Study On The Role Of Mitochondrial Homeostasis Imbalance And Its Regulatory Factors In Neuronal Injury Induced By Aluminum

Objective:Aluminum（Al）is the most abundant metal element in the earth’s crust.It is widely used in our daily life and is widely considered as an important environmental pathogenic factor of neurodegenerative diseases.Previous studies have shown that aluminum can cause mitochondrial dysfunction and mitochondrial damage.In this study,we established the in vivo model of aluminum chloride drinking water exposure and the in vitro model of primary neuron to explore the possible mechanism of aluminum chloride exposure leading to the imbalance of neuronal mitochondrial homeostasis through SIRT1/PGC-1αpathway,so as to provide experimental basis for aluminum induced neurodegenerative changes.Methods:In vivo,After 7 days of adaptive feeding of Wistar rats,the female and male rats are in the same cage 1:1.If a vaginal plug is observed in the next morning,the female is considered to be pregnant.Pregnant rats were randomly divided into control group,0.2%Al Cl₃,0.4%Al Cl₃,0.6%Al Cl₃and 0.6%Al Cl₃+RES groups.After giving birth,the mothers drink different concentrations of Al Cl₃solution,and the pups are infected by the mother’s milk.After weaning,the animals drink directly to drink the poison,until the end of 12 weeks after birth.0.6%Al Cl₃+RES group was intervened from 9 to 12 weeks for 4 weeks.At the end of the exposure,Morris water maze test was performed,and then the cerebral cortex of rats was taken.In vitro,the primary neurons were cultured and exposed to aluminum chloride,and added SIRT1 activator（RES）,inhibitor（EX-527）and PGC-1αactivator（ZLN005）.The cell survival rate was detected by CCK8 method.The level of MDA was determined by kit method.The contents of ROS and mitochondrial respiratory chain complex I were detected by ELISA.Mito Sox ^TMRed fluorescent probe was used to detect the content of Mitochondrial ROS,TMRM probe was used to detect the level of mitochondrial membrane potential,and chemiluminescence was used to detect the level of ATP.The mitochondrial DNA copy number was detected by RT q PCR.The mRNA levels of Sirt1、Pgc-1α、Nrf1、Tfam、Drp1、Mfn1、Opa1 were detected by RT-q PCR.Western blot analysis was used to detect the expression levels of COXⅣ、SIRT1、PGC-1α、NRF1、TFAM、DRP1、p-DRP1（S616）、MFN1、OPA1.The morphology of mitochondria was observed by confocal laser scanning,and the co localization level of SIRT1 and PGC-1αwas detected by immunofluorescence.Statistical analysis was performed by SPSS 22.0 software.Results:1.In vivo experiments showed that the learning and memory abilities of aluminum exposed rats were damaged,and RES intervention could significantly improve the long-term spatial memory of aluminum exposed rats.2.The content of MDA in cerebral cortex increased and the content of COX IV decreased significantly（P<0.05）.After RES intervention,the content of MDA decreased and the content of COX IV increased significantly.3.With the increase of aluminum chloride concentration,the mitochondrial DNA copy number in cerebral cortex of rats was significantly decreased（P<0.05）,and increased after RES intervention（P<0.05）.The mRNA levels of Sirt1,Pgc-1α,Nrf1,Tfam,Mfn1 and Opa1 in cerebral cortex of rats were significantly decreased（P<0.05）,and the above gene levels were restored after RES intervention,but the mRNA level of Drp1 had no significant change.The protein levels of SIRT1,PGC-1α,NRF1,TFAM,MFN1 and OPA1 decreased with the increase of exposure concentration（P<0.05）.After RES intervention,the above protein levels increased again,but the protein level of p-DRP1（S616）increased significantly（P<0.05）without significant change after aluminum exposure.The protein level of p-DRP1（S616）decreased significantly after RES intervention（P<0.05）.4.In vitro,the cell survival rate decreased significantly with the increase of aluminum chloride concentration（P<0.05）.5.After aluminum exposure,ROS and mitochondrial ROS in neurons increased significantly（P<0.05）,mitochondrial respiratory chain complex I content decreased,mitochondrial membrane potential decreased,and ATP level also decreased significantly（P<0.05）.6.After adding RES,the mitochondrial DNA copy number increased significantly（P<0.05）.The contents of SIRT1,PGC-1α,NRF1,TFAM,MFN1,OPA1 were significantly increased（P<0.05）,the level of DRP1 had no change,and the protein content of p-DRP1（S616）was decreased（P<0.05）.And after adding EX-527,the mitochondrial DNA copy number decreased significantly（P<0.05）.The contents of SIRT1,PGC-1α,NRF1,TFAM,MFN1,OPA1 were significantly decreased（P<0.05）,the level of DRP1 had no change,and the protein content of p-DRP1（S616）was increased.7.After adding ZLN005,the mitochondrial DNA copy number increased significantly（P<0.05）.The contents of SIRT1,PGC-1α,NRF1,TFAM,MFN1 and OPA1 were significantly increased（P<0.05）,the level of DRP1 had no change,and the protein content of p-DRP1（S616）was decreased（P<0.05）.8.There was co localization of SIRT1/PGC-1α.The results of 3m M Al Cl₃group showed that the co localization of SIRT1/PGC-1αdecreased and the Pearson correlation coefficient decreased.RES intervention increased the co localization and EX-527 intervention decreased the co localization.9.Laser confocal observation of mitochondrial network showed that the number of mitochondria decreased and the average length of mitochondria shortened in Al group;RES and ZLN005 could improve the mitochondrial network structure and prolong the length of mitochondria;EX-527 could aggravate the fragmentation of mitochondria.Conclusions:1.Aluminum exposure can lead to learning and memory impairment in rats.2.Aluminum exposure can lead to oxidative damage and mitochondrial damage in cerebral cortex of rats,which can be improved by RES intervention.3.Aluminum exposure can reduce the mitochondrial biosynthesis,increase the mitochondrion division and decrease the mitochondrial fusion in rat cerebral cortex,which can be restored after RES intervention.4.Aluminum exposure can cause morphological damage and decrease the survival rate of primary neurons.5.Aluminum exposure induced oxidative damage and mitochondrial dysfunction in primary neurons of rats.6.Aluminum exposure leads to mitochondrial biogenesis disorder,mitochondrial division increase,mitochondrial fusion decrease and mitochondrial homeostasis imbalance in primary rat neurons through SIRT1/PGC-1α.