Pulsed Electromagnetic Fields Inhibit Osteoclast Differentiation In RAW264.7 Macrophages Via Suppression Of The Protein Kinase B/mammalian Target Of Rapamycin Signaling Pathway

BackgroundAs the world's population ages,the incidence of senile diseases,especially osteoporosis,also shows a trend of simultaneous growth.Bone metabolism under physiological conditions is the dynamic balance of bone resorption and formation.Accelerated absorption and lack of formation caused by various causes lead to break the balance,resulting in decreased bone mass or osteoporosis.The treatment of osteoporosis diseases has caught much attention since recent years.The main treatment principles include slowing decrease in bone mineral density and bone remodeling.The main clinical treatments include mineralized preparations,bone resorption inhibitory drugs and bone formation promoting drugs,which can relieve symptoms and prevent complications such as fractures.However,drug treatment often brings some side effects which is not conducive to the patient's health.Therefore,the research on new osteoporosis treatment methods has always been a hot spot in the field of orthopedics.Osteoclast are the main cells involved in bone metabolism balance.Osteoclast activation is an important cause of osteoporosis.Therefore,the molecular mechanism of this process has become a key research object in recent years.Previous studies have shown that RANKL/RANK/0PG signal axis is an important signaling pathway in the process of osteoclast differentiation.Following these signals,the downstream signal changes,such as Src activation,which affects the function of cells.Akt/mTOR signaling pathway may be the downstream molecule of Src pathway.RANKL acts on osteoclast precursor cells,which may activate Src protein,and the downstream Akt and mTOR protein molecules may also be activated,causing subsequent signal changes,eventually leading to osteoclast differentiation.As a new type of physical therapy,pulsed electromagnetic fields has been used for the study of osteoporosis treatment.It has the advantages of non-invasive,low-cost,simple and easy to implement.Pulsed electromagnetic fields may affect cell proliferation,apoptosis,and differentiation.Some experiments have shown that pulsed electromagnetic fields can inhibit the differentiation of osteoclast-like cells and may be beneficial for the treatment of osteoporosis,but the specific mechanism is not clear yet.In this study,RAW264.7 cells were induced by receptor activator of nuclear factor-xB ligand(RANKL)to obtain osteoclasts in vitro.The aim of this research was to investigate the effect of pulsed electromagnetic fields on the differentiation of RAW264.7 macrophages into osteoclasts.Meanwhile,according to the cell signaling pathway,to explore Whether the pulsed electromagnetic fields affects the differentiation of osteoclasts through the Akt/mTOR signaling pathway and further to elucidates the mechanism of the pulsed electromagnetic field on osteoclast differentiation.Objective1.Whether pulsed electromagnetic fields could inhibit RANKL-induced differentiation of RAW264.7 macrophages into osteoclasts.2.Whether pulsed electromagnetic fields inhibit to osteoclast differentiation in RAW264.7 macrophages by regulating Akt/mTOR signaling pathway.Materials and Methods1.The effect of various stimuli on the proliferation of RAW264.7 macrophages detected by CCK-8 kit.2.RAW264.7 macrophages were treated with RANKL to obtain osteoclasts in vitro.To investigate the effect of PEMF on osteoclast formation and function,Tartrate-resistant acid phpsphatase(TRAP)staining and TRAP activity were performed.Bone slices were stained with toluidine blue.Osteoclastic specific gene were tested by qRT-PCR and marker protein was performed by western blotting.3.Following serum-starvation for 6 h,RAW264.7 cells were incubated for 15 min with RANKL(50 ng/ml),RANKL(50 ng/ml)+ PEMF or RANKL(50 ng/ml)with pharmacological inhibitors[perifosine(Akt inhibitor,2.5 ?M,dissolved in water)and rapamycin(mTOR and p70S6K inhibitor,1?M,dissolved in dimethyl sulfoxide)].Total protein was extracted from each group and evaluated by western blotting.ResultsThe experimental results showed that various stimulatory factors had no significant effect on the proliferation activity of the cells.When treated with RANKL,the number of giant osteoclasts containing multiple nuclei was markedly increased compared with the control.However,the number of multinuclear osteoclasts induced by RANKL was significantly decreased by PEMF application.Results of the TRAP activity assay also demonstrated that RANKL treatment resulted in enhancement of TRAP activity,while this facilitating effect was attenuated by PEMF application.RANKL-induced osteoclasts resulted in resorption pits on bovine bone slices.However,the bone resorption of osteoclasts induced by RANKL was markedly attenuated by exposure to 50 Hz PEMFs.Simultaneously,PEMF inhibited the expression level of osteoclast marker genes and proteins.Western blot results showed that RANKL can activate intracellular Akt,mTOR and p70S6K proteins by promoting the protein phosphorylation.Conversely,the upregulation of these proteins induced by RANKL was suppressed by pharmacological inhibitors,perifosine(Akt inhibitor)and rapamycin(mTOR and p70S6K inhibitor).In accordance with the inhibitory effects of antagonists,PEMF application also distinctly decreased RANKL-induced upregulation of these phosphorylated proteins.Conclusions1.Pulsed electromagnetic fields inhibits RANKL-induced RAW264.7 macrophage differentiation to osteoclasts and suppresses its bone absorption activity.2.Pulsed electromagnetic fields inhibits osteoclast differentiation in RAW264.7 macrophages by inhibiting RANKL-activated Akt/mTOR signaling pathway.