The Role Of Resveratrol In The Mechanism Of Oxidative Stress Injury In Central Nerve System Induced By Experimental Fluorosis

Objective:It has been reported that long-term intake of excessive fluoride can result in whole-body damages.The key pathogenesis mechanism of these damages was correlated with oxidative stress?OS?,which induced by fluoride ion.Researches also fund that resveratrol?3,5,4'-trihydroxystilbene,RSV?,agonist of silent mating type information regulation 2 homolog?SIRT1?,exert anti-OS effect in many diseases.In present study,brain tissues of experimental chronic-fluorosis rats and its offspring and primary cultured neurons cultured in vitro were used as models to test the protective effect of RSV on the central nerve system,which injured by injured chronic fluorosis.Methods:1.Establish of animal model:Thirty-six Sprague-Dawley?SD?rats were randomly divided into four groups?8 rats for each group?.Different experimental conditions were applied to each group:1)rats drank tape-water?F<1 mg/L?and ate the normal forage was set as Control group;2)rats drank same tap-water and feed same forage for 4 months and force-feeding 50 mg/kg/day RSV for 3 months was named as RSV group;3)rats expose to NaF?50 mg/L?in drinking water was fluoride-expose group;4)fluoride-exposure&RSV group:rats,which raise conditions were same as fluoride-expose group,except for force-feeding RSV?50mg/kg/day?after 4 months and the feeding of RSV lasted for 3 months.After 6months,female and male rats were caged together?2:1?,which allowed them breeding progeny,offspring of 1,7,14,21 and 28 days old of rats from different groups were subjected to further experiments.2.Establish of Cell model:Moreover,primary cultured neurons and SHYH-5Y cells were also used as cell models,both models were expose to NaF?4 mmol/L?,RSV?10 or 20?mol/L?and Suramin?inhibitor of SIRT1,200 or 300?g/mL?for 48 h,alone or with different combination.The degree of dental fluorosis was observed and recorded.The fluorine electrode method was used to determine the amount of fluoride in urine,serum,brain tissues and bone tissues of rats from different groups.Morris Water Maze test was used to determine the spatial learning and memory of these rats.Protein abundance of SIRT1,PGC-1??Peroxisome proliferator-activated receptor?coactivator-1??,?7 nAChRs?Nicotinic Acetylcholine Receptors?were determined by using western blot analysis.Neural cell survival rate under different conditions were measured by CCK-8 test.The activities ofSOD?Superoxidedismutase?,?-GCS??-Glutamylcysteinesynthetase?,CAT?Catalase?and the concentration of MDA?Malondialdehyde?,H₂O₂,carbonyl were measured by using corresponding biochemical kits.Finally,the concentration of8-OHdG?8-Hydroxy-2 deoxyguanosine?was determined by ELISA kit?DNA/RNA Oxidative Damage ELISA Kit?.Results:1.In animal model:Chronic-fluorosis model of rats was established successfully,as evidenced by exhibiting dental fluorosis and increased fluoride concentration in the serums,urine,brains and bone tissues of rats.The seriousness of dentalfluorosisandconcentrationoffluorideinfluoride-exposeand fluoride-exposure&RSV groups were increased compare to Control and RSV groups,this result suggest that RSV has no effect on reduce the absorbance of fluoride in our model.The ability of learning and memory were decreased in fluoride-expose group compare to Control group,while these cognitive abilities were partially restored in fluoride-exposure&RSV group.The expression levels of SIRT1,PGC-1?and?7nAChR proteins in hippocampus and cortex regions of fluoride-expose group and its offspring?28 days old?were significantly decreased compare to Control group,however this trend was partially reversed in fluoride-exposure&RSV group.In addition,compare to Control group,the OS level?concentrations of MDA,carbonyl,8-OHdG and H₂O₂?were increased in fluoride-expose group and its offspring.Whereas,the concentrations of SOD,?-GCS and CAT were decreased significantly.In comparison,OS level in fluoride-exposure&RSV group was decreased.Correlation analysis showed that the increase of OS level in fluoride-expose group and its offspring was negatively correlated with expression of SIRT1 protein,while the decreased activity of SOD,?-GCS and CAT was positively correlated with the expression of SIRT1 protein.2.In cell model:The protein levels of SIRT1,PGC-1?,?7 nAChR and?4 nAChRs were decreased in NaF exposed primary cultured hippocampus cells and SH-SY5Y cells?addition of suramin will further decrease the expression of these proteins?.However,the addition of RSV could attenuate the decreasing of SIRT1,PGC-1?,?7nAChR and?4 nAChRs proteins,which may cause by OS of fluoride.Conclusions:?1?The expression of SIRT1,PGC-1?and?7 nAChR proteins,and the anti-OS level in brains and serums were decreased in fluoride-expose group and its offspring?28 days of age?;?2?The expression of SIRT1,PGC-1?and?7 nAChR proteins and the anti-OS level were also decreased in cells exposed to high dose NaF;?3?The treatment by RSV along did not attenuated the the contents of fluorideof the rats,but RSV treatment could attenuated the decreased expression level of SIRT1 and the increased level of OS.Thus,partially reversed the declined ability of learning and memory,which resulted from chronic fluorosis.These results suggesting that RSV could protect neuronal cell form OS damage?caused by fluorosis?.?4?The reduced protein levels of SIRT1 were significant correlated with the increased the levels of OS of the rats with fluorosis.The pretreatment with RSV attenuated the reduced SIRT1and the raised level of oxidative stress in rat brains with chronic fluorosis,suggesting that RSV may play a antioxidant effect to fight against chronic fluorosis via stimulating SIRT1,and PGC-1?may be involved in the occurrence of chronic fluorosis.