The Role Of ATP5B In Bone Destruction Of Rheumatoid Arthritis And Its Mechanisms

Rheumatoid arthritis(RA)is a chronic inflammatory disease which can cause systemic inflammation and bone loss.Osteoclasts are polar multi-nuclear macrophage uniquely undertake bone resorption function.In RA,osteoclasts are excessively activated,causing severe bone destruction.Bone resorption by osteoclast was a complex process.Once attaching to bone surface,osteoclast reorganize cytoskeleton to form a sealing zone,separating bone from the extracellular space,actively release H+and proteolytic enzymes such as MMP9 though ruffled border to keep an acid resorption environment,degrade bone matrix,whilst mobilize degradation products by endocytosis.The vesicle transport process which are highly energy-intensive is critical in the differentiation and bone resorption of osteoclast.There are abundant mitochondria in osteoclast.The abnormal expression of mitochondrial-related proteins is tightly linked to osteoclast and its function.ATP5B,a gene that encodes Fo-F1 ATP synthase β subunit is a catalytic core for ATP synthesis.Our preliminary experiment showed that the expression of ATP5B was up regulated during osteoclast differentiation,supposing it may play a crucial role in the differentiation and activation of osteoclast.In this study,we confirmed that intervention with ATP5B significantly impaired osteoclast mitochondrial function,leading to an inhibition of osteoclast bone resorption function,protecting against bone destruction of collagen induced arthritis mice.OBJECTIVE:To study the role of ATP5B in osteoclast differentiation and bone resorption function,explore the effect of ATP5B on bone erosion in collagen-induced arthritis mice.METHODS:1.Lentivirus-mediated gene silencing down-regulated the expression of ATP5B in vitro.To identify the effect of ATP5B on osteoclast formation and bone resorption activity using TRAP staining and bone resorption assay.The expression of osteoclast-related genes and adhesion-related proteins were detected by RT-qPCR and Western blot,respectively.To observe the formation of osteoclast actin ring by immunofluorescence staining.To evaluate the ability of H+secretion by acridine orange staining.To assess the activity of MMP9 secretion by Gelatin zymography.2.In order to investigate the effect of ATP5B on mitochondrial function,intracellular ATP content and ROS level were detected by testing kit,RT-qPCR was used to detect the expression of mitochondrial biogenesis relative genes.An ATPase inhibitor was used to explore the effect of blocking energy supply on the expression of osteoclast-related genes.3.A collagen-induced mice arthritis model was established,the expression of ATP5B was knocked down by local injection of joints with lentivirus.The inflammation score was recorded.Micro-CT and HE staining were used to evaluate joint bone destruction.ELISA was used for the detection of inflammatory cytokines in serum and bone degradation products CTX-I in joint tissue.RT-qPCR and Western blot were used to evaluate osteoclast-related genes and proteins level.RESULTS:1.In vitro,knocking down of ATP5B down-regulated the expression of osteoclast-related genes,disturbed the formation of F-actin ring as well as significantly down-regulated the protein level of Integrin β3 and p-FAK/p-Src.ATP5B knockdown blocked the secretion of H+and MMP9,inhibiting osteoclast bone resorption activity.2.Knocking down of ATP5B reduced cellular ATP and increased intracellular ROS level,promoting cell autophagy.RT-qPCR showed that ATP5B knockdown can significantly suppressed the mRNA level of PGC-lα,NRF-1/2,TFAM.5 μM and 10μM Oligomycin A down-regulated the expression of osteoclast-related genes.3.In vivo,knocking down of ATP5B reduced the inflammation score,ameliorated joint bone destruction in CIA mice.Compared with negative control CIA mice,the mRNA level of TRAP,CtsK,MMP9,Integrin β3 and NFATc1 as well as the protein level of V-ATPase,MMP9,Integrin β3,p-FAK/p-Src and CTX-I from arthritic paw in ATP5B knockdown CIA mice were significantly decreased.The level of TNF-α,IL-1β and IgG-2α in serum showed no significant difference between this two group.CONCLUSIONS:1.In vitro,knocking down of ATP5B significantly reduced osteoclast maturation and activation,suppressed its bone resorption activity by disturbing the formation of sealing zone and destroying the secretion of proton and MMP9.2.Knocking down of ATP5B disturbed mitochondria stability leading to deficient supply of ATP,causing osteoclast bone resorption dysfunction.3.In vivo,ATP5B knockdown of joints effectively reduced bone destruction in CIA mice by inhibiting the differentiation and activation of osteoclast.