A study based on reverse network pharmacology of kidney stones identified that quercetin targets the myosin light chain kinase/myosin light chain 2 pathway in human renal HK-2 cells.

Objective: Kidney stones are a global health issue with high recurrence rates. This study aimed to apply reverse network pharmacology to identify and validate components of traditional Chinese medicines effective against kidney stones and to explore their therapeutic mechanisms. Methods: We utilized the GSE73680 gene expression profile from the Gene Expression Omnibus database and employed the weighted gene co-expression network analysis (WGCNA) method to identify modules containing kidney stone-related genes. The protein-protein interaction network was constructed. A reverse network pharmacology approach was employed to create a "TCM-components-targets" network to find the most effective small-molecule monomer. Western blot was used to detect myosin light chain kinase/myosin light chain 2 (MLCK/MLC2)-related protein expression, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to detect cell apoptosis. Molecular docking, molecular dynamics simulation, and ultraviolet spectroscopy experiments were used to predict and validate the interaction between quercetin and MLCK. Results: The WGCNA analysis identified 913 genes, and six candidate targets were further obtained from protein-protein interaction analysis. Reverse network pharmacology results showed that six candidate key targets were highly correlated with three active ingredients, namely quercetin, ursolic acid, and puerarin, as well as four traditional Chinese medicines. The prediction of quercetin targets and the integration of WGCNA results showed that quercetin may exert therapeutic effects on kidney stones through the MLCK/MLC2 pathway. In HK-2 cells, quercetin could inhibit the calcium oxalate monohydrate-activated MLCK/MLC2 pathway and reduce cell apoptosis. Molecular docking and molecular dynamics simulation results predicted that quercetin could directly interact with MLCK through a mechanism similar to ML-9, thereby inhibiting MLCK activity, and this direct interaction was demonstrated by ultraviolet absorption spectroscopy. Conclusion: Our findings suggest that quercetin may play a role in preventing and treating kidney stones by targeting the MLCK/MLC2 pathway.