SC79 Rescues Osteoblasts From Dexamethasone Though Activating Akt-Nrf2 Signaling

purposes: To observe the effect of dexamethasone on osteoblast cell injury and to explore the mechanism of inhibition of Akt-Nrf2 signaling pathway.Methods:1.MC3T3-E1 cells,primary mouse osteoblasts and human OB-6 osteoblasts were pretreated with SC79 at the indicated concentration gradient(0.1-25 μg / mL)for 1 hour.1μM dexamethasone for 24 hours.MTT assay was used to detect the viability of the cells.LDH release assay was used to detect cell death;2.MC3T3-E1 cells,mouse primary osteoblasts and human OB-6 osteoblasts were pretreated with SC79(10μg/mL)for 1 hour and then treated with 1μM dexamethasone for different times.Caspase-3,Annexin V,MMP,ELISA,mitochondrial damage and cytochrome release assay were used to detect the apoptosis;3.MC3T3-E1 osteoblasts were pretreated with three different Akt inhibitors LY294002,perifosine and MK-2206 for 30 minutes,then treated with SC79(10μg/mL)for 1 hour,and then treated with 1μM dexamethasone.Western blot was used to detect the protein expression.MTT and ELISA assay were used to detect cell survival and apoptosis.Primary mouse osteoblasts were treated with SC79(10μg/mL)for 1 hour and then treated with perifosine at a concentration of 10μM for 30 minutes;4.ShRNA was used to knockdown and block Nrf2 to construct the Nrf2 shRNA and dn-Nrf2 stably transfected cells.ROS assay was used to detect ROS concentration,RT-qPCR was used to detect mRNA expression and MTT assay were used to detect cell survival.Result: 1.MTT survival assay showed that SC79 at 1-25 mg / mL significantly reduced Dex-induced MC3T3-E1 cell viability.In addition,SC79 can inhibit Dex-induced cell LDH release.2.SC79 significantly attenuated Dex-induced caspase-3 activation,increased annexin V detection rate and increased histone DN apoptosis ELISA OD.SC79 inhibited Dex-induced activation of MC3T3-E1 cells.SC79 inhibited MMP-induced CyPD-ANT-1 complex and cytochrome C release in Dex-induced MC3T3-E1 cells and primary mouse osteoblasts.SC79 attenuated Dex-induced apoptosis and programmed necrosis.3.Western blot results showed that Akt inhibitors(LY294002,perifosine and MK-2206)could block SC79-induced Akt phosphorylation.MTT assay and ELISA test showed that Akt inhibitor significantly reduced the effect of SC79 on dex-induced apoptosis.4.Real-time quantitative PCR(RT-qPCR)assay demonstrated that SC79 can increased the expression of Nrf2-regulated genes(HO-1 and NQO-1 and GCLC)mRNA of MC3T3-E1 cells,but Nrf2 mRNA did not change.However,the use of Akt inhibitors almost blocked SC79-induced HO-1 mRNA expression.Nrf2 shRNAs or mutations did not affect SC79-induced Akt activation in Nrf2 shRNA and dn-Nrf2 stably transfected cells,but SC79-mediated HO-1 mRNA expression was almost knocked down or mutated by Nrf2 knockdown,and SC79-induced anti-Oxidant activity and cytoprotection were significantly attenuated.Conclusion: SC79 can inhibit the apoptosis and process necrosis of osteoblasts induced by Dex.The effect is mediated by the activation of Nrf2 signal in Akt downstream of osteoblasts,and the activation of Nrf2 signaling may play an antioxidative and cytoprotective role.