The Role Of Peroxisome Proliferator-activated Receptor ? In The Oxidative Stress Mechanism Of Nerve System Injury Induced By Experimental Fluorosis

Objective Peroxisome proliferator-activated receptor ?(PPAR?)is a member of the PPARs family in the nuclear receptor superfamily and has anti-oxidative stress effects.In order to further explain the injury mechanism of oxidative stress in chronic fluorosis nervous system,this study selected chronic fluorosis rats and fluorine treated neurons in vitro to measure the expression of PPAR?,Peroxisome proliferator-activated receptor ? coactivator-1?(PGC-1?),nuclear factor-E2-related factor 2(Nrf2)and ?-glutamylcysteine synthetase(?-GCS)and the level of oxidative stress;to investigate the effect of PPAR? in the mechanism of oxidative stress injury in chronic fluorosis nerve system.Methods:(1)The animal model with chronic fluorosis was successfully estabolished.60 Sprague Dawley(SD)rats weighing 100-120 g were divided randomly into a control group(drinking fluoride content <0.5 mg/L),low fluoride group(drinking fluoride content at 5.0 mg/L),high fluoride group(drinking water fluoride content at 50.0 mg/L),10 in each group(equal number in both genders).The experiment periods were 3 and 6 months.(2)SH-SY5 Y cells were treated with fluoride(NaF)and 15d-PGJ2.The cells were divided into normal control group,fluoride group(cells were treated with 4 mmol/L NaF),PPAR? agonist group(cells were treated with 20 ?mol/L 15d-PGJ2),and intervention group(cells were treated with 20 ?mol/L 15d-PGJ2 first and then exposed to 4 mmol/L NaF),the cultivating time was 48 h.(3)Determination method: Dental fluorosis was tested by Three-degree method,and the fluoride content in urine,serum and brain tissue were detected by Fluoride-ion selective electrode;the ability of learning and memory of rats was examined by Morris water maze test;the activity of 15d-PGJ2 in SH-SY5 Y cell was detected by CCK-8 Method;the levels of PGC-1?,PPAR?,NrF2 and ?-GCS mRNA and protein expression level in brain tissue were detected by real-time fluorescence quantitative PCR and Western blotting,respectively;the activities of Glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)and content of malondialdehyde(MDA)in serum were detected by biochemical method,serum 8-OHDG levels was detected by ELISA;the correlation between PPAR? protein expression and SOD activities and MD content was analyzed.Result: The results obtained from animal investigation showed that the fluorosis groups had different degrees of dental fluorosis,especially obvious in 6-month high fluoride rats;in the rats exposed to low or high fluoride in experimental period of 3 months and 6 months,the fluorine contents in urine,serum and brain tissue were significantly higher than those in the control group,which was in direct proportion to the fluoride content.The escape latency of rats was significantly longer than that of the control group,the total number of space exploration was significantly reduced,and the time of the quadrant of the platform stay was significantly shortened,indicating that the learning and memory ability was significantly reduced,and the rats at the 6-month experiment were more obvious than those at 3 months.The expression of PPAR?,PGC-1?,NrF2 and ?-GCS mRNA and protein in the hippocampus and cortex of rats treated with fluoride for 3 months showed a decreasing trend with the increase of fluoride concentration,but there was no statistical significance.The level of the above-mentioned indicators in the rat brain tissues treated with fluoride for 6 months were significantly reduced,which was statistically significant.The serum SOD and GSH-Px activities in fluorosis rats were significantly lower than those in normal control rats,and the serum MDA content was significantly increased.The results of correlation analysis showed that there was a negative correlation between PPAR? protein and brain fluoride contentin in fluorisis rats brain tissue,and a positive correlation with SOD expression in rat serum and negative correlation with MDA.The results of in vitro culture of SH-SY5 Y cells of CCK-8 showed that the cell survival rate of SH-SY5 Y cells treated with 30 ?mol/L or more of 15d-PGJ2 cultivating 2 h was significantly lower than that of the control group(P <0.05),so the concentration of 20 ?mol/L was selected as follow-up.And compared with the control group,the levels of PPAR? mRNA and protein were significantly decreased in the fluorosis group,and the activities of SOD and GSH-Px were significantly decreased,and the MDA content was significantly increased.The activity of SOD and GSH-Px in nerve cells of 15d-PGJ2+NaF group and NaF+15d-PGJ2 group were still lower than that of normal control group,and the content of MDA was higher than that of normal control group,but compared to the the fluorine-treated group,the activities of SOD and GSH-Px increased,MDA content decreased significantly.The expression of PPAR? protein in SH-SY5 Y cells was positively correlated with SOD activity and negatively correlated with MDA content.Conclusion: The expression of PGC-1?/PPAR? mRNA and protein have been decreased in rat brain tissues exposed to excessive fluoride for a long time and in vitro exposed to fluorine,and the level of oxidative stress has been increased,resulting in brain tissue and cell damage.Treatment with PPAR? agonists reduces the level of oxidative stress and thus reduces the toxic effects of fluorine.Activation of the PGC-1?/PPAR? pathway reduces elevated levels of oxidative stress due to fluorosis.The attenuation of the PGC-1?/PPAR? pathway is one of the mechanisms of chronic fluorosis in the oxidative stress of the nervous system.