| Objective: Osteoporosis and osteoarthritis are two degenerative bone diseases with a high incidence in clinical practice,and although there are differences in their pathologic changes,there is a strong correlation between them in terms of age,weight,and nutrition.Especially in postmenopausal women,these two diseases tend to occur together,seriously affecting the quality of life of patients,but there is still a lack of effective treatment.Based on the theory of "treating the kidney is also treating the bone",Bone Paralysis and Pain Relief Formula has been widely used in the treatment of paralysis of various chronic orthopedic related diseases.Although Bone Paralysis Pain Relief Formula is widely used in clinical practice,the molecular mechanism of its regulation of osteoblasts and chondrocytes is still unclear,and the specific pharmacological substances are also still unclear.Therefore,the present study firstly investigated the biological mechanism of GBZTF in promoting the repair of OP-OA damage through a series of in vivo and in vitro experiments,which provided important clues to further elucidate its therapeutic effects.Methods: The OP-OA rat model was established to observe the weight changes and joint swelling in each group of rats,the changes in physiological and motor functions were analyzed by gait analysis and weight-bearing experiments,three-point bending experiments of bone tissues and Micro-CT were used to assess the changes in bone structure and density,and biomarkers related to bone metabolism and inflammation were detected in serum by ELISA.In addition,histopathological testing of articular cartilage and lower limb bone tissues was further performed to assess the degree of pathological damage.At the molecular level,techniques such as polymerase chain reaction(q RT-PCR)and protein blotting were used to analyze the expression levels of key genes and proteins.CCK-8,q RT-PCR and various cell staining techniques were used to detect the effects of GBZTF on the proliferation,differentiation and mineralization of osteoblasts;a cell model of injury was established by inducing bone marrow MSCs through hydrogen peroxide(H2O2)(in vitro),and molecular biology techniques,such as CCK-8 and flow cytometry,were used to detect the effects of GBZTF on the proliferative and differentiation activities of bone marrow MSCs.Effects.The active ingredients and related targets of Bone Paralysis Pain Relief Formula were screened by TCMSP database and Traditional Chinese Medicine Efficacy and Drug Interaction Database,and the potential therapeutic targets of OP-OA were further collected by Gene Cards and OMIM database.Protein-protein interaction(PPI),compound-target-disease(C-T-D)and compound-targetpathway(C-T-P)networks were established using Cytoscape,and the main components,core targets and pharmacological pathways of Bone Paralysis Pain Relief Formula against OP-OA were identified by data mining techniques and topological parameters,and Metascape database was used for GO and KEGG pathway analysis,and molecular docking technique was used to estimate the binding between components and key genes.Results: GBZTF can effectively alleviate the phenotypic symptoms of bone loss,trabecular bone reduction,and osteomalacia and cartilage detachment in OP-OA rats.In vitro experiments showed that Bone Paralysis Analgesic Formula could promote the pluripotent differentiation of bone marrow mesenchymal stem cells to osteoblasts and chondrocytes,and inhibit apoptosis induced by hydrogen peroxide,but did not have a significant effect on osteoblasts.Network pharmacological screening of the active ingredients Formononetin,Kaempferol,Luteolin,Naringenin,Quercetin,Epicatechin and three disease critical protein targets(JAK-1,STAT-3 and BCL-2),the molecular docking results showed that Formononetin had the best binding effect on JAK-1.48 signaling pathways were obtained from KEGG pathway enrichment analysis,mainly including IL6-JAK-STAT signaling pathway,apoptosis signaling pathway and PI3K-AKT signaling pathway.q RT-PCR and protein immunoblotting further validated the IL6-JAK-STAT signaling pathway and the PI3K-AKT signaling pathway.q RT-PCR and protein immunoblotting further verified that IL-6/JAK/STAT signaling pathway might be the key pathway for the anti-OP-OA effect of Bone Paralysis Pain Relief Formula.Conclusion:1.Bone paralysis analgesic formula can also reduce knee swelling and pathological weight gain,adjust painful gait,reduce bone fragility,improve serum inflammation and bone metabolism factor levels in rats with OP-OA,and show protective and repairing effects on the pathological state of joint and bone tissue damage caused by OP-OA.2.GBZTF in vitro experiments did not demonstrate to promote the proliferation,differentiation and mineralization of osteogenic precursor cells,but can promote the differentiation of bone marrow mesenchymal stem cells to osteoblasts and chondroblasts,respectively,and can inhibit apoptosis induced by hydrogen peroxide(H2O2).3.Network pharmacology GBZTF intervenes in OP-OA active ingredients include Formononetin,Kaempferol,Luteolin,Naringenin,Quercetin,Epicatechin;JAK-1,STAT-3 and BCL-2 may be the key proteins in the regulation of OP-OA by bone paralysis and pain relief formula.The molecular docking results showed that Formononetin binds best to JAK-1.The gene and protein validation experiments indicated that GBZTF may promote the pluripotent differentiation of bone marrow mesenchymal stem cells and inhibit apoptosis by regulating IL-6/JAK/STAT pathways,thus improving the symptoms of arthritis and alleviating the loss of bone mass in rats with OP-OA. |
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