Abstract: BACKGROUND: Animal experiments have demonstrated that Bushen Qiangjin capsule can significantly improve subchondral bone metabolism and abnormal bone remodeling, and delay the progression of knee osteoarthritis, providing experimental evidence for elucidating the mechanism of action of this drug in treating knee osteoarthritis.
OBJECTIVE: To systematically explore the mechanism of action of Bushen Qiangjin capsule in regulating inflammatory signaling pathways to improve knee osteoarthritis using network pharmacology, Mendelian randomization based on pooled data, two-sample Mendelian randomization, molecular docking, and cell experiments.
METHODS: (1) The effective components, core targets, and signaling pathways of Bushen Qiangjin capsule in treating knee osteoarthritis were obtained using network pharmacology tools. Potential targets were imported into the STRING platform to construct a protein interaction network for further screening of core targets. Mendelian randomization analysis based on pooled data was conducted to assess the causal relationship between core targets and the risk of knee osteoarthritis. Two-sample Mendelian randomization was used to analyze the causal relationship between mitogen-activated protein kinase 8 and knee osteoarthritis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on potential targets of Bushen Qiangjin capsule in intervening in knee osteoarthritis. Molecular docking was used to further clarify the interaction between the key active ingredients of Bushen Qiangjin capsule and its core target protein. (2) Five Sprague-Dawley rats were administered Bushen Qiangjin capsule at a dose of 0.243 g/kg per day via gavage for 7 consecutive days. Venous blood was collected after the last administration, and serum containing the drug was separated. Chondrocytes from the rat knee were cultured in three groups: a blank group received no treatment; a model group was treated with interleukin-1β for 24 hours to establish an osteoarthritis cell model; and a serum-containing group was treated with interleukin-1β for 24 hours followed by treatment with 10% drug-containing serum (volume fraction) for 24 hours. After treatment, chondrocyte morphology was observed by alicin blue and toluidine blue staining. The mRNA and protein expression of mitogen-activated protein kinase 8 and type II collagen were detected by RT-qPCR and western blot assay.
RESULTS AND CONCLUSION: (1) A total of 85 active ingredients related to Bushen Qiangjin capsule were obtained, among which kaempferol and ferulic acid were key components. Core targets included serine/threonine protein kinase 1 and phosphatidylinositol 3-kinase catalytic subunit α. Mendelian randomization and two-sample Mendelian randomization analyses based on the aggregated data indicated a direct causal relationship between mitogen-activated protein kinase 8 and knee osteoarthritis, and this relationship was positive. Gene Ontology enrichment results mainly involved biological processes such as phosphorylation, response to heterobiotic stimuli, and negative regulation of apoptosis. The Kyoto Encyclopedia of Genes and Genomes pathways mainly included signaling pathways such as phosphatidylinositol 3-kinase-serine/threonine protein kinase, tumor necrosis factor α, Toll-like receptor 4, hypoxia-inducible factor 1, and mechanosensory-related axes. Molecular docking results showed that kaempferol and ferulic acid exhibited good binding activity to core targets such as serine/threonine protein kinase 1, phosphatidylinositol 3-kinase catalytic subunit α, tyrosine kinase C, signal transduction and transcription activator 3, and tumor protein p53. (2) Alcian blue and toluidine blue staining showed that Bushen Qiangjin capsule promoted the synthesis of extracellular matrix in an osteoarthritis cell model. RT-qPCR and western blot analysis showed that, compared with the model group, the mRNA and protein expression levels of type II collagen were increased in the serum-containing group (P < 0.05), while the mRNA and protein expression levels of mitogen-activated protein kinase 8 were decreased (P < 0.05). (3) The results indicate that Bushen Qiangjin capsule may improve knee osteoarthritis-related damage by synergistically regulating inflammatory responses and extracellular matrix homeostasis through a multi-component, multi-target, and multi-pathway approach.

Key words: knee osteoarthritis, Bushen Qiangjin capsule, network pharmacology, Mendelian randomization, molecular docking, cell experiment