RESUMO
Objective To explore the effect of Bajitianwan（BJTW）formula on bone formation of Aβ-injured osteoblasts and its mechanism. Methods Osteoblasts isolated from neonatal 24-hour Wistar rats were used for the study, and osteoblasts were subjected to damage with Aβ1-42 oligomers, and pharmacological intervention was performed with the aqueous extract of BJTW formula. The MTT assay, alkaline phosphatase（ALP）activity assay, catalase（CAT）activity assay, superoxide dismutase（SOD）activity assay, glutathione（GSH）activity assay and malondialdehyde（MDA）activity assay were carried out respectively. The expression levels of bone morphogenetic protein 2（BMP2）, osteogenic specific transcription factor（RUNX-2）and osteoprotective protein（OPG）were detected by Western blotting. After confirming the effect of BJTW formula on Aβ-injured osteoblasts, the network pharmacology method was used to predict the potential pathways. Results The BJTW formula significantly promoted the proliferation of Aβ-injured osteoblasts, increased ALP, SOD and GSH activity, inhibited MDA activity, and promoted the expression of bone formation-related proteins BMP2, RUNX-2 and OPG. Network pharmacological analysis showed that the effect of ameliorating of Aβ-injured osteoblasts by BJTW formula was mainly mediated by AGE-RAGE, PI3K-Akt, MAPK and neuroactive ligand-receptor interaction signaling pathways. Conclusion In this study, the effect of BJTW formula on improving the osteoblasts damaged by Aβ was confirmed for the first time, and its related mechanism was explored based on network pharmacology method. The results lay a strong foundation for the clinical application of traditional formula BJTW against osteoporosis.
RESUMO
Objective To explore the effect and mechanism of Bajitianwan on preventing D-galactose (D-gal)-induced osteoblast bone loss. Methods Osteoblasts isolated from 24 h old Wistar rats were injured by D-gal and intervened with Bajitianwan extract. The osteoblastic proliferation and differentiation were determined by MTT and alkaline phosphatase (ALP), respectively. The cell reactive oxygen species (ROS) levels were detected by DCFH-DA fluorescent probes. The expression of cellular oxidation-related protein nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase 1 (NQO1) were detected by Western blotting. The intranuclear expression of Nrf2 protein was measured by immunofluorescence. Results Bajitianwan extract had significantly increased the osteoblastic proliferation and differentiation, and significantly reduced the intracellular ROS level. Bajitianwan extract had activated the PI3K/AKT pathway via activating the phosphorylation of AKT in osteoblasts, and promoted NQO1 and HO-1 expression. In addition, Bajitianwan had significantly promoted the expression of Nrf2 in the nucleus of osteoblasts, activating Nrf2 signaling pathway, and further promoted bone formation. Conclusion This study confirmed that Bajitianwan could prevent D-gal injured osteoblastic bone loss for the first time. The mechanism might be related to the regulation of oxidative stress associated PI3K/AKT and Nrf2 signaling pathway.