)-induced BALB/c mice was utilized for evaluating inflammatory responses. H&E staining was performed to assess the histopathological changes. RESULTS: The best anti-inflammatory active fractions of WXD were found to be WXD-PE and WXD-EA.123 compounds from WXD-PE and 7 representative compounds with high content from WXD-EA were successfully identified. The in vitro anti-inflammatory activity studies confirmed that both WXD-PE and WXD-EA can significantly inhibit the production of NO, TNF-α, IL-6, and IL-1β in LPS-induced RAW264.7 cells, leading to a downregulation of COX-2 and iNOS expression, as well as a reduction in the phosphorylation levels of ERK, p38, and JNK proteins. Notably, the nuclear expression levels of phosphorylated STAT3 were significantly downregulated. Additionally, WXD-H2O and WXD-EA significantly alleviated DNCB-induced acanthosis, lymphocyte and neutrophil infiltration, and collagen over-deposition in the skin of BALB/c mice. CONCLUSION: WXD-PE and WXD-EA, are identified as the most potent anti-inflammatory constituents of WXD as these fractions can effectively inhibit the MAPK signaling pathway and prevent the nuclear translocation of phosphorylated STAT3 in LPS-stimulated RAW264.7 cells. They also showed significant anti-inflammatory activity in the DNCB-induced inflammatory mouse model.