The Role Of Endoplasmic Reticulum Stress And The Resulting Autophagic Flux Dysfunction In Fluoride-induced Neurotoxicity

Part 1: The role of Endoplasmic reticulum stress and autophagy in fluoride-induced neurotoxicity of SD ratsObjective: Excessive fluoride exposure leads to neurotoxicity,but the exact mechanisms are still unknown.The present study aimed to explore the possible effects and mechanisms of Endoplasmic reticulum stress(ERS)and autophagy in sodium fluoride(NaF)-induced neurotoxicity of SD rats,thus providing a basis for elucidating the mechanism of fluoride-induced neurotoxicity.Methods: Forty SPF Sprague-Dawley(SD)rats weighing 180-220 g were randomly divided into 4 groups: control group,10,50 and 100 mg/L NaF group,the NaF were administrated by drinking water for 2 months.The Morris water maze was carried out to examine the learning and memory ability of rats.The nissl staining and transmission electron microscopy were applied to detect the morphology changes of rat's hippocampus.The Western blot was utilized to determine the expression of key protein of ERS and apoptosis,as well as autophagy key indicators,such as glucose-regulated protein 78(GRP78),inositol-requiring enzyme 1?(IRE1?),cysteine containing aspartate specific protease-12(Caspase-12)and cysteine containing aspartate specific protease-3(Caspase-3),P62 / sequestosome 1,Beclin1 and microtubule-associated protein 1 light chain3-?(LC3)expression in the rat's hippocampus and cortex.Furthermore,the immunohistochemical technique was operated to determine the location and expression of the key protein of ERS and autophagy in the rat's hippocampus.Result: The results demonstrated that in the place navigation test of Morris water maze,compared with the control,the rats of 100 mg/L NaF group needed more distance to find the platform at the same time point(P < 0.05),and the average latency of the 100 mg/L NaF group was significantly longer than that of the control group(P < 0.05).Furthermore,in the spatial probe test,the rats of 100 mg/L NaF group performed fewer times to find the location where the platform was(P < 0.05).In addition,compared with the control,the rats of 50 and 100 mg/L NaF group performed the decreased ratio of the time spent in the target quadrant / the time spent in all quadrant,and the ratio of the distance travelled in the target quadrant / the distance travelled in all quadrant(P < 0.05).The nissl staining results showed that,compared with the control,the quantity of nissl body were reduced in the CA3 region of the hippocampus of NaF treated rats.NaF exposure resulted in ultrastructural abnormalities in rat hippocampus,as manifested by shrinkage,jagged,and irregular nucleus appearance,as well as chromatin homogenization.Furthermore,the autophagosomes and swollen endoplasmic reticulums were also discerned.Moreover,elevated GRP78 protein expression were detected in 50 and 100 mg/L NaF treated hippocampus and in all NaF treated cortex(P < 0.05);concurrently,elevated IRE1? protein expression were detected in all NaF treated hippocampus and in 50 and 100 mg/L NaF treated cortex(P < 0.05);elevated cleaved caspase-12 protein expression were detected in all NaF treated hippocampus and in 50 and 100 mg/L NaF treated cortex(P < 0.05);in addition,cleaved-caspase-3 were increased in all NaF treated hippocampus and cortex(P < 0.05).Furthermore,defective autophagy was observed,as shown by increased Beclin1,LC3-II and P62 expression in all NaF treated hippocampus and in 50 and 100 mg/L NaF treated cortex(P < 0.05).The results of immunohistochemical staining showed that the number of cells with positive expression of GRP78,Caspase-12,and LC3 in the hippocampus and cortex of fluoridetreated rats was higher than that in the control group,and the number of positive cells of these proteins was increased with the increase of NaF dose.Conclusions: Fluoride lead to neurotoxicity of SD rats,inducing damaged learning and memory ability.In addition,fluoride induces excessive ERS and activates the ERSmediated apoptotic pathway in the hippocampus and cortex of SD rats.Concurrently,fluoride can either promote autophagosome formation or inhibits autophagosome degradation,resulting in impaired autophagic flux in hippocampus and cortex of SD rats.In summary,ERS and autophagy were involved in the damage of the nervous system of SD rats induced by fluoride.Part 2: The role of ERS and its mediated autophagic flux dysfunction in fluoride-induced toxicity of SH-SY5Y cellsObjective: To explore the role of autophagy mediated by ERS in fluoride-induced toxicity in the human neuroblastoma SH-SY5Y cells,and provide clues for exploring the mechanisms of neurotoxicity of fluoride.Methods: SH-SY5Y cells were exposed to different doses of NaF(0,10,20,30 mg/L)for 24 h.The Western blot was applied to determine the expression of the key protein of ERS,apoptosis as well as autophagy,such as GRP78,IRE1?,C/EBP homologous protein(CHOP),Caspase-3,Beclin1,P62 and LC3 in SH-SY5Y cells which exposed to different NaF doses.Furthermore,the cells were pretreated with the 4-Phenylbutyric acid(4-PBA),Wortmannin(Wort),chloroquine(CQ)as well as the Rapamycin(Rapa)for 1 h,and then exposed to NaF for 24 h,the key protein expression of ERS and autophagy,the LC3 dots,as well as the cell viability of each group were tested by Western blot,immunofluorescence and Cell Counting Kit-8(CCK-8),respectively.Result: Compared with the control group,the GRP78,IRE1?,cleaved caspase-3,Beclin1 and LC3-(40)(40)protein expression in 30 mg/L NaF-treated cells were significantly increased(P < 0.05),while the CHOP and P62 protein expression in 20 and 30 mg/L NaF-treated cells were significantly increased(P < 0.05);Furthermore,compared with the NaF group,the IRE1?,CHOP,cleaved caspase-3,P62 and LC3-(40)(40)protein expression were significantly decreased in NaF+4-PBA group(P < 0.05);concurrently,compared with the NaF group,the P62 and LC3-(40)(40)protein expression were significantly decreased in NaF+wort group(P < 0.05),while increased in NaF+CQ group(P < 0.05),interestingly,the P62 protein expression was significantly decreased,while the LC3-(40)(40)protein expression was significantly increased in NaF+Rapa group(P < 0.05).Similarly,the results of immunofluorescence also verified that the aggregation of LC3 dots was in accordance with the LC3-(40)(40)protein expression level in each group.More importantly,the results of CCK-8 showed that 4-PBA intervention significantly increased NaF group cell viability(P < 0.05).In addition,the intervention of Wort,CQ was significantly decreased while the Rapa intervention significantly increased the cell survival rate of the NaF group(P < 0.05).Conclusions: A certain dose of NaF induces ERS and autophagy in SH-SY5Y cells,and autophagy suppression leads to decreased SH-SY5Y cell viability,while autophagy activation can promote SH-SY5Y cell viability.Furthermore,the impaired autophagic flux in NaF-treated SH-SY5Y cells is mediated by ERS,and inhibition of ERS can improve SH-SY5Y cell viability.