OBJECTIVE To investigate the pharmacodynamic material basis and molecular mechanisms of Guiqi Baizhu prescription(GQBZP) in the treatment of uveal melanoma(UM) through weighted network pharmacology, molecular docking, and in vitro and in vivo experiments.

METHODS The effect of GQBZP on the progression of UM was observed using nude mouse tumor xenograft models. Network pharmacology was applied to screen intersecting key targets between GQBZP and UM. The weights were assigned based on the sovereign-minister-assistant-messenger formula. A “herbal ingredient-potential active component-key target” network was constructed. GO and KEGG pathway enrichment analysis was performed on key targets within the network. K-means clustering was applied for target clustering. Molecular docking and clustering analysis were performed between GQBZP components and MEK to identify potential active constituents. CCK8 assays were utilised to screen effective and safe concentrations of quercetin, kaempferol, aloe emodin, emodin and calycosin against UM cells. The effects of representative components quercetin and kaempferol on UM cell apoptosis were assessed by Hoechst staining. The impact of quercetin and kaempferol on the UM cell cycle was evaluated by means of flow cytometry. The effects of representative components on the protein expression of MEK, p-MEK, ERK, p-ERK, Bax, Bcl-2, CDK2, and CDK4 were detected by Western blotting analysis. RT-qPCR was utilised to detect the effects on the mRNA expression of Bax, Bcl-2, CDK2, and CDK4.

RESULTS GQBZP could delay the progression of UM by promoting tumor cell apoptosis and inhibiting tumor cell proliferation. Network pharmacology analysis suggested that MEK may be a key therapeutic target for GQBZP in treating UM. The molecular docking results indicated the potential active components may include quercetin, kaempferol, aloe-emodin, emodin and calycosin. In vitro experiments demonstrated that representative components of GQBZP, quercetin(40 μmol·L⁻¹) and kaempferol(20 μmol·L⁻¹), inhibited UM cell viability(P<0.001) while exhibiting protective effects on HEK293T cells(P<0.05). The Hoechst staining method revealed that quercetin and kaempferol promoted apoptosis in UM cells. The results of the flow cytometry analysis demonstrated that the components in question induced UM cell cycle arrest at the G0/G1 phase. The results of Western blotting analysis indicated that quercetin and kaempferol were able to reduce the levels of p-MEK/MEK, p-ERK/ERK, Bcl-2, CDK2, and CDK4 protein expression, whilst increasing the level of Bax protein expression. RT-qPCR analysis demonstrated that quercetin and kaempferol led to a decrease in the mRNA levels of Bcl-2, CDK2, and CDK4, whilst concurrently increasing Bax mRNA levels.

CONCLUSION In vivo experiments demonstrate that GQBZP effectively delays the progression of UM. Its representative components, quercetin and kaempferol, target the MEK pathway by inhibiting MEK phosphorylation. This has been demonstrated to promote apoptosis in UM cells and to cause cell cycle arrest at the G0/G1 phase, thereby suppressing UM cell viability.