Effect Of Mitochondrial Pathway In The NaF-induced Apoptosis Of Mouse Renal Cells

The objective of the present study was to explore the molecular mechanism of apoptosis induced by sodium fluoride(NaF)in the mouse kidney by using methods of flow cytometry,quantitative real-time polymerase chain reaction(qRT-PCR),western blotting,and experimental pathology.240 four-week-old ICR mice were randomly divided into 4 groups and exposed to different concentrations of NaF(0 mg/kg,12 mg/kg,24 mg/kg and 48 mg/kg)for a period of 42 days.At 21 and 42 days of the experiment,the histopathological changes,the percentage of apoptotic cells,mitochondrial membrane potential(MMP)and relative expression of apoptotic regulatory genes and proteins in the mouse kidney of every group were observed.Results were as follow:1.Pathological changes of sodium fluoride-damaged kidney: the renal volume was reduced.Histopathological observation showed that the lesions were mainly characterized by granule degeneration,vacuolar degeneration,necrosis of the tubular epithelial cells and glomerular enlargement in a dose-and time-dependent manner.2.Effect of NaF on the apoptotic rate of renal cell: the results of flow cytometry showed that the apoptotic rate of renal cells was significantly increased with the increase of sodium fluoride intragastric time and dose(p < 0.05 or p < 0.01).3.Effect of NaF on MMP: The results of flow cytometry showed that the MMP showed a significant depolarization trend with the increase of sodium fluoride intragastric time and dose(p < 0.05 or p < 0.01).4.Effect of sodium fluoride on gene expression of mitochondrial apoptotic pathway-related markers: the results of real-time PCR showed that sodium fluoride up-regulated the mRNA expression of Cyt c,Smac/Diablo,AIF,Endo G,Bax,Bak,Bim,P53,Caspase-9,PARP and Caspase-3 in mitochondrial-mediated apoptotic pathway,and down-regulated the mRNA expression of XIAP,Bcl-2 and Bcl-xl.At the 21 st day of the experiment,the mRNA expression levels of Cyt c,Bak,Bax,Bim,Caspase-3,AIF and p53 in the three experimental groups,as well as Bax/Bcl-2,were significantly higher than the control group(p < 0.01).The mRNA expression levels of Caspase-9,Endo G,PARP and Diablo in the 24 and 48 mg/kg groups were significantly increased in comparison to those in the control group(p < 0.05 or p < 0.01).The mRNA expression levels of Bcl-2,Bcl-xl and XIAP in the three experimental groups were significantly lower than those of the control group(p < 0.05 or p < 0.01).At the 42 st day of the experiment,the mRNA expression levels of Cyt c,Bak,Bax,Bim,Caspase-3,Caspase-9,Endo G,PARP,Diablo and p53 in the three experimental groups,as well as Bax/Bcl-2,were significantly higher than those of the control group(p < 0.05 or p < 0.01),and the AIF mRNA expression level in the 24 and 48 mg/kg groups were significantly increased when compared to that in the control group(p < 0.01),while the mRNA expression levels of Bcl-2,Bcl-xl and XIAP were the same as those on the 21 st day of the experiment(p < 0.05 or p < 0.01).5.Effect of sodium fluoride on protein relative expression of mitochondrial apoptotic pathway-related markers: at the 21 st day of the experiment,western blot results showed that protein expression levels of cytosolic Cyt c,Bak,Bim,cleaved-Caspase-3,Endo G,p-p53 and Smac of the three experimental groups were significantly increased(p < 0.05 or p < 0.01).Protein expression levels of Bax,AIF,PARP and Omi in the 24 and 48 mg/kg group were also significantly elevated(p < 0.05 or p < 0.01),and Caspase-9 protein expression levels were significantly increased in the 12 and 48 mg/kg groups(p < 0.05 or p < 0.01).However,the XIAP protein expression levels in the 24 and 48 mg/kg groups were significantly decreased(p < 0.01),and protein expression levels of Bcl-2 and Bcl-xl were significantly decreased in the experimental group(p < 0.05 or p < 0.01).Bax/Bcl-2 was significantly increased in the 24 and 48 mg/kg groups(p < 0.05 or p < 0.01).At the 42 st day of the experiment,protein expressions levels of Cyt c,Bak,Bim,cleaved-Caspase-3,Endo G,p-p53,Smac,Bax,AIF,Caspase-9 and Omi were all significantly increased in the three experimental groups(p < 0.05 or p < 0.01),and PARP protein expression levels were significantly increased in the 24 and 48 mg/kg groups(p < 0.05 or p < 0.01).XIAP Protein expression levels were significantly decreased in the 24 and 48 mg/kg groups(p < 0.01),and protein expression levels of Bcl-2 and Bcl-xl in the three experimental groups were significantly decreased(p < 0.05 or p < 0.01).Bax/Bcl-2 were significantly increased in the three experimental groups(p < 0.05 or p < 0.01).In conclusion,the sodium fluoride at a concentration of 12 mg/kg or more can cause apoptosis of the kidney cells in mice.Sodium fluoride induces renal cell apoptosis through a mitochondria-mediated apoptotic pathway.Changes in gene and protein expression in mitochondrial pathway-associated markers are the molecular basis for renal cell apoptosis induced by sodium fluoride.