Effects Of Aluminum Exposure On The Regulation Of Nrf2 Level And Stability By BRCA1 In SH-SY5Y Cells

Objective:Aluminum?Al?is an important neurotoxicant.The brain is the main accumulation site and the important target organ of Al.Chronic Al exposure is closely related to the occurrence of neurodegenerative diseases.Previous studies have demonstrated that Al could induce oxidative stress in CNS and breast cancer susceptibility gene1 may play a role in it.This study investigated oxidative damage,mitochondrial damage and BRCA1 and Nrf2 expression in SH-SY5Y cells to confirm the possible mechanism of neurodegeneration induced by Al.Methods:SH-SH5Y cells were identified by NSE immunofluorescence.Cell viability was measured by the CCK-8 method to determine the Al exposure dose and time,the antioxidant quercetin dose.The morphology of the cells in different dose groups was observed by light microscopy.The cell MDA and GSH levels were determined by the kit method.Cellular ATP levels were determined by ELISA.Apoptosis/necrosis rate and mitochondrial membrane potential were determined by flow cytometry.The mRNA levels of Brca1 and Nrf2 and their downstream Gclc,Ho-1 and Nqo1 were detected by RT-PCR.Western blot analysis was used to detect the protein levels of BRCA1,Nrf2 and its downstream GCLC,HO-1 and NQO1.The interaction between BRCA1 and NRF2 protein and the level of Nrf2 ubiquitination were determined by immunoprecipitation.Statistical analysis was performed by SPSS 18.0 software.Results:1.Effects of Al exposure on cell morphology.With the increase of aluminum exposure dose,the number of cells decreased,the cell body gradually became round,the synapse became shorter,and the intercellular connections became loose.2.Effects of Al exposure on oxidative stress.With the increase of Al,the MDA levels were increased,and the GSH contents decreased significantly.After the intervention of the antioxidant QUE,the above results could be reversed.3.Effects of Al exposure on apoptosis/necrosis rate.With the continuous increase of Al concentration,the apoptosis/necrosis rate gradually increased,and it was relieved after QUE intervention.4.Effects of aluminum exposure on mitochondrial function.Compared with the control group,the intracellular ATP level and mitochondrial membrane potential showed a downward trend with the increasing dose of Al,and QUE could reverse the mitochondrial dysfunction.5.Effects of Al exposure on the mRNA and protein levels of BRCA1 and Nrf2 pathway.The levels of BRCA1,Nrf2,GCLC,HO-1 and NQO1 mRNA and protein in cells gradually decreased with the increase of Al,after the addition of QUE,these levels were increased.6.Effects of FSK on BRCA1 and Nrf2 pathways.The results showed that after the intervention of FSK,BRCA1 mRNA and protein levels increased,and NRF2 and HO-1 protein levels also increased.7.The co-localization of BRCA1 and Nrf2.There was co-localization between BRCA1 and Nrf2.The results of the 5 mM AlCl₃ group showed that the co-localization of the two proteins was weakened.Also,the Pearson correlation coefficient was reduced.8.Effects of Al on the interaction between BRCA1 and Nrf2.There was an interaction between BRCA1 and Nrf2 in the control group,and it was weakened by Al exposure.9.Effects of Al exposure on Nrf2 ubiquitination levels.The results of MG-132 addition in cells suggested that Nrf2 degradation might be achieved through the ubiquitin-proteasome pathway,and Nrf2 ubiquitination levels in the 5mM AlCl₃ group increased significantly.Conclusions:1.Al exposure induced morphological damage in SH-SY5Y cells,caused oxidative stress and mitochondrial dysfunction,increased the apoptosis/necrosis rate.Al could down-regulate BRCA1 levels and inhibit Nrf2 expression.The injuries mentioned above were alleviated after QUE intervention.2.The intervention of FSK rescued the decrease of BRCA1 level and increased the level of Nrf2 pathway proteins.3.Al reduced the interaction between BRCA1 and NRF2 proteins,meanwhile,increased Nrf2 ubiquitination levels and lowered its stability.From these results,we draw a conclusion that BRCA1 regulating Nrf2/ARE pathway impairment may be an important mechanism in Al-induced neuronal damage.