Exosomes From Adipose-derived Stem Cells Alleviate Dexamethasone-induced Apoptosis Of Osteoblasts By Regulating The Nrf2/HO-1 Axis

Objective: The oxidative damage model of MC3T3-E1 osteoblasts was established to observe and analyze the effects of adipose derived mesenchymal exosomes(ADSCs-Exos)on the proliferation,apoptosis and oxidative stress of osteoblasts induced by dexamethasone(Dex),and to explore the molecular mechanism of ADSCs-Exos in treating osteoporosis.Methods: ADSCs were extracted from bilateral inguinal region of 5 week old SD rats and identified by flow cytometry.ADSCs-Exos were extracted from the supernatant,the expression of its surface protein markers was identified by WB,its shape and size were identified by NTA and TEM,and cell uptake test was conducted with PKH-26 dye.MC3T3-E1 cells with logarithmic growth were taken to construct Dex induced oxidative stress model.MC3T3-E1 cells were divided into five groups: PBS group,Exo(50 μ G/ml)group,Dex group,Dex+Exo(25 μ G/ml)and Dex+Exo(50 μ g/ml).The effects of ADSCs-Exos on apoptosis and oxidative stress in Dex induced osteoblasts were analyzed in vitro.Flow cytometry and fluorescence microscope were used to observe the expression of ROS in different groups of cells;The changes of mitochondrial membrane potential were observed by fluorescence microscope.Osteogenic activity and mineralization capacity were detected by alkaline phosphatase(ALP)and alizarin red(ARS)staining,respectively.The potential molecular mechanism was explored by WB and immunofluorescence staining.In addition,glucocorticoid induced osteoporosis(GIOP)model was established in vivo.TUNEL staining,Micro CT,HE staining,Masson staining,immunohistochemistry and immunofluorescence staining were used to evaluate the effect of ADSCs-Exos on Dex induced osteoporosis.Results: ADSCs-Exos restored the viability and osteogenic potential of MC3T3-E1 cells by reducing apoptosis,oxidative damage,intracellular ROS production and mitochondrial dysfunction.In addition,Nrf2 expression was up-regulated in Dex stimulated osteoblasts after pretreatment with ADSCs-Exos.Inhibition test showed that silencing Nrf2 partly eliminated the protective effect of ADSCs-Exos.The rat model test confirmed that ADSCs-Exos reduced the increase of Dex induced oxidation level,restored the bone mass of distal femur,and increased the expression of Nrf2 and osteogenic markers in bone tissue.Conclusion: The results indicate that ADSCs-Exos can reduce Dex induced apoptosis and oxidative damage by regulating the expression of Nrf2/HO-1,and prevent the development of GIOP in vivo.