OBJECTIVE To explore the effect and mechanism of Tibetan medicine Gentiana urnula H.Smith in improving chemical liver injury based on network pharmacology combined with experimental verification.

METHODS Potential active components of Gentiana urnula H.Smith were screened by literature retrieval combined with the Swiss ADME database platform. Targets of potential active components were obtained using the TargetPrediction and TCMSP databases. Venn analysis was performed with liver injury targets obtained from the Genecards platform, and a “drug-component-target” network was established to identify its hepatoprotective active components and core targets. The protein-protein interaction(PPI) network of core target proteins was constructed using the String platform and CytoScape software, and GO and KEGG pathway enrichment analysis was performed through the DAVID database. Forty-eight C57 mice were randomly divided into blank group, model group, positive drug group, as well as high, medium, and low-dose Gentiana urnula H.Smith groups. All mice received continuous intragastric administration for 7 d, and an acute liver injury model in mice was established by intraperitoneal injection of 0.5% CCl₄ olive oil solution. The liver appearance was observed and pathological HE staining was performed. Serum and liver tissues of mice were collected to detect the levels of liver function indicators(ALT, AST, ALB, LDH), inflammatory indicators(IL-6, IL-1β, TNF-α), and oxidative stress indicators(MDA, SOD, GSH). Western blotting and RT-qPCR were used to detect the expression of proteins and genes related to the PI3K/AKT/NF-κB signaling pathway, and immunohistochemistry was used to detect NF-κB protein expression. A CCl₄-induced Hep-G2 cell injury model was established to screen the hepatoprotective active fractions of Gentiana urnula H.Smith. Cells were treated with high, medium, and low doses of ethanol fractions of Gentiana urnula H.Smith for 24 h before model establishment, and cell survival rate was detected using the CCK-8 method. Biochemical methods were used to detect ALT, AST, and LDH levels. Western blotting and RT-qPCR were used to detect the expression of proteins and genes related to the PI3K/AKT/NF-κB signaling pathway, flow cytometry was used to detect cell apoptosis rate.

RESULTS A total of 29 components and 177 potential hepatoprotective targets were screened out from Gentiana urnula H.Smith. The core targets including AKT1, ALB, EGFR and so on, which were involved in multiple pathways including PI3K/AKT/NF-κB. Animal experiments showed that Gentiana urnula H.Smith could improve CCl₄-induced extensive necrosis and structural damage of liver tissue in mice, reduce levels of ALT, AST, LDH, TNF-α, IL-1β, IL-6, and MDA, and elevate the contents of SOD, and GSH. Meanwhile, it regulated the PI3K/AKT/NF-κB pathway and inhibited the expression of apoptosis-related genes and proteins. Cell experiments confirmed that the ethanol extract fraction of Gentiana urnula H.Smith rigescens could relieve CCl₄-induced Hep-G2 cell damage, reduce the levels of ALT, AST, and LDH, and modulate the PI3K/AKT/NF-κB signaling pathway, thereby decreasing the cell apoptosis rate.

CONCLUSION Gentiana urnula H.Smith can alleviate oxidative stress and inflammatory responses, improving chemical liver injury, and its mechanism may be related to the inhibition of cell apoptosis via regulating the PI3K/AKT/NF-κB signaling pathway.