Fluid Shear Stress Suppresses Osteoclast Differentiation In RAW264.7 Cells Through ERK5 Signaling Pathway

Objective: To study the effects of fluid shear stress(FSS)on the differentiation of osteoclasts and bone resorption by regulating extracellular-regulated kinase 5(ERK5)signaling pathway in RAW264.7 cells.Materials and Methods: Set time gradient to load RAW264.7 cells with FSS of 0,15,30,45,60,90 min,and detect the effect of FSS on ERK5 and the optimal time by Western blot,and verify the inhibitory effect of XMD8-92 on ERK5 phosphorylation;Then designed the experiment grouping into blank control group,RANKL group,FSS group and FSS + RANKL group.After 4 days of treatment,observe and detect osteoclast differentiation,bone resorption function,and detect TRAP enzyme activity;CCK-8 assay was used to detect cell viability;Western blot was used to detect and analyze the protein expression of phosphorylated ERK5(p-ERK5),NFATc1,CTSK,TRAP,MMP-9?Results: Western blot analysis showed that RANKL had no obvious phosphorylation activation effect on ERK5,but loaded FSS for 30 min had significant activation effect on ERK5,and XMD8-92 can specifically inhibit ERK5 phosphorylation activated by FSS;Cell count,TRAP enzyme activity,and bone resorption experiments showed that compared with the RANKL group,the TRAP-positive multinuclear cells and the TRAP enzyme activity in the FSS group were significantly reduced,and FSS significantly inhibits RANKL-activated osteoclast differentiation.Meanwhile,CCK-8 detection showed that FSS had no toxic effect on RAW264.7 cells and did not affect cell viability.Western blot analysis showed that the expression of NFATc1,CTSK,TRAP and MMP-9 in the FSS group was significantly reduced,and FSS could inhibit RANKL-activated expression of these proteins;XMD8-92 could inhibit ERK5 phosphorylation in all subgroups.At the same time,it was found that the addition of XMD8-92 significantly reversed the inhibitory effect of FSS on RANKL activation of NFATc1,and increased the expression of NFATc1 and osteoclast differentiation-specific proteins CTSK,MMP9 and TRAP.Conclusions: FSS inhibits the expression of NFATc1 and its downstream factors MMP9,CTSK and TRAP by activating the ERK5 signaling pathway,and then affects osteoclast differentiation and bone resorption in RAW264.7 cells.