OBJECTIVE To reveal the activity and mechanism of the Tibetan medicine Jiejinabao(Gentiana crassicaulis Duthie ex Burk, GC) against hepatocellular carcinoma using UHPLC-MS/MS, network pharmacology, molecular docking combined with in vivo and in vitro experiments.

METHODS UHPLC-MS/MS was employed to identify the chemical constituents of GC and their categories. Active components of GC were screened through TCMSP, TCM-ID, HERB, HIT 2.0, and SymMap databases, and their potential targets were obtained using SwissTargetPrediction database. Disease targets of hepatocellular carcinoma were screened via GeneCards and OMIM databases. A “drug-component-target” network was constructed using Cytoscape 3.9.1 software to screen core components of GC. The STRING database was used to construct the protein-protein interaction network of intersecting targets for screening core targets. GO and KEGG enrichment analyses were performed using the DAVID database. Molecular docking was performed using ChemBio3D, AutoDockTools 1.5.6 and PyMol 3.1.0 software to determine the binding stability between core components of GC and key targets. In vitro, CCK-8 assay, Transwell invasion assay, wound healing assay and flow cytometry were used to detect the effects of GC on proliferation, invasion, migration, apoptosis and cell cycle of HepG2 hepatocellular carcinoma cells. In vivo, a subcutaneous H22-LUC transplanted tumor mouse model was established, and the in vivo anti-hepatocellular carcinoma effect of GC was evaluated by animal imaging, HE staining and immunohistochemistry. Finally, qPCR and Western blotting were adopted to verify the anti-tumor effect and molecular mechanism of GC.

RESULTS Five major active components of GC including hydroxyevodiamine, rutaecarpine, and diosgenin, as well as 10 core targets such as STAT3, PIK3CA, and AKT1 were identified and screened, and the PI3K/AKT pathway was recognized as the key pathway. Molecular docking results showed that targets PIK3CA, AKT1, BAX, BCL-2 and CDK4 exhibited strong binding activity with core components including rutaecarpine, diosgenin, oleanolic acid and evodiamine. In vitro experiments showed that GC could inhibit the proliferation, invasion and migration of hepatocellular carcinoma cells, and exert anti-hepatocellular carcinoma effects by inducing cell apoptosis and arresting cell cycle. In vivo experiments demonstrated that GC could effectively reduce tumor fluorescence intensity, tumor weight and volume, inhibit Ki-67 proliferation in tumor cells, with favorable in vivo safety. The positive staining areas of BAX and Cleaved Caspase-3 were significantly increased(P<0.001), accompanied by significant reductions of BCL-2, Cyclin D1, and p-AKT(P<0.001). Both in vivo and in vitro assays showed that GC upregulated the gene and protein expression of Caspase-3 and BAX, downregulated the expression of BCL-2, CDK4, and Cyclin D1, and reduced the protein ratios of p-PI3K/PI3K and p-AKT/AKT.

CONCLUSION GC can exert anti-hepatocellular carcinoma effects by inhibiting the PI3K/AKT signaling pathway, inducing hepatocellular carcinoma cell apoptosis and arresting cell cycle progression.