Effects Of Autophagy On Fluoride-induced Cytotoxicity In Human Neuroblastoma SH-SY5Y Cells

PartⅠ Effects of fluoride on autophagy in human neuroblastoma SH-SY5 Y cellsObjective: Fluoride is an essential trace elements for the human body,which is widely present in nature.Excessive fluoride intake not only damages the skeletal system,but also damages the central nervous system.Studies have shown that autophagic dysfunction is associated with the effects of fluoride on male reproductive system damage,but the role of autophagy in fluoride-induced neurotoxicity is rarely reported.Therefore,the aim of this study was to investigate the effect of Na F on the autophagy in SH-SY5 Y cells,and to provide a theoretical basis for the further study of the mechanisms of fluoride neurotoxicity.Methods : SH-SY5 Y cells were exposed to sodium fluoride(NaF)at different concentrations(20,30,40,50,60,80,100 mg/L)for 24 hours(h)and cell viability was measured using CCK-8 kit.Appropriate Na F concentrations were selected duce to cell viability test.Transmission electron microscope(TEM)was used to detect the ultrastructure of autophagosomes.Acridine orange staining was used to examine the formation of autophagic vesicles and Western blotting was used to determine the expression levels of autophagy relevant proteins autophagy related gene 5(Atg5),microtubule-associated protein 1 light chain 3(LC3)and P62 in SH-SY5 Y cells.Result: With the Na F dosese increased,the cell viability was decreased gradually,and showed in a dose-response manner.When the concentration of Na F was no more than 30 mg/L,the toxic effects of Na F on SH-SY5 Y cells was not obvious.When the concentration of Na F was above 40 mg/L,the cell viability was significantly decreased as compared with control(P < 0.05).For the further studuies the final concentrations of Na F were 20,40 and 60 mg/L.The TEM results showed the presence of autophagic ultrastructure in SH-SY5 Y cells and fluoride could decrease the amount of autophagosomes.Acridine orange staining results showed that the amount of autophagic vesicles were obviously decreased in 40 and 60 mg/L Na F-treated groups compared with control.The protein expression levels of Atg5 and LC3-II were dose-dependently decrease and P62 was dose-dependently increased in Na F-treated groups compared with those in control(P < 0.05).Conclusion: Fluorine can inhibit the autophagy activity of SH-SY5 Y cells.PartⅡ Effects of autophagy on fluoride-induced oxidative stress and apoptosis in SH-SY5 Y cellsObjective: The first part of the study found that a certain dose of fluoride can reduce the survival rate of SH-SY5 Y cells and inhibit its autophagic activity.Whether up-regulation of autophagy level can alleviate the cytotoxicity caused by fluoride? In this part using autophagic activator rapamycin(RAP)to up-regulate the autophagy level,the study aimed to investigate the effects of autophagy on fluoride-induced oxidative stress and apoptosis in SH-SY5 Y cells.Methods:SH-SY5 Y cells were exposed to different concentrations(20,40,60 mg/L)of Na F for 24 h.Flow cytometry was used to detect the production of reactive oxygen species(ROS)and apoptosic rate in SH-SY5 Y cells.Western blotting was used to detect the expression levels of apoptosis-related proteins Cleaved-caspase-3 and Poly ADP-ribose polymerase(PARP).The RAP intervention experiments were divided into four groups: Control,Na F(60 mg/L),RAP(100 nmol/L)and Na F + RAP.SH-SY5 Y cells were exposed to the above four groups for 24 h.Western blotting was used to determine the protein expression levels of LC3 and P62.Flow cytometry was used to detect the production of ROS and apoptosic rate in SH-SY5 Y cells.Result: With the increase of NaF concentrations the levels of ROS and apoptotic rate of cells were significantly increased in 40 and 60 mg/L Na F-treated groups(P < 0.05).The protein expression levels of Cleaved-caspase-3 and Cleaved-PARP were significantly increased in 40 and 60 mg/L Na F-treated groups(P < 0.05).The result of RAP intervention experiments showed that compared with the control group,the protein expression levels of LC3-II was significantly decreased and P62 was significantly increased in 60 mg/L Na F-treated group(P < 0.05).Compared with the 60 mg/L Na F-treated group,the protein expression levels of LC3-II was significantly increased and P62 was significantly decreased in Na F + RAP group(P < 0.05).The production of ROS and apoptotic rate was significantly decrease in Na F + RAP group compared with the 60 mg/L Na F-treated group(P < 0.05).Conclusion: RAP treatment can alleviate fluoride-induced oxidative stress and apoptosis in SH-SY5 Y cells,that is,up-regulation of autophagy protects against fluoride-elicited cytotoxicity in SH-SY5 Y cells.