Role Of Mitophagy In Bone Formation Inhibition Induced By Aluminum And The Regulatory Mechanism Of ROS/PINK1/Parkin Signaling Pathway

Aluminum is a toxic metal element,which mainly accumulates in bone tissue after entering the body,inhibits bone formation and causes aluminum bone disease.Oxidative stress is the pathological basis of aluminum osteotoxicity,and alumin um exposure causes excessive production of reactive oxygen species（ROS）,damages mitochondria,and leads to cellular osteogenic dysfunction.The body can initiate mitophagy to specifically remove excessive ROS and damaged mitochondria to maintain intracellular environment stability,PINK1/Parkin signaling pathway is the most classical way to regulate mitophagy,but the role of mitophagy in bone formation inhibition induced by aluminum and the regulation mechanism of ROS/PINK1/Parkin signaling pathway remain unclear.Based on the close relationship between mitophagy,oxidative stress and bone formation,this study takes"Mitophagy is involved in the pathological process of aluminum-inhibited bone formation and is regulated by the ROS/PINK1/Parkin signaling p athway"as the theoretical hypothesis and entry point,and intends to carry out research from the following six aspects in vivo and in vitro.（1）Mice AlCl₃ median lethal dose(LD₅₀)determination:4-week-old male wild-type C57BL/6N mice were given AlCl₃ solution with gradient concentration by intragastric administration,and the LD₅₀ of AlCl₃ to them was determined.（2）Mice AlCl₃ poisoning experiment:mice were given 0,44.8 mg/kg,89.7 mg/kg and 179.4 mg/kg B.W.AlCl₃ solution for 90 d,the growth and development of mice,bone formation,oxidative stress,and mitophagy were detected to determine the dose-effect relationship between aluminum exposure and bone formation,oxidative stress and mitophagy in mice,and to screen the optimal aluminum dose for subsequent intervention tests.（3）Experiment of N-Acetyl Cysteine（NAC）intervention in AlCl₃ poisoning mice:4-week-old male wild-type C57BL/6N mice were given 0,100 mg/kg NAC solution,179.4 mg/kg AlCl ₃solution,179.4 mg/kg AlCl₃ solution and 100 mg/kg B.W.NAC solution for 90 d,the oxidative stress,bone formation and mitophagy of the mouse femora were detected to clarify the effects of ROS clearance on aluminum inhibiting bone formation and PINK1/Parkin signaling pa thway;（4）Experiment of Parkin knockout(Parkin^-/-)intervention in AlCl₃ poisoning mice:4-week-old male wild-type and Parkin^-/-C57BL/6N mice were given 0 and 179.4 mg/kg B.W.AlCl₃ solution for 90d,the mitophagy of femoral tissue,bone formation and oxidative stress were detected to determine the role of mitophagy in aluminum inhibiting bone formation.（5）Experiments on AlCl₃ poisoning in mouse embryonic osteoblasts（MC3T3-E1）:MC3T3-E1 cells were added with 0 m M,0.5 m M,1m M and 2 m M AlCl₃ solution for 24 h,the cell morphology,osteogenic function,oxidative stress and mitophagy were detected to determine the dose-effect relationship between aluminum exposure and osteogenic function,oxidative stress and mitophagy of MC3T3-E1 cells,and to screen the optimal aluminum dose for subsequent intervention tests.（6）Experiment of NAC intervening in AlCl₃ poisoning MC3T3-E1 cells:MC3T3-E1 cells were divided into blank control group,NAC intervention control group（5 m M NAC）,aluminum exposed group（2 m M AlCl ₃）and NAC intervention aluminum exposed group（2 m M AlCl₃+5 m M NAC）.After incubation for 24 h,oxidative stress and related indicators of PINK1/Parkin signaling pathway were det ected to clarify the effect of ROS clearance on PINK1/Parkin signaling pathway in aluminum exposed MC3T3-E1cells.（7）Experiment of Parkin gene silencing（si Parkin）intervention in AlCl₃ poisoning MC3T3-E1 cells:MC3T3-E1 cells with normal and si Parkin were divided into blank control group,si Parkin control group,aluminum exposed group（2 m M AlCl₃）and si Parkin aluminum exposed group（2m M AlCl₃）and cultured for 24 h,the mitophagy,cell viability,osteogenic function,ROS content and mitochondrial injury were detected to clarify the role of mitophagy in aluminum induced osteogenic dysfunction of MC3T3-E1 cells.The experimental results are as follows:（1）Determination of the LD₅₀ of AlCl₃ in miceThe LD₅₀ of AlCl₃ on male C57BL/6N mice is 1793.64 mg/kg.（2）Experimental results of AlCl₃ poisoned miceAluminium poisoned led to depression and slow weight gain in mice;inhibition of bone formation（decreased bone mineral density,impaired femoral microstructure,increased aluminium contents and decreased calcium,phosphorus,BGP and B-ALP contents in serum and femora）;femoral oxidative stress（increased ROS and MDA contents,decreased CAT and T-AOC activities）and mitophagy activation（decreased MMP,increased PINK1,Parkin,and LC3 expression,and decreased p62 expression）.（3）Experimental results of NAC intervention on AlCl₃ poisoned miceNAC intervention alleviated the femoral oxidative stress,growth inhibition,osteogenic dysfunction and femoral mitophagy induced by aluminum poison ed in mice;indicating that oxidative stress was the main pathological mechanism of bone formation inhibition caused by aluminum,and ROS mediated the PINK1/Parkin signaling pathway activation by aluminum poisoned.（4）Experimental results of Parkin^-/-in AlCl₃ poisoned miceParkin^-/-inhibited mitophagy in the femora of aluminum poisoned mice;aggravated growth inhibition,osteogenic dysfunction and oxidative stress in mice;indicating that mitophagy played a protective role in aluminum inhibiting bone formation by alleviating oxidative stress.（5）Effects of AlCl₃ exposured on MC3T3-E1 cellsAluminum exposure resulted in abnormal cell morphology,osteogenic dysfunction,oxidative stress and activation of mitophagy.（6）Experimental results of NAC intervention on AlCl₃ exposed MC3T3-E1 cellsNAC intervention inhibited the oxidative stress and the expression of PINK1 and Parkin in MC3T3-E1 cells exposed to aluminum;indicating that ROS mediated the activation of PINK1/Parkin signaling pathway in aluminum exposed MC3T3-E1 cells.（7）Experimental results of si Parkin intervention in AlCl₃ exposed MC3T3-E1 cellssi Parkin inhibited mitophagy in aluminum exposed MC3T3-E1 cells;exacerbated cellular osteogenic dysfunction,ROS overproduction,and mitochondrial damage;indicating that mitophagy played a protective role in aluminum-induced MC3T3-E1 cells injury by scavenging ROS and damaged mitochondria.In conclusion,aluminum exposure can lead to injury of femoral tissue and MC3T3-E1 cells,osteogenic dysfunction,oxidative stress and activation of PINK1/Parkin signaling pathway mediated mitophagy.ROS is the upstream signal of the PINK1/Parkin signaling pathway activated by aluminum.Mitophagy plays a protective role in aluminum inhibiting bone formation by alleviating oxidative stress and mitochondrial damage.