Advanced Oxidation Protein Products Induce Osteoblast Apoptosis Through A Redox-dependent MAPKs Mediated Intrinsic Apoptosis Pathway

BackgroundOsteoporosis is a chronic,progressive disease characterized by low bone mass,bone deterioration,and decreased bone strength,which is increasingly being recognized as an important cause of morbidity and mortality in the elderly population.Age related bone loss is characterized by the reduction of bone formation,which mainly results from the decreased number and activity of osteoblasts.Apoptosis is thought to be one of the primary regulators of osteoblast homeostasis.Osteoblast apoptosis increases with age,and is responsible for reduction in osteoblast number and activity,which contributes to decreased bone formation.Oxidative stress occurs as a consequence of an imbalance between the formation and inactivation of reactive oxygen species(ROS),which plays roles in aging and associated complications.Advanced oxidation protein products(AOPPs)are considered to be biomarkers of oxidant mediated protein damage.They are a group of carbonyl-,dityrosine-,and pentosidine-containing protein products generated by reaction of plasma proteins with hypochlorous acid and chloramines during oxidative stress and generally considered to be carried mainly by albumin in the blood circulation.AOPPs are also inducers of ROS generation.Previous studies have shown that AOPPs can mediate multiple pathogenic processes by inducing a redox-dependent apoptosis in diverse cells.Previous studies have found that oxidative stress and AOPPs levels increased with age,and AOPPs accumulation was correlated with age-related bone loss.AOPPs treatment aggravated bone loss and deteriorated bone microstructure in the aged rats.However,the effect of AOPPs on the osteoblast apoptosis still remains unknown.Materials and MethodsMurine osteoblastic MC3T3-E1 cells were used as in-vitro models,and treated with AOPPs to detect the ROS production,cell apoptosis rate,and the potential mechanism.Forty male SD rats aged 18 months were used as in-vivo modles.All of the rats were randomly divided into five groups containing eight rats per group and received the following treatments:group 1,daily intraperitoneal injection of vehicle(PBS,pH =7.4);group 2,daily intraperitoneal injection of unmodified RSA(50 mg/kg);group 3,daily intraperitoneal injection of AOPPs(50 mg/kg);group 4,daily intraperitoneal injection of AOPPs(50 mg/kg)together with apocynin(NADPH oxidase inhibitor)at 100 mg/kg in drinking water and changed the water daily to quantify the volume of water intaking;and group 5,apocynin at 100 mg/kg/day in drinking water.At the end of 16 weeks,animals were sacrificed,the blood,tibias and L4 vertebral bodies samples were collected to conduct the further research.ResultsWe found that AOPPs administration significantly increased plasma AOPPs level,while decreased plasma bone specific alkaline phosphatase(B-ALP)level.In addition,the Tb.Th(Trabecular Thickness),Tb.N(Trabecular Number),BV/TV(Bone Volume/Total Volume)of proxima tibias and L4 vertebral bodies markedly decreased after AOPPs administration,nevertheless,the Tb.Sp(Trabecular Space)increased.The results above revealed that AOPPs could destroy the microstructure of the trabecular bone.Flow cyrtometry and Live and Dead Cell Staining results showed that AOPPs could induce MC3T3-E1 cell apoptosis in a concentration-and time-dependent manner.Further study showed that AOPPs administration could activate NADPH oxidase and induce ROS overproduction.The excessive ROS then lead to the activation of MAPKs signalling(JNK,p38,ERK1/2),decreased the mitochondrial membrane potential,increased mitochondrial membrane permeability,induced endoplasmic reticulum(ER)stress in combination with Ca2+ overload,finally activated the intrinsic apoptosis pathway and resulted in cell death.By in-vitro studies using TUNEL stainining,we found that AOPPs stimulation significantly increased the apoptotic rate of osteoblast in proximal tibias and L4 vertebral bodies.Furthermore,immunohistochemical staining also showed the increased expression of NADPH oxidase subunits(Nox2,Nox4,p47phox,p22phox),pro-apoptosis protein of intrinsic apoptosis pathway(BAX,cleaved caspase3)and MAPKs(JNK,p38,ERK1/2)phosphorilation in proximal tibis and L4 vertebral bodies of AOPPs stimulated aged rats.ConclusionsBy in-vitro and in-vivo studies,we found that AOPPs take part in the progression of age related osteoporosis by inducing osteoblast apoptosis through a NADPH oxidase-dependent,MAPKs-mediated intrinsic apoptosis pathway.