Advanced Oxidation Protein Products Induce The Osteoclast Differentiation And Bone Resorption

BackgroundOsteoclasts are multinucleated cells that are responsible for bone resorption,and play an important role in bone homeostasis.Enhanced osteoclastogenesis and activity of these cells result in an imbalance in skeletal turnover,cause bone loss due to excessive bone resorption,and ultimately lead to a risk of osteoporosis.Advanced oxidation protein products(AOPPs)are dityrosine cross-linked and carbonyl-containing protein products mainly formed by reaction of plasma proteins with chlorinated oxidants(hypochloric acid and chloramines)deriving from the activity of myeloperoxidase released by activative phagocyte.Our previouly studies have demonstrated that plasma level of AOPPs was negatively correlated with lumbar bone mineral density(BMD)in postmenopausal osteoporotic women,and positively correlated with bone turnover markers,such as tartrate-rsistant acid phosphatase(TRAP).The accumulation of AOPPs greatly promoted degeneration of bone microstructure and accelerates bone deterioration in aged rats.Furthermore,AOPPs can inhibit proliferation and differentiation of rat osteoblast-like cells.However,the effect of AOPPs in osteoclast differentiation and activation is still unknown.ObjectivesTo investigate the effect of AOPPs in osteoclast differentiation and activation,and its molecular mechanism.MethodsIn this study,rat bone marrow mononuclear cells(BMMs)were used as in vitro model.After BMMs were treated by AOPPs,we performed the investigations as the following:1.TRAP staining to detected positive TRAP multinucelear cells formation;2.TRAP activity analysis;3.Real-Time PCR to detect the gene expression of osteoclast differentiation markers;4.Rhodamine-phalloidin stains to visualize F-actin ring;5 Bone resorption pit assay;6.DCFH-DA probe to detect the generation of intracellular reactive oxygen species(ROS);7.Immunoblotting to detect the expression of nicotinamide-adenine dinucleotide phosphate(NADPH)oxidase,NFATc1,and the phosphorylation of MAPKs subunits.We utilized repeat injections of AOPPs for our in vivo bone destruction model.Rats were treated with local injection of AOPPs.Micro CT was used to rebuild skull 3D structure and evaluate the area of bone resorpt pits;TRAP staining was used to detect positive TRAP multinucelear cells formation in skull.Results1.The effect of AOPPs on osteoclast differentiation and activation(1)AOPPs induced the formation of TRAP-positive multinuclear cells and gene expression of osteoblast differentiation markers,and increased the activation of TRAP;(2)AOPPs induced phophorylation of c-fos and the expression of NFATc1;(3)AOPPs induced the formation of F-actin ring and the bone resorption pits;(4)Local injection of AOPPs aggravated osteoclast differentiation and bone resorption in vivo.2.The molecular mechanism of AOPPs-induced osteoclast differentiation and activation.(1)AOPPs increased intracellular ROS generation in a time-dependent manner;(2)AOPPs increased intracellular ROS generation via the activation of NADPH oxidaseAOPPs treatment significantly increased the expression of NADPH oxidase subunits,such as Nox1,Nox4,p22phox and p47phox.AOPPs induced intracellular ROS production was inhibited by antioxidant treatment,such as NADPH oxidase hibitor Apocynin and superoxide dismutase(SOD);(3)AOPPs activated MAPKs by NADPH oxidase-dependent ROS generationAOPPs treatment significantly increased the phosphorylation of JNK,p38 and p38 in BMMs in 120min.Apocynin and SOD significantly blocked AOPPs-induced phosphorylation of JNK,p38 and ERK;(4)AOPPs triggered c-fos/NFATc1 pathway by NADPH oxidase-dependent,MAPKs signalingAOPPs treatment significantly increased the expression of NFATc1 and phosphorylated c-fos.Pretreated with apocynin,and SOD significantly decreased expression of NFATc1 and phosphorylated c-fos induced by AOPPs.JNK inhibitor SP600125,ERK inhibitor U0126 and p38 inhibitor SB203580 significantly decreased expression of NFATc1 and phosphorylated c-fos induced by AOPPs;(5)AOPPs induced osteoclast differentiation and function through a NADPH oxidase/ROS/MAPK pathwayTRAP staining shown that Apocynin,SOD,SP600125,U0126 and SB20358 block AOPPs-induced the formation of TRAP positive cells differentiation,the gene expression of osteoblast differentiation markers,the formation of F-actin and bone resorption pits.Conclusion1.AOPPs can induce BMMs differentiated into osteoclast and activate bone resorption.2.AOPPs induced osteoclast differentiation and activation via NADPH oxidase/ROS/MAPK pathways.