Action mechanisms of Bushen Qiangjin capsule in regulating inflammatory signaling pathways and improving knee osteoarthritis

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) Network pharmacology tools were used to obtain the active components, core targets, and signaling pathways of Bushen Qiangjin capsule in treating knee osteoarthritis. Potential targets were imported into the STRING platform to construct a protein-protein interaction network and further screen core targets. Mendelian randomization based on pooled data was conducted to evaluate 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 (MAPK8) and knee osteoarthritis. GO function and KEGG pathway enrichment analyses were performed on potential targets of Bushen Qiangjin capsule intervention in knee osteoarthritis. Molecular docking was used to further clarify the interaction between key active components of Bushen Qiangjin capsule and core target proteins. (2) Five SD rats were given Bushen Qiangjin capsule by gavage at 0.243 g/(kg·d) for 7 consecutive days. After the last administration, venous blood was collected and serum was separated to obtain drug-containing serum. Rat knee chondrocytes were divided into three groups for culture: blank group (no treatment), model group (treated with interleukin-1β for 24 h to establish an osteoarthritis cell model), and drug-containing serum group (treated with interleukin-1β for 24 h, then treated with 10% drug-containing serum for 24 h). After treatment, Alcian blue and toluidine blue staining were used to observe chondrocyte morphology. RT-qPCR and western blot were used to detect the mRNA and protein expression of MAPK8 and type II collagen. RESULTS AND CONCLUSION: (1) A total of 85 active components related to Bushen Qiangjin capsule were obtained, among which kaempferol and ferulic acid were key components. Core targets included serine/threonine-protein kinase 1 (AKT1), phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA), etc. Mendelian randomization based on pooled data and two-sample Mendelian randomization indicated that MAPK8 had a direct causal relationship with knee osteoarthritis and was positively correlated. GO enrichment results mainly involved biological processes such as phosphorylation, response to xenobiotic stimulus, and negative regulation of apoptotic process. KEGG pathways mainly included phosphatidylinositol 3-kinase-serine/threonine-protein kinase, tumor necrosis factor alpha, Toll-like receptor 4, hypoxia-inducible factor 1, and mechanosensory-related axes. Molecular docking results showed that kaempferol and ferulic acid had good binding activity with core targets such as AKT1, PIK3CA, tyrosine kinase C, signal transducer and activator of transcription 3, and tumor protein p53. (2) Alcian blue and toluidine blue staining showed that Bushen Qiangjin capsule could promote the synthesis of cartilage extracellular matrix in the osteoarthritis cell model. RT-qPCR and western blot showed that compared with the model group, the mRNA and protein expression of type II collagen increased (P < 0.05), while the mRNA and protein expression of MAPK8 decreased (P < 0.05) in the drug-containing serum group. (3) These results indicate that Bushen Qiangjin capsule may improve knee osteoarthritis-related damage through 'multi-component, multi-target, multi-pathway' synergistic regulation of inflammatory response and extracellular matrix homeostasis.

1. Introduction

Knee osteoarthritis is a degenerative disease characterized by articular cartilage degeneration, subchondral bone changes, osteophyte formation, and synovial lesions. Its clinical manifestations mainly include knee pain and tenderness, joint swelling, and stiffness, leading to decreased mobility and quality of life. Knee osteoarthritis has become one of the most common disabling diseases. Currently, non-steroidal anti-inflammatory drugs are mainly used to improve clinical symptoms in early and mid-stage knee osteoarthritis; however, they have significant gastrointestinal adverse effects and fail to fundamentally delay the progression of the disease. For advanced stages, total knee arthroplasty is often chosen, but surgical risks and treatment costs often deter patients. Traditional Chinese medicine (TCM) therapy has advantages such as fewer adverse reactions, high patient acceptance, high safety, and significant efficacy, playing an important role in the treatment of knee osteoarthritis.

In TCM, knee osteoarthritis is classified under the category of 'knee bi syndrome', with the pathogenesis considered as 'deficiency of liver and kidney as the root, and invasion of wind, cold, and dampness as the manifestation'. Bushen Qiangjin capsule, a TCM formula, has been used clinically for years. Animal experiments have shown that it can significantly improve subchondral bone metabolism and abnormal bone remodeling, and delay the progression of knee osteoarthritis. However, the underlying molecular mechanisms remain to be fully elucidated. This study aims to systematically explore the mechanism of Bushen Qiangjin capsule in regulating inflammatory signaling pathways to improve knee osteoarthritis, using network pharmacology, Mendelian randomization, molecular docking, and cell experiments, providing a scientific basis for its clinical application.