The Protective Mechanisms Of Chlorogenic Acid On Dexamethasone-induced Apoptosis In Mouse Osteoblastic MC3T3-E1 Cells

Dexamethasone represents a widely used glucocorticoid for the treatment of inflammatory diseases,rheumatic diseases and autoimmune disorders.However,long term or excessive dexamethasone application has substantial adverse effects,including inhibiting osteoblastic cell proliferation and inducing apoptosis of osteoblasts via generation of reactive oxygen species(ROS).Chlorogenic acid(CGA)is the major index ingredient found in Eucommia Ulmoides,which exhibits a wide range of diverse pharmacological effects,including anti-inflammatory,anti-oxidative,anti-apoptosis and neuroprotection activities.The present study aims to investigate the molecular mechanisms of apoptosis induced by dexamethasone in MC3T3-E1 cells,and to clarify the protective mechanisms of CGA against dexamethasone-induced oxidative stress and apoptosis,which contributes to provide a theoretical basis for clinical application of CGA.MC3T3-E1 cells were used to explore the molecular mechanisms of dexamethasone-induced apoptosis and the regulatory role of both PI3K/Akt pathway and p21 during the apoptotic process.Results demonstrated that dexamethasone activated caspase-dependent mitochondrial signaling pathway and induced cell damage and apoptosis in MC3T3-E1 cells.In order to investigate the mechanism of PI3K/Akt signaling pathway in dexamethasone-induced apoptosis,LY294002 and IGF-1 were used to inhibit or promote Akt activation,respectively.Results showed that dexamethasone downregulated the protein expression of p-Akt,decreased the p21 protein level by destabilizing the p21 protein,and altered the subcellular localization of p21 in MC3T3-E1 cells.Western blot results indicated that dexamethasone upregulated ratio of Bax/Bcl-2,promoted cyt c release to cytosol,increased the protein expression of cleaved caspase-9 and cleaved caspase-3,and thus induced apoptosis in MC3T3-E1 cells.To study the influence of p21 on dexamethasone-induced cell death and oxidative stress,knockdown experiments were performed by siRNA.Results demonstrated that p21 knockdown by siRNA in dexamethasone-treated cells significantly reduced cell viability,and enhanced activation of caspase-3 and apoptosis.Western blot analysis results showed that deletion of p21 efficiently suppressed dexamethasone-induced upregulation of nuclear Nrf2 and HO-1.Furthermore,level of intracellular ROS and superoxide in the mitochondrial were obviously increased by p21 knockdown after incubation with dexamethasone.In the present study,we evaluated the protective effect of CGA on dexamethasone-induced osteoblasts apoptosis via measuring cell viability,LDH release,cell morphology,apoptotic rate as well as apoptosis-related proteins expression and caspase activities,including Bax,Bcl-2,cyt c,caspase-9 and caspase-3.Results demonstrated that CGA treatment could promote cell proliferation and significantly attenuate dexamethasone-induced reduction of cell viability,LDH release and cell ultrastructural lesions.Additionally,CGA treatment was also found to inhibit dexamethasone-induced activation of caspase-9 and caspase-3,upregulation of Bax/Bcl-2 ratio,release of cyt c and apoptosis in MC3T3-E1 cells.To further investigate the anti-oxidation potentials of CGA in osteoblast cell and uncover the underlying mechanisms,experiments were carried out by using MTT assay,fluorescence microscopy and Western blot.Here,CGA treatment increased HO-1 expression together with its upstream mediator Nrf2 and upregulated the protein level of p-Akt.However,ZnPP(a specific HO-1 inhibitor)and LY294002(a specific PI3K/Akt inhibitor)dramatically blocked CGA-induced HO-1 expression.Moreover,the increased cell viability by CGA after exposure to dexamethasone was significantly inhibited by ZnPP,indicating that the protective role of CGA was through PI3K/Akt pathway,a vital upstream signaling pathway that mediated the Nrf2-related cytoprotective effect.Furthermore,CGA inhibited dexamethasone-induced oxidative stress in MC3T3-E1 cells by reducing mitochondrial superoxide and promoting the activation of Nrf2/HO-1 signaling pathway.In conclusion,CGA protected MC3T3-E1 cells against apoptosis and oxidative damage via PI3K/Akt-mediated activation of Nrf2/HO-1 pathway,which may be an effective drug in treatment of osteoporosis.