The Effect And Mechanism Of NIPA2 Regulating Osteoblast Function By Modulating Mitophagy In Type 2 Diabetic Osteoporosis

Objective:The prevalence of type 2 diabetes mellitus increases with economic development and aging,exerting a substantial economic burden on the healthcare system.Loss of bone mass is a well-known risk factor associated with osteoportic fractures in T₂DM patients,leading to significant impairment in their daily functions and quality of life.Although large studies have been carried out in the past decade to study the possible pathological mechanism of type 2 diabetes osteoporosis,the main active pathological mechanism is complicated and remains controversial.Non-imprinted in Prader-Willi/Angelman syndrome region protein 2?NIPA2?was found the only transporter that is highly selective for Mg²⁺and helps maintain Mg²⁺influx.We believe that NIPA2 may be an important factor in the pathogenesis of type2 diabetic osteoporosis.In the microenvironment of type 2 diabetes,the level of intracellular mitophagy changes,but its specific role and regulation mechanism in osteoblasts is still unclear.Magnesium ions can stabilize the mitochondrial membrane potential,thereby preventing mitochondrial dysfunction caused by decreased mitochondrial membrane potential,and the decrease of mitochondrial membrane potential is an important factor in inducing mitophagy.Therefore,we hypothesized that changes in NIPA2 in a high-glucose microenvironment result in changes in intracellular magnesium levels,which in turn affect osteoblast osteogenesis through mitophagy.Through this study,we will conduct an in-depth study on the pathogenesis of type 2 diabetic osteoporosis,and provide a strong theoretical basis and a new therapeutic target for clinical treatment of type 2 diabetic osteoporosis.Methods:We first observed the changes in bone microstructure of db/db mice in type2 diabetes by micro-CT and established a model of type 2 diabetic osteoporosis.The differential expression of NIPA2 in bone tissue was detected by immunohistochemistry.Then,we used the method of in vitro experiments to determine the cell viability of human osteoblast hFOB1.19 at different glucose concentrations and time of treatment using CCK8 cell activity assay.The expression of NIPA2 in osteoblasts was detected by PCR and Western blot.The difference of intracellular magnesium ions was detected by magnesium ion fluorescent probe and the level of mitophagy was determined.After being silenced and overexpressing NIPA2 by lentivirus in high glucose environment,the effects of it on magnesium ion,mitochondrial membrane potential,mitophagy and osteogenesis in hFOB1.19 cells were detected.After pharmacological regulation of mitophagy and knockdown of Parkin,the key mitophagy gene,the effects of mitophagy on apoptosis and expression of osteogenesis-associated proteins were examined.After clarifying the above phenomenon,we determined the regulation mechanism of NIPA2 on mitophagy and osteogenic function of osteoblasts by detecting and interfering with PGC-1?/FoxO3a signaling pathway.Results:?1?Compared with normal mice,bone mineral density,trabecular bone volume per tissue volume,trabecular bone number and thickness were significantly reduced in type 2 diabetes model db/db mice.?2?The expression of NIPA2 in human osteoblast hFOB1.19 was significantly inhibited in high glucose environment,and the level of intracellular magnesium was decreased.?3?Mitochondrial membrane potential/PINK1/Parkin-mediated mitophagy occurs in osteoblasts in a high glucose environment.?4?NIPA2 has a negative regulation on mitophagy in osteoblasts under high glucose conditions.?5?Mitophagy caused by decreased expression of NIPA2 in high glucose environment has an inhibitory effect on osteogenic function of osteoblasts,which may be achieved through its promotion of apoptosis.?6?The decrease of NIPA2 caused by high glucose environment may reduce the mitochondrial membrane potential of osteoblasts through PGC-1?/FoxO3a signaling pathway,and then induce mitophagy and damage osteoblast osteogenesis.Conclusion:?1?NIPA2 plays an important role in the pathogenesis of type 2 diabetic osteoporosis and may be a potential target for clinical treatment.?2?In high glucose environment,NIPA2 affects the mitochondrial membrane potential of osteoblasts through PGC-1?/FoxO3a signaling pathway,and then regulates mitophagy.?3?Osteoblast mitophagy in a high glucose environment negatively regulates osteoblast function and may be achieved by promoting apoptosis.