Salvianolic Acid B Promotes Osteogenesis Of Mesenchymal Stem Cells By Protecting Dexamethasone-induced Mitochondrial Dynamics Disorder

Objective: Excessive and long-term steroid-induced femur head necrosis is a major challenge for clinicians.However,the specific mechanism is unclear and there is no good treatment.The dynamic balance of mitochondria plays an important role in disease.We speculated that excessive or persistent glucocorticoid using changed the microenvironment in bone marrow.by interfering the function of SIRT3,glucocorticoid could cause mitochondrial dynamics disorder,induce the increase of ROS production and led to the abnormal proliferation and differentiation of BMSC.Finally,development of femoral head necrosis can occur.Salvianolic acid B,a reactive oxygen scavenging agent,can restore the proliferation and differentiation abnormalities of BMSC caused by dexamethasone-induced mitochondrial dynamics disorder.Methods: Mesenchymal stem cells were extracted from human bone marrow.the surface markers of stem cells were identified first by flow cytometry,and then the differentiation ability of the three lines was tested.The effects of different concentrations of dexamethasone and salvianolic acid B on cell activity at different time points were detected by CCK-8 and the live and dead staining kit.Apoptosis was detected through flow cytometry using the Annexin V-PI Commercial kit.Westeron blot was used to detect the relevant indicators of mitochondrial dynamics,apoptosis and osteogenesis.Transmission electron microscopy was used to observe the mitochondrial morphology.The changes of mitochondrial membrane potential were detected by JC-1 probe.Alizarin red,alkaline phosphatase and oil red O staining kits were used to detect the osteogenesis.DCFH-DA was used to detect the intracellular ROS levels.Results: Flow cytometry results showed that bone marrow mesenchymal stem cells with three-line differentiation ability were successfully extracted from bone marrow.Cell activity test showed that high concentration of dexamethasone could induce apoptosis of stem cells after 72 hours.WB test found that high concentration of dexamethasone promoted the increase of cell mitochondrial division.DCFH-DA test showed a decrease in intracellular ROS.Osteogenesis related experiments showed that high concentration of dexamethasone inhibited bone formation and promoted the adipogenesis.After salvianolic acid B treatment,ROS in cells were significantly cleared and osteogenesis was promoted.Conclusion: High concentration of dexamethasone inhibits osteogenesis by promoting the production of ROS in mitochondrial dynamics disorder,while Salvianolic acid B can promote osteogenesis by clearing intracellular ROS level.