The Mechanism Of Osteoblast On Osteoclastogenesis By Regulating The Mitophagy In A High Glucose Environment

Objective:In recent years,with the rapid development of the economy and the aging of the population,the incidence of type 2 diabetes is increasing,and it has become a global problem which cannot be ignored.It causes various complications such as cardiovascular disease and neurological diseases and it is a serious threat to human health.It is also recognized that diabetes can lead to osteoporotic fractures,causing serious consequences of disability and even death.A prominent feature of diabetes is the increase of blood sugar,which can disrupt the homeostasis of bone homeostasis by affecting osteoblasts and osteoclasts,leading to osteoporosis.Osteoblasts and osteoclasts are key effector cells that maintain bone homeostasis in humans.The main function of osteoblasts is to promote bone formation,while osteoclasts mainly promote bone resorption.It is currently believed that the occurrence of diabetic osteoporosis is mainly due to the breaking of the homeostasis of bone homeostasis in a high glucose environment,which leads to redistribution of bone mass and rearrangement of bone structure.In human body,osteoblasts and osteoclasts coexist,and there are many ways of communication between them.The researches so far are mostly limited to the biological effects of high glucose on osteoblasts or osteoclasts alone,and the results are still controversial.The relationship between the two cells under high glucose conditions remains to be unclear.Recent studies have showed that the co-culture environment can more accurately simulate the human body than the single culture,which is more conducive to the exploration of related effects.As highly developed cells,the communication between osteoblasts and osteoclasts is mainly in a paracrine form,which is cytokine exchange.During this process,the cytokine RANKL?receptor activator of NF-KB ligand?plays a central role.RANKL is a member of the TNF family and is secreted mainly by osteoblasts in vivo.RANKL can not only bind to its receptor RANK,but also cause intracellular high iron,which leads to mitophagy of osteoclast.As a multinucleated giant cell,the formation and function of osteoclasts require enormous energy support,and mitochondria plays a key role in this process as a major site for intracellular energy metabolism.However,its specific role and mechanism in osteoclasts is still unclear.Therefore,we hypothesized that osteoblasts in high glucose environment may regulate mitophagy of osteoclasts by regulating the secretion of RANKL.This study will use the in vitro co-culture system of osteoblasts and osteoclasts to explore the mechanism of osteoporosis in type 2 diabetes,and provide a powerful theoretical basis for the clinical treatment of type 2 diabetic osteoporosis.Methods:We used osteoblast-like cell line MC3T3-E1 and co-cultured with osteoclast precursor cell line RAW264.7,after 10-8 mol/L of 1?,25?OH?2 vitamin D3?1??,25?OH?2D₃)and 50 ng/mL macrophage colony-stimulating factor?M-CSF?,induced differentiation of osteoclasts in vitro,and co-culture with Transwell chamber?PET membrane,pore size 0.4um?system.After co-culture for 5 days,the proliferation and differentiation activities of osteoblasts in the co-culture system were identified by alkaline phosphatase?ALP?activity assay and osteogenic function-related proteins OPG,OCN.Anti-tartaric acid phosphatase staining),bone resorption assay,and CtsK expression is used to measure osteoclast function.The co-culture system was stimulated by the configured high-sugar?-MEM medium to detect the osteogenesis and RANKL secretion function of MC3T3-E1.The effects of RANKL on mitophagy and function in osteoclasts were examined by controlling the content of RANKL in supernatant.Finally,the mitophagy was regulated by chemical methods to detect its effect on osteoclast function.Results:?1?Compared with the singleculture group,the ALP activity of co-cultured bone cells did not change significantly,while the TRAP activity of osteoclasts in the co-culture group was significantly enhanced,and the number of TRAP-positive cells was significantly increased.The bone resorption experiment showed that the co-culture group was broken.Bone cell bone resorption activity is also greatly enhanced.?2?Compared with the single culture,the osteoblasts in the co-culture group showed a significant inhibition of osteogenesis in the high-glycemic environment,but showed increases in the secretory function?RANKL?.In the high glucose environment,the osteoclast function of the co-culture group was significantly more active than that of the single culture,and even more active than the co-culture group with normal sugar concentration.?3?Compared with the single culture group or the normal glucose concentration co-culture group,the mitophagy-related protein lc3of osteoclasts in the co-culture group was significantly up-regulated and p62 was down-regulated.After the addition of recombinant OPG inhibited RANKL,the corresponding mitophagy level was significantly reduced.?4?After activation/inhibition of mitophagy by chemical means,the function of osteoclasts in the co-culture system is enhanced/decreased.Conclusion:?1?Both osteoblasts and osteoclasts in Transwell co-culture system are alive,and osteoclast precursors can be induced to differentiate into mature osteoclasts and have bone resorption function.This system can be used to study the mechanism between OBs and OCs in high glucose environment.?2?High glucose environment can inhibit the osteogenic function of osteoblasts,promote the RANKL secretion function of osteoblasts,and then promote the bone resorption function of osteoclasts.?3?After the high glucose environment acts on the co-culture system,it can promote the mitophagy of osteoclasts in the co-culture system by promoting the secretory function of osteoblasts.Mitophagy plays a protective role in osteoclasts.