The Role Of AMPK In Regulation Of Osteogenesis And Adipogenesis And Prevention Of Cell Senescence In MSC

Objective:1.To clarify the effect of aging on osteogenesis,cell senescence and AMPK activation.2.To clarify the regulation role of AMPK in osteogenesis and adipogenesis and to explore the underlying mechanism.3.To explore the functional differences between AMPKα1 andα2 subunits.4.To clarify the effect of salicylate on oxidative stress-induced cell senescence.Methods:1.To determine the osteogenic potential of rat and human BMSCs in different age,osteogenesis associated staining and realtime PCR were performed.Then SA-β-Gal staining and realtime PCR were performed in order to evaluate the cell senescence.The intracellular ROS production was measured using DCFH-DA fluorescence dye.Western blot was used to detect the activation of AMPK.2.AMPKα1 was overexpressed in pre-osteoblast cell line MC3T3-E1 and pre-adipocyte cell line 3T3-L1.Osteogenesis and adipogenesis associated staining and realtime PCR were performed to examine the effect of AMPKα1 overexpression on osteogenesis and adipogenesis.To verify the role of OPN in AMPKα-induced osteogenesis and adipogenesis,neutralization antibody and si RNA were used to downregulate the expression of OPN and rescure experiment was performed.Ch IP,luciferase reporter assay and overexpression were performed to verify AMPKαregulation on osteogenesis and adipogenesis and underlying AMPK-Gfi1-OPN pathway.Furthermore,ectopic bone formation in nude mice were evaluated.3.Lentivirus vectors were used to overexpress AMPKα1 andα2 subunit respectively in MC3T3-E1 cells,primary calvarial osteoblasts and BMSCs.To detect the difference in osteogenesis between AMPKα1 andα2 overexpression,alizarin red staining and realtime PCR were performed.TRAP staining,bone absorption assay and realtime PCR were used to examine the difference in MC3T3-E1 cell associated osteoclastogenesis between AMPKα1 andα2 overexpression.Lentivirus vectors were used to overexpress AMPKα1 andα2 subunit respectively in 3T3-L1 cells and BMSCs.Oil Red O staining and realtime PCR were performed to detect the difference in adipogenesis between AMPKα1 andα2 overexpression.4.Salicylate,an AMPK activator,was used in the H₂O₂-induced cell senescence model of BMSCs.Then SA-β-Gal staining and realtime PCR were performed.The intracellular ROS production was measured using DCFH-DA fluorescence dye and ALP staining was performed.Aged rat BMSCs were treated with salicylate and all tests above were repeated.Results:1.Along with aging,increased of SA-β-Gal staining and upregulation of senescence-associated genes were observed in BMSCs.ALP and alizarin red staining were inhibited,while downregulation of osteogenesis-asssociated genes was found.Increase of ROS production and inhibition of AMPK activation were detected.2.Overexpression of AMPKα1 promoted osteogenesis of MC3T3-E1 cells and inhibited adipogenesis of 3T3-L1 cells.Overexpression of AMPKα1 upregulated the OPN expression.Downregulation of OPN in AMPKα1-overexpressed cells inhibited osteogenesis and promoted adipogenesis,which was rescured by recombinant OPN treatment.Overexpression of AMPKα1 upregulated OPN expression by downregulating Gfi1 expression and inducing Gfi1 disassociation from the OPN promoter.3.Overexpression of AMPKα2 promoted osteogenesis compared with over-expressed AMPKα1,which AR and osteoactivin were involved.Furthemore,MC3T3-E1 cell associated osteoclastogenesis was inhibited by Overexpression of AMPKα2compared with AMPKα1.Overexpression of AMPKα2 inhibited adipogenesis of 3T3-L1 cell and BMSCs compared with AMPKα1,which AR was involved.4.In H₂O₂-induced cell senescence model of BMSCs,salicylate,an AMPK activator,prevented senescence associated phenotypes,including promotion of SA-β-Gal staining,upregulation of senescence-associated genes,increased ROS production and inhibited ALP staining.Aged rat BMSCs were treated with salicylate,ALP staining was enhanced and senescence-associated genes were downregulated.Furthemore,ROS production was decreased.Conclusion:1.Aging was associated with cell senescence,inhibited osteogenesis,increased ROS production and downregulated AMPK activation in BMSCs.2.AMPK promotes osteogenesis and inhibits adipogenesis through AMPK-Gfi1-OPN axis.3.AMPKα2 and AMPKα1 have several functional differences,withα2 conferring stronger osteogenic potential and a lower adipogenic potential as well as conferring a weaker ability to induce osteoblast-associated osteoclastogenesis.4.Salicylate,an AMPK activator,prevented H₂O₂-induced cell senescence,increased ROS production and inhibited osteogenesis.