Effects And Mechanisms Of Advanced Oxidation Protein Products In Accelerating Age-Related Bone Loss By Fluctuating Sclerostin Expression In Osteocytes

BackgroundAdvanced oxidation protein products(AOPPs)are a kind of cross-linked proteins enriched in dityrosine,which are formed mainly by the oxidation of plasma proteins during oxidative stress(OS).Recent studies suggest that AOPPs are not only the consequence of OS,but also a kind of important mediators involved in the process of many diseases.AOPPs is closely related to osteoporosis.As the age increasing,there is AOPPs accumulation.Our previous study demonstrated that AOPPs accelerate bone mass loss and bone microstructure degeneration in aged rats.However,the underlying mechanisms remained unknown.Sclerostin is a kind of glycoprotein mainly secreted by osteocytes,which specifically inhibits the Wnt/β-catenin signaling and shows significant influence on both osteoblasts and osteoclasts.Sclerostin displays remarkable negative effects on bone metabolism by suppressing bone formation but promoting bone absorption.Our previous study also showed that AOPPs-treated rats displayed decreased bone forming activities but increased bone absorbing activities,indicating a possibility involvement of sclerostin in AOPPs-induced bone loss.Besides,plasma sclerostin level increases with age.Therefore,in this study,we proposed the hypothesis that AOPPs may accelerate age-related bone loss by fluctuating the expression of sclerostin in osteocytes.Materials and methodsAn AOPPs interventional experiment was firstly carried out.Twenty-eight 12-month-old male C57BL/6 mice were randomized into four groups and received the following treatments:intraperitoneal(ip)injection of vehicle(PBS)in group 1;ip injection of native BSA at 50 mg/kg/d in group 2;ip injection of AOPPs-BSA at 50 mg/kg/d in group 3;ip injection of AOPPs-BSA at 100 mg/kg/d in group 4.16 weeks later,effects of AOPPs on bone mass,bone microstructure,bone turnover,expression of sclerostin and Sirtl on bone tissue,and OS status in bone were analyzed.Then,an osteocytic cell line,MLO-Y4 cell,was employed to test the effects and mechanisms of AOPPs on sclerostin expression in osteocytes in vitro.Finally,a blocking experiment was done.Another 35 12-month-old male mice were randomized into five groups and subjected to treatments as follows:ip injection of PBS alone at 100mg/kg/d in group 1,ip injection of AOPPs-BSA alone at 100 mg/kg/d in group 2;ip injection of AOPPs-BSA at 100 mg/kg/d and apocynin at 100 mg/kg/d in drinking water in group 3;ip injection of AOPPs-BSA at 100 mg/kg.d and NAC at 100mg/kg/d in drinking water in group 4;ip injection of AOPPs-BSA at 100 mg/kg.d and SRT3025 at 50mg/kg.d in drinking water in group 5.After a treatment duration of 16 weeks,bone mineral density(BMD),bone microstructure and sclerostin expression in each group were analyzed and compared.Results50 mg/mL of AOPPs-BSA significantly decreased the BMD of L4 vertebral body,and this effect was more remarkable in 100mg/mL AOPPs-treated aging mice.However,no marked difference was observed in tibial BMD among different groups.Micro-CT analysis revealed that treatment with AOPPs-BSA prominently aggravated the trabecular microstructure in aging mice.Compared to PBS or BSA-treated mice,AOPPs-treated mice displayed a marked decrease in BV/TV,Tb.N,and Tb.Th but an significant increase in Tb.Sp in both L4 vertebral bodies and proximal tibias.Also,the plasma PINP concentration was greatly depressed in mice treated with AOPPs-BSA,while CTX-1 concentration was highly elevated.Corresponding to this finding,there was a significant decrease in the osteoblast number and an increase in osteoclast area observed in the cortical diaphysis of mice treated with AOPPs-BSA.Both sost mRNA(encoding sclerostin)and sclerostin protein levels in AOPPs-treated groups were higher than those in PBS-or BSA-treated groups.Conversely,sirt1 mRNA and Sirt1 protein levels in mice treated by AOPPs were lower.Further,the plasma AOPPs and bone malondialdehyde(MDA)levels were elevated by AOPPs,while bone total superoxide dismutase(t-SOD)activity was largely suppressed.In vitro study showed that AOPPs triggered ROS production by stimulating the NADPH oxidases,which then decreased Sirtl abundance and finally elevated sclerostin expression in MLO-Y4 cells.And compared to mice treated by AOPPs alone,the sost mRNA and sclerostin protein levels in tibial bone tissues were lower in mice co-treated by apocynin,NAC,or SRT-3025.The decrease of BMD in L4 vertebral bodies and degeneration of bone microstructure in both L4 vertebral bodies and proximal tibias were improved in mice of co-treated groups.ConclusionThe present study demonstrates that AOPPs increase sclerostin expression in osteocytes via an NADPH oxidase-dependent,downregulation of the Sirt1 pathway.The elevated sclerostin then suppresses bone formation but promotes bone resorption and contributes to age-related bone loss.