Analysis of the Potential Angiogenic Mechanisms of BuShenHuoXue Decoction against Osteonecrosis of the Femoral Head Based on Network Pharmacology and Experimental Validation.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease with a high disability rate. The clinical effect of BuShenHuoXue decoction (BSHX) for ONFH is satisfactory. We aimed to elucidate the potential angiogenic mechanisms of BSHX in a rat femoral osteonecrosis model and bone marrow mesenchymal stem cells (BMSCs). METHODS: With in vivo experiments, we established the steroid-induced osteonecrosis of the femoral head (SONFH) model using Sprague-Dawley (SD) rats (8-week-old). The rats were randomly divided into five group of 12 rats each and given the corresponding interventions: control, model (gavaged with 0.9% saline), BSHX low-, medium- and high-dose groups (0.132 3, 0.264 6, and 0.529 2 g/mL BSHX solution by gavage). After 12 weeks, haematoxylin and eosin (H&E) staining was preformed to evaluate rat osteonecrosis. the expression of angiogenic factors (CD31, VEGFA, KDR, VWF) in rat femoral head was detected by immunohistochemistry, qPCR and western blotting. In cell experiment, BMSCs were isolated and cultured in the femoral bone marrow cavity of 4-week-old SD rats. BMSCs were randomly divided into eight groups and intervened with different doses of BSHX-containing serum and glucocorticoids: control group (CG); BSHX low-, medium-, and high-dose groups (CG + 0.661 5, 1.323, and 2.646 g/kg BSHX gavage rat serum); dexamethasone (Dex) group; and Dex + BSHX low-, medium-, and high-dose groups (Dex + 0.661 5, 1.323, and 2.646 g/kg BSHX gavaged rat serum), the effects of BSHX-containing serum on the angiogenic capacity of BMSCs were examined by qPCR and Western blotting. A co-culture system of rat aortic endothelial cells (RAOECs) and BMSCs was then established. Migration and angiogenesis of RAOECs were observed using angiogenesis and transwell assay. Identification of potential targets of BSHX against ONFH was obtained using network pharmacology. RESULTS: BSHX upregulated the expression of CD31, VEGFA, KDR, and VWF in rat femoral head samples and BMSCs (p < 0.05, vs. control group or model group). Different concentrations of BSHX-containing serum significantly ameliorated the inhibition of CD31, VEGFA, KDR and VWF expression by high concentrations of Dex. BSHX-containing serum-induced BMSCs promoted the migration and angiogenesis of RAOECs, reversed to some extent the adverse effect of Dex on microangiogenesis in RAOECs, and increased the number of microangiogenic vessels. Furthermore, we identified VEGFA, COL1A1, COL3A1, and SPP1 as important targets of BSHX against ONFH. CONCLUSION: BSHX upregulated the expression of angiogenic factors in the femoral head tissue of ONFH model rats and promoted the angiogenic capacity of rat RAOECs and BMSCs. This study provides an important basis for the use of BSHX for ONFH prevention and treatment.

Molecular mechanism of Wutou Decoction in the treatment of osteoarthritis: a bioinformatics and molecular docking study.

BACKGROUND: Osteoarthritis (OA) is a chronic joint disease characterized by cartilage destruction and periarticular osteophyte formation. One therapeutic option for this condition, the Wutou Decoction (WTD) Chinese medicine formula, is satisfactory in its efficacy. Here, we used bioinformatic and molecular docking techniques to investigate the mechanism of action of WTD in the treatment of OA. METHODS: The active compounds (and their target proteins) of 5 Chinese herbs in WTD were obtained by searching the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. The action targets of WTD for OA were obtained by searching the Therapeutic Target Database and by mining the microarray data in the Gene Expression Omnibus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to identify key targets for OA treatment with the help of Database for Annotation, Visualization, and Integrated Discovery. Based on the Cytoscape software version 3.6.1, the visual networks of the "TCM drugs-Active Compounds-Targets-Diseases" and protein-protein interaction of the key targets of WTD for the treatment of OA were constructed. The core active compounds and the key targets obtained were molecularly docked and validated. RESULTS: Analyses revealed 140 active compounds in WTD, 123 of which had a total of 163 corresponding targets. In addition, 331 differentially expressed genes and 227 OA-related targets were obtained. The interaction networks among 32 key targets were identified. The biological processes of WTD in treating OA mainly involved regulation of inflammatory factors, transcription of genetic materials, cell cycle, angiogenesis, and endocrine regulation. The signaling pathways involved mainly included TNF signaling pathway, rheumatoid arthritis signaling pathway, cancer-related signals, vascular endothelial growth factor signaling pathway, and osteoclast differentiation signaling pathways. Molecular docking showed that 7 core compounds including quercetin and kaempferol had strong affinities with key target proteins for the WTD treatment of OA. CONCLUSIONS: WTD with multi-component can treats OA through multi-pathway. Its active compounds, including quercetin and kaempferol, can exert their therapeutic effects on OA by acting on TNF, PTGS2, MMP2, IL-6, IL-1ß, and other key targets to regulate inflammation, immunity, autophagy, and endocrine-related signaling pathways.

Study on the molecular mechanism of BuShenHuoXue capsule in treatment of steroid-induced osteonecrosis of the femoral head.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is the pathological process caused by the death of the active components of the head of the femur due to the high dose of hormones, which has become a common public health problem. BuShenHuoXue capsule (BSHXC) has been clinically proven to be effective against the SONFH, the main pharmacological action of BSHXC is tonifying kidney and promoting blood circulation, but the mechanism remains to be explored. METHODS: We established a rat SONFH model by injecting Methylprednisolone (MPS) into the right gluteus muscle 30 mg/kg/d, 3 days of continuous injection every week, 4 weeks in total. According to the clinical dosage of BSHXC (Herba epimedium 3 g, Eucommia ulmoides 15 g, Salvia miltiorrhizae 30 g, Chuanxiong 15 g, Paeonia lactiflora Pall 15 g, Poria cocos 12 g, Achyranthes bidentata 12 g, antler gum 10 g, Cyperus rotundus L. Nine g and Radix Glycyrrhizae 9 g), it was converted into the equivalent dose of rats, and gavage was performed at the weight of 10 mL/kg, once per day. The BSHXC was subjected to experiments in vivo, SONFH pharmacodynamics, bioinformatics, and network of pharmacology to determine the active ingredients, and its protective role against SONFH, Enrichment analysis was performed to explore the possible mechanism of BSHXC, and cell experiments were undertaken to analyze the impact of BSHXC on the hormones associated with bone marrow mesenchymal stem cells (BMSCs) between osteogenesis and apoptosis. RESULTS: Experiments confirmed that BSHXC could effectively reduce bone loss in SONFH rat models. From bioinformatics and a network constructed from 10 drugs-208 pharmacology-126 targets, the enrichment analysis showed that the core targets were inflammatory reaction, steroid hormones, estrogen receptors, osteoporosis, and adjustment of osteogenesis and osteoclast differentiation, among others. The cell proliferation and staining supported that the mechanism of BSHXC promoted osteogenesis and intervening in apoptosis. CONCLUSIONS: The BSHXC reduced the inflammatory response, changed steroid response, regulated estrogen receptors, delayed osteoporosis, regulated osteoblast and osteoclast differentiation by regulating related targets, and improved the local microenvironment by a multi-component, multi-target, and multi-link process to delay or reverse the progression of SONFH.