The Role Of Mitochondrial Biogenesis In Fluoride-induced Neurotoxcity

Part Ⅰ: The role of mitochondrial biogenesis in fluoride-induced neurotoxicity of SD ratObjective: The fluoride-induced neurotoxicity has attracted considerable attention,yet litte is known about the exact mechanisms.The results of previous studies revealed that the mitochondrial dysfunction contributes to the fluoride neurotoxicity,and the mitochondrial biogenesis was related to the mitochondrial recovery.The present study is aimed to explore the effect of mitochondrial biogenesis in fluoride-induced neurotoxicity of SD rats,and to provide a theoretical basis for the further study of the mechanisms of fluoride neurotoxicity.Methods: After one week acclimation,60 Sprague-Dawley rats were exposed to NaF at 10,50 and 100 mg/L via drinking water from 2 months before mating to the weaning of offspring,during which the corresponding NaF exposure still continued.Rats in each group were chosen randomly for mating according to the ratio of male-to-female as 1:2.The offspring rats were exposed to the same NaF concentration as the adult rats did until 2 months old.The offspring rats were sacrificed within 24 h.Both the brain and hippocampal tissue were harvested at low temprature.The protein and DNA of the hippocampal tissue was extracted.The relative mitochondrial DNA(mtDNA)content of the hippocampal tissue was determined by Quantitative Real-time PCR.Western blot was used to determine the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α),nuclear respiratory factor 1(NRF1),mitochondrial transcription factor A(TFAM),CO2 and ATP6 in the hippocampal tissue.The immunehistochemical method was utilized to determine the location and expression of mitochondrial biogenesis key regulator PGC-1α and NRF1 in the hippocampus CA1 region of the offspring rat.Results: The protein levels of PGC-1α,NRF1 and TFAM were dose dependently decreased after NaF treatment in the hippocampal tissue(P < 0.05).The relative mtDNA contents and mRNA expression of complexes subunit CO1 and ATP8 were also significantly decreased compared with control(P < 0.05).Compared with the control group,the expression of PGC-1α and NRF1 was decreased significantly in the hippocampus CA1 region.Conclusions: Fluoride can cause a decrease of relative mtDNA content in the hippocampal tissue of offspring SD rat.The expression of mitochondrial biogenesis key regulator and respiratory chain complex subunits also decreased in 100 mg/L NaF group.In summary,mitochondrial biogenesis was involved in the fluoride-induced neurotoxicity.Part Ⅱ: The effect of mitochondrial biogenesis in fluoride-induced cytotoxicity in SH-SY5 Y cellsObjective: To investigate the influence of NaF on mitochondrial biogenesis in SH-SY5 Y cells,and provide theoretical basis for further clarifying the fluorine neurotoxic mechanism.Methods: SH-SY5 Y cells in exponential phase were treated with different concentrations(20,40,60 mg/L)of sodium fluoride(NaF)for 24 h.The TFAM overexpression adenovirus was used to transfect SH-SY5 Y cells.The mRNA levels of PGC-1α,NRF1 and TFAM were determined by Quantitative Real-time PCR in SH-SY5 Y cells,as well as the relative mitochondrial DNA(mtDNA)contents and mRNA expression of mitochondrial respiratory chain complexes subunit CO1,CO2,CO3,ATP6 and ATP8.Western blot was used to determine the expression levels of mitochondrial biogenesis key regulating factor PGC-1α,NRF1 and TFAM in SH-SY5 Y cells.The expression of mitochondrial respiratory chain complexes subunit CO2 and ATP6 is also determined by Western blot.We have used the flow cytometry to test mitochondrial membrane potential and mitochondrial ROS in SH-SY5 Y cells.Cell Counting Kit-8 was utilized to test cell viability.Results: Both the protein and mRNA levels of PGC-1α,NRF1 and TFAM were dose dependently decreased after NaF treatment in SH-SY5 Y cells(P < 0.05).Compared with control group,the relative mtDNA contents and mRNA expression of complexes subunit CO1,CO2,CO3,ATP6 and ATP8 were also significantly decreased(P < 0.05).The protein expression levels of CO2 and ATP6 were significantly decreased in 40 mg/L and 60 mg/L NaF group(P < 0.05).The cell viablity was significantly decreased in 40 mg/L and 60 mg/L NaF group(P < 0.05).After overexpression of TFAM,compared with Vector+NaF group,both the protein and mRNA levels TFAM were increased after TFAM overexpression in SH-SY5 Y cells(P < 0.05).The relative mtDNA contents and mRNA expression of complexes subunit CO1,CO2,CO3,ATP6 and ATP8 in TFAM overexpression+NaF group were also significantly increased compared with Vector+NaF group(P < 0.05).The protein levels of CO2 and ATP6 were significantly increased compared with Vector+NaF group(P < 0.05).Compared with Vector+NaF group,the mitochondrial membrane potential and cell viability were significantly increased,while the mitochondrial ROS was significantly decreased in TFAM overexpression+NaF group(P < 0.05).Conclusions: A certain dose of NaF induces suppression of mitochondrial biogenesis in SH-SY5 Y cells,leads to mitochondrial dysfunction and decrease SH-SY5 Y cell viability.While the overexpression of TFAM can activate mitochondrial biogenesis and alleviate fluoride-induced mitochondrial dysfunction restores the cell viability of SH-SY5 Y cells.