OBJECTIVE To explore the molecular mechanism of Xijiao Dihuang Decoction (XJDHT) in inhibiting inflammatory response in sepsis based on network pharmacology, molecular docking and in vitro and in vivo experiments.
METHODS Active components of XJDHT were screened using the TCMSP and HERB databases. Sepsis-related targets and histone H3K36 trimethylation (H3K36me3)-associated targets were retrieved from GeneCard, OMIM, and DisGeNet databases. A protein-protein interaction (PPI) network was constructed using the STRING database, and core targets were identified via Cytoscape 3.9.1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict potential mechanisms. Molecular docking validated ligand-receptor binding affinity. A cecal ligation and puncture (CLP) model was established in mice to evaluate 24-hour sepsis scores (MSS) and survival rates. Blood routine parameters were analyzed using an automated hematology analyzer. Serum IL-1β and TNF-α levels were measured by ELISA. Liver histopathology was assessed via HE staining, and H3K36me3 expression in Kupffer cells was detected by immunofluorescence. In vitro, LPS-induced THP-1 cells were used as an inflammatory model. ChIP-qPCR evaluated H3K36me3 enrichment at IL-1β and TNF-α gene loci. Western blot analyzed HIF-1α, EGFR, and AKT1 pathway proteins.
RESULTS A total of 28 active components of XJDHT were identified, corresponding to 987 gene targets, with 189 overlapping sepsis-related targets. Core targets included TNF, IL1B, and GAPDH. Enriched pathways included EGFR tyrosine kinase inhibitor resistance. Molecular docking confirmed strong binding between core components and key targets. In vivo, compared to the sham group, the CLP group exhibited significantly reduced 24-hour survival (P < 0.01), elevated MSS (P < 0.01), immune imbalance, and increased serum IL-1β and TNF-α levels (P < 0.01). High- and low-dose XJDHT intervention improved survival (P < 0.01), reduced MSS (P < 0.01), restored immune homeostasis, and dose-dependently suppressed IL-1β and TNF-α (P < 0.01). CLP mice showed elevated H3K36me3 in Kupffer cells and severe hepatic inflammation, while XJDHT dose-dependently reduced H3K36me3 (P < 0.05) and attenuated liver injury. In peritoneal macrophages, CLP upregulated H3K36me3, IL-1β, TNF-α, HIF-1α, p-AKT1/AKT1, and EGFR (P < 0.01), which were reversed by XJDHT (P < 0.05, P < 0.01). In vitro, LPS increased H3K36me3 and IL-1β and TNF-α expression (P < 0.01), with ChIP-qPCR confirming H3K36me3 enrichment at IL-1β loci (P < 0.01). Treatment with 15% XJDHT-containing serum for 24 h reduced H3K36me3 (P < 0.01), diminished its recruitment to IL-1β loci(P < 0.01), and inhibited LPS-induced activation of EGFR, HIF-1α, and p-AKT1/AKT1 (P < 0.05, P < 0.01). HIF-1α agonist Dimethyloxallyl Glycine(DMOG) further validated that XJDHT suppressed H3K36me3-mediated epigenetic remodeling via HIF-1α-related pathways.
CONCLUSION XJDHT inhibits inflammatory responses, regulates immune homeostasis, and improves sepsis prognosis, potentially by modulating H3K36me3 epigenetic modifications at IL-1β loci.
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