Bisphosphonates Regulate The Molecular Mechanism Of Diabetic Osteoporosis Via Adenylate Activated Protein Kinase(AMPK)Pathway

In presedent Osteoporosis,a common disease among middle-aged and elderly people,is characterized by excessive bone resorption,resulting in decreased bone mass and increased bone fragility,which are prone to fracture.Osteoporosis is mainly caused by the imbalance between bone resorption and bone formation,which is mainly dependent on the activity of osteoblasts(bone formation effector cells)and osteoclasts(bone resorption effector cells).In the stomatology,the osseointegration success of dental implants is affected by the imbalance of bone remodeling in patients with diabetic osteoporosis.It has been reported that high glucose level can reduce the mineralization of osteoblasts,increase the expression of RANKL and reduce the expression of OPG,which affect the differentiation of osteoblasts,and inhibit the differentiation and bone resorption of osteoclasts.BisPhosphonate,as the main therapeutic drug for osteoporosis,has been proved to be effective in preventing bone loss in diabetic osteoporosis.As the main type of bisphesphonates,the molecular mechanism of the effect of zoledronate(ZOL)on the differentiation and function of osteoblasts and osteoclasts in high glucose environment is still unclear.For example,whether the AMPK pathway plays a role in the effects of zoledronate needs to be confirnied by further experiments.Adenosine monophosphate-activated protein kinase(AMPK)pathway can regulate cell energy homeostasis and participate in cell apoptosis,cell proliferation and cell differentiatioa Current studies have shown that the AMPK pathway also affects bone metabolism.AMPK stimulated the proliferation,differentiation and osteogenesis of osteoblasts,but inhibited osteoblasts apoptosis.Activation of AMPK pathway promoted bone formation and inhibits bone resorption.However,whether AMPK pathway is involved in the regulation of the effect of zoledronate on the differentiation and function of osteoblasts and osteoclasts in high glucose environment has not been reported.Mothod:According to the current research results,our study was divided into two parts to explore the effects of zoledronate on the differentiation and function of osteoblasts and osteoclasts in high glucose environment.Part Ⅰ,Zoledronate promoted osteogenic differentiation and osteogenesis in MC3T3-E1 cells via regulating the AMPK/OPG/RANKL signaling pathway under high glucose.In this part MC3T3-E1 cells were used to induce osteoblast differentiation The experiment was divided into four groups:low glucose group,high glucose group,low glucose+ZOL group and high glucose+ZOL group.Cell proliferation,cell migration,osteogenic differentiation and osteogenic function were detected.The mRNA and protein expressions of AMPK,phosphorylated of AMPK(p-AMPK),osteoprotegerin(OPG),ligand of receptor activator of NF-kB(RANKL),and expressions of bone morphogenetic protein 2(BMP2)and Collagen I(COL I)were detected by real-time PCR and western blot analysis under the conditions of AMPK antagonist Compound C and AMPK agonist AICAR.Part Ⅱ,Zoledronate and high glucose levels affect osteoclast differentiation and bone resorption through the AMPK/NFATc1/SYK pathway.In this study,RAW264.7 cells were used to induce osteoclast differentiation.The experiment was divided into four groups:low glucose group,high glucose group,low glucose+ZOL group and high glucose+ZOL group.Cell proliferation cell migration,integrity of osteoclast seal,osteoclast differentiation and bone resorption were detected.The mRNA and protein expressions of AMPK,p-AMPK,nuclear fector of activated T cells cytoplasmic 1(NFATc1),spleen tyrosine kinase(SYK),cathepsin K(CTSK),and tartrate-resistant acid phosphatase(TRAP)were detected by real-time PCR and western blot analysis under the conditions of AMPK antagonist Compound C and AMPK agonist AIC ARResults:1)High glucose promoted the migration of MC3T3-E1 cells,inhibited osteoblastic differentiation and osteogenesis,and inhibited the egression of AMPK,p-AMPK,OPG,BMP2,COLI.2)In high glucose condition,ZOL promoted osteoblastic differentiation and osteogenesis by activating AMPK,p-AMPK,OPG,BMP2,COL I,which regulated via AMPK/OPG/RANKL pathway.3)High glucose promoted the egression of AMPK and p-AMPK,and inhibited the egression of NFATc1,SYK,CTSK and TRAP,thereby inhibited osteoclast differentiation and bone resorption.4)ZOL further promoted the expression of AMPK and p-AMPK,and inhibited the egression of NFATc1,SYK,CTSK and TRAP,which regulated via AMPK/NFATc1/SYK pathway,thereby further inhibited osteoclast differentiation and bone resorption.Conclusion:This study confirmed the regulatory effect of AMPK pathway on the differentiation and function of osteoblasts and osteoclasts under the condition of high glucose.The results provided new insights into the effects of bisphosphonates on the differentiation and function of osteoblasts and osteoclasts under high glucose and the regulatory mechanism of AMPK pathway,which provided research basis for the clinical study of bisphosphonate on the treatment of diabetic osteoporosis and the improvement of implants osseointegration success rate of diabetic osteoporosis,and suggested that AMPK pathway was a new potential target for the treatment of diabetic osteoporosis.