Low-frequency Pulsed Electromagnetic Field Inhibits RANKL-induced Osteoclastic Differentiation In RAW264.7 Cells By Scavenging Reactive Oxygen Species

Background:The balance of the body’s bone mass is maintained by bone resorption and bone formation.Osteoclasts are the only cells in the body with bone resorption functions,and their increased number and activity will lead to osteoporosis.Statistics show that osteoporosis increases the chance of elderly people suffering from medullary fractures by 50%,which places a heavy burden on society and families.At present,osteoporosis is mainly based on estrogen replacement and drug treatment,but the side effects are obvious,and the risk of tumors is even increased.It is necessary to seek new treatments.Osteoclast differentiation is regulated by multiple signaling pathways.The classic RANK/RANKL signaling pathway is the most important for osteoclast differentiation.Low frequency pulsed electromagnetic field(PEMF)has been used as a new therapeutic method to treat bone and soft tissue diseases.Many basic studies have also confirmed that LPEMF can affect osteoclast differentiation.Intracellular reactive oxygen species(ROS)is a by-product of related enzymatic reactions during the transfer of mitochondrial electron chains in cells.Recent studies have shown that changes in intracellular ROS are closely related to osteoclast differentiation and maturation.Some literatures have reported that pulsed electromagnetic fields can reduce ROS levels in other cells,so we suspect that PEMF may inhibit osteoclast differentiation by regulating intracellular ROS levels.Objective:(1)To investigate whether PEMF can inhibit RANKL-induced osteoclast differentiation;(2)To investigate whether PEMF can inhibit RANKL-induced osteoclast differentiation by regulating intracellular reactive oxygen species;Materials and Methods:(1)The effect of PEMF on the proliferation activity of RAW264.7 cells was detected using the CCK-8 kit;(2)Cells were treated with RANKL as an inducer for 4 days.Cells were treated in groups according to different experiments.After TRAP staining,the number of osteoclasts induced by each group was compared to detect changes in the expression levels of marker genes and proteins during osteoclast differentiation.(3)To explore the effect of PEMF on ROS during cell differentiation.After 2 days of induction,DCFH-DA was used as a ROS probe to detect changes in ROS levels under different intervention conditions.(4)Investigate the relationship between ROS and osteoclast differentiation.RANKL-induced osteoclasts were treated with ROS scavenger DPI.The number of osteoclast formation was counted after TRAP staining,and the levels of related proteins in each group were detected to verify ROS and osteoclasts Differentiated relationship.Results:Our experiments confirm that PEMF does not affect the proliferative activity of RAW264.7 cells.PEMF can inhibit RANKL-induced osteoclast differentiation.Compared with RANKL group cells,the number of mature osteoclasts and TRAP enzyme activity in PEMF+RANKL group were significantly reduced,and the expression levels of osteoclast differentiation-related marker genes and proteins(CTSK,TRAP,NFATc1)were decreased.PEMF has antioxidant properties and can reduce the level of intracellular reactive oxygen species during osteoclast differentiation.ROS is closely related to osteoclast differentiation.Using ROS scavenger DPI to reduce intracellular ROS levels can significantly inhibit RANKL-induced osteoclast differentiation.Conclusion:1.In vitro culture conditions,PEMF can inhibit RANKL-induced RAW264.7 cells to differentiate into osteoclasts.2.Changes in intracellular ROS levels are closely related to osteoclast differentiation.Decreasing intracellular ROS levels can significantly inhibit osteoclast differentiation.ROS may become a potential target for the treatment of osteoporosis.3.PEMF can inhibit RANKL-induced osteoclast differentiation by reducing intracellular ROS levels.