Effects Of Aluminum Exposure On SIRT1/SIRT3-FOXO3A-PINK1/PARKIN Pathway Regulating Neuronal Mitophagy

Objective:Aluminum（Al）,as a neurotoxic substance harmful to the human body,mainly accumulates in the brain.Long-term aluminum exposure can cause neurodegenerative diseases.Previous studies have shown that mitophagy dysfunction is closely related to many neurodegenerative diseases,and Al can cause neuronal mitophagy;other studies have shown that Al can reduce the expression of SIRT1,suggesting that SIRT1 may play a significant role in Al-induced neuronal mitophagy.However,how Al regulates mitophagy through SIRT1 is still unclear.This study examines the possible mechanism of aluminum exposure on SIRT1/SIRT3-FOXO3A-PINK1/PARKIN pathway to regulate mitophagy,and provides new clues and experimental basis for finding molecular targets for early intervention in aluminum-related neurodegenerative diseases.Methods:The method used in this study is a combination of vivo and vitro methods.First,a 3-month rat model was set up by drinking water stained with aluminum.Then,the learning and memory ability of rats was tested by the dark test.Then,SH-SH5Y cells,as vitro cellular aluminum exposure model were selected for testing,and neuron characteristics were identified by NSE immunofluorescence.The MTS method was used to ensure cell viability of exposed aluminum and the intervention agents resveratrol and EX-527.Observe the morphological changes of SH-SH5Y cells under the microscope;GSH kit to detect GSH level;ELISA kit to detect ATP level;Mito SOX?Red method to measure mitochondrial ROS level;RT-PCR to detect Sirt1,Foxo3a,Pink1 Lc3 m RNA level;Western blot to detect the protein levels of SIRT1,SIRT3,FOXO3A,PINK1,PARKIN,LC3,P62.Immunofluorescence method was used to determine the expression level of Ace-FOXO3A protein;laser confocal method was used to determine the colocalization of mitochondrial lysosomes.Use SPSS 18.0 software to perform statistical analysis on the above results.Results:1.In vivo experiments showed that:Learning and memory ability.In the shuttle box test of subchronic aluminum-exposed rats,the number of errors at 6 h and24 h increased,and the escape latency was significantly reduced.2.The effect of Al exposure on the m RNA and protein levels of SIRT1,SIRT3,FOXO3A,PINK1,PARKIN,LC3,and P62 in rat cortex.With the increase of aluminum concentration,the level of Sirt1 m RNA in rat cortex decreased,and the level of Foxo3a,Pink1,Lc3m RNA increased;SIRT1,SIRT3 protein levels was down-regulated,and FOXO3A,PINK1,PARKIN,LC3,P62 protein levels was up-regulated with the dose of aluminum dyed increased gradually,and the above results were all opposite after the addition of RES.3.In vitro experiments,with increased Al concentration,the survival rate of SH-SY5Y cells gradually reduced.4.Aluminum will cause the GSH level in the cells to decrease significantly.After adding RES,the above results play different.After the intervention of EX-527,the GSH level decreased.5.Effects of Al exposure on mitochondrial function.Compared with the control group,with the continuous increase of aluminum dose,the intracellular ATP level showed a downward trend,and the active oxygen in the mitochondria showed an upward trend.RES can reverse mitochondrial function damage caused by Al exposure,and EX-527 can aggravate mitochondrial damage.6.With the increased concentration of Al chloride,the protein content of SIRT1 and SIRT3 in the cells was down-regulated significantly,increased after adding RES,and decreased after the intervention of EX-527;FOXO3A,PINK1,PARKIN and LC3,P62 m RNA and protein level was up-regulated with the increase of Al dye dose,decreased after adding RES,and increased after the intervention of EX-527.7.Effects of Al exposure on FOXO3A acetylation levels.The results of immunofluorescence indicated that the acetylation level of FOXO3A in the 4 m M Al Cl₃group increased significantly,the acetylation level decreased after the intervention of RES,and the acetylation level was upward after adding EX-527.8.Effects of Al exposure on mitochondrial-lysosomal colocalization.Laser confocal results showed that Al exposure caused an increase in mitochondrial-lysosomal co-localization,which decreased after the addition of RES,and increased after the intervention of EX-527.Conclusions:1.Aluminum exposure reduces the learning and memory abilities of rats.2.Al exposure led to morphological damage in SH-SY5Y cells.3.Al exposed cells could cause mitochondrial dysfunction.4.Al exposure could down-regulate SIRT1,SIRT3 levels,which in turn causes the expression levels of FOXO3A,PINK1,PARKIN,LC3 and P62 to increase and mitophagy lysosomes increase,leading to abnormal activation of mitophagy.5.Al exposure can increase the levels of FOXO3A acetylation.6.The SIRT1 agonist RES has a protective effect on the above process after intervention,and the SIRT1 inhibitor EX-527 has an aggravated effect on the above process after intervention.It suggests that the SIRT1/SIRT3-FOXO3A-PINK1/PARKIN pathway plays an important regulatory role in mitophagy caused by Al.