Integrated network pharmacology and experimental validation reveal the anti-inflammatory mechanisms of Wuwei Shaji Powder in hepatic ischemia–reperfusion injury

Abstract: Background: Hepatic ischemia–reperfusion injury (HIRI) is primarily mediated by oxidative stress and inflammatory activation, yet effective pharmacological interventions remain limited. Objectives: This study aimed to elucidate the key active ingredients, hub targets and molecular pathways through which Wuwei Shaji Powder may exert anti-inflammatory effects in HIRI using integrated network pharmacology and experimental validation. Methods: Active components of WSP and their predicted targets were obtained from TCMSP and HERB, while HIRI-related genes were obtained from GeneCards, OMIM and analysis of differential expression for the GEO dataset GSE228782. Differentially expressed genes were identified using the thresholds of |log2FC| ≥ 1 and adjusted P-value, also known as false discovery rate (FDR), < 0.05. Common targets underwent GO and KEGG enrichment, PPI network construction and molecular docking. Subsequently, functional verification was performed using a hypoxia/reoxygenation (H/R) model in AML12 cells treated with low- or high-dose WSP. Cell viability, inflammatory cytokines, ROS levels and protein expression of TLR4, AKT1, p-AKT (Ser473) and p-NF-κB were evaluated. Results: Network analysis highlighted inflammation- and immunity-related pathways, with AKT1, TNF, JUN, STAT3 and TP53 characterized as potential core regulatory targets. Docking results indicated favorable binding between selected WSP components and AKT1. Experimental data demonstrated that WSP significantly restored cell viability (P < 0.001), reduced IL-6, TNF-α and IL-1β secretion (all P < 0.001), attenuated ROS accumulation (P < 0.001) and suppressed activation of the TLR4–AKT1–NF-κB axis, as indicated by reduced protein levels of TLR4, p-AKT (Ser473) and p-NF-κB (all P < 0.001). Conclusion: WSP mitigates HIRI-induced inflammatory injury mainly by suppressing TLR4-mediated activation of the AKT1/NF-κB signaling cascade, thereby attenuating ROS accumulation and pro-inflammatory cytokine secretion (IL-6, TNF-α and IL-1β). The data furnish mechanistic evidence for its therapeutic potential against ischemia–reperfusion–triggered hepatic inflammation.