Dachaihu decoction alleviates septic intestinal epithelial barrier disruption via PI3K/AKT pathway based on transcriptomics and network pharmacology

Ethnopharmacological relevance: Dachaihu decoction (DCH) is a famous and ancient TCM formula, extensively utilized for over 1800 years in treating gastrointestinal and inflammatory conditions. Our previous study showed that DCH ameliorated intestinal damage and modulated the gut microflora in septic rats. However, the material basis for these effects and the underlying mechanism of action remains ill-defined. We aimed to explore the pharmaceutical ingredients of DCH and its mechanism in mitigating sepsis-induced intestinal epithelial barrier disruption (IEBD).

Materials and methods: Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) was used to identify DCH composition. A septic rat model and Caco-2 cells were employed to investigate DCH's effects on IEBD. Transcriptomics and network pharmacology were used to predict potential mechanisms, which were further validated by molecular docking and dynamics simulations. The Modified Murine Sepsis Score (mMSS) and histological assessments were performed. Serum fluorescence intensity of FD4 and the expression of Occludin were evaluated to assess intestinal barrier integrity. And p-PI3K P85, PI3K P85, p-AKT, Akt, Bax and Bcl-2 were determined by Western blot. Cell viability was determined using CCK-8 assay, IL-6 and TNF-α by ELISA and quantitative Real-Time PCR (RT-qPCR). The integrity and permeability of single layer of Caco-2 cells were assessed via transepithelial resistance (TEER), Alkaline Phosphatase (ALP) activity and FD4 permeability.

Results: UHPLC-HRMS identified 180 compounds in DCH. DCH significantly reduced mMSS, improved pathological conditions in the ileum, decreased FD4 serum fluorescence, and enhanced Occludin expression. Transcriptomic and network pharmacology analyses identified the PI3K/Akt pathway as a critical mechanism of action. Molecular docking and dynamics simulations confirmed strong binding of DCH components to PIK3R1. DCH upregulated p-PI3K and p-AKT in ileum tissue of septic rats. DCH improved cell viability, decreased IL-6 and TNF-α, promoted cell survival and Occludin level, and upregulated p-PI3K and p-AKT in LPS-stimulated Caco-2 cells. DCH also maintained TEER, ALP activity and decreased FD4 permeability and these effects were reversed by PI3K Inhibitor, LY294002. DCH also downregulated Bax expression and increased Bcl-2 levels in both septic rats and LPS-stimulated Caco-2 cells.

Conclusion: DCH ameliorates sepsis-induced IEBD via PI3K/Akt pathway activation, offering a novel therapeutic perspective for sepsis-related intestinal dysfunction.

Dachaihu decoction; Intestinal epithelial barrier; Network pharmacology; PI3K/AKT pathway; Sepsis; Transcriptomics.