| Mitochondrial fusion/fission process is crucial for mitochondrial function maintenance.Studies have shown that mitochondrial fusion/fission imbalance participates in neurotoxicity induced by fluoride,however,the underlying mechanisms of mitochondrial fusion/fission imbalance caused by excessive fluoride in neuronal cells have not been clarified.In diverse tissues,Silent mating type information regulation 2 homolog 1(SIRT1)plays protective roles in diverse tissues through regulating mitochondrial fusion/fission process by peroxisome proliferator-activated receptor ? coactivator 1?(PGC-1?).In addition,more and more studies have shown that micro RNA(miRNA)can affect SIRT1 expression at transcriptional level and exist pivotal roles in a variety of diseases through regulating mitochondrial fusion/fission process.Part 1.Roles of SIRT1 in fluoride-induced neurotoxicityObjective: to investigate roles of SIRT1 in fluoride neurotoxicity and its possible regulatory mechanisms.Methods:(1)In vivo experiments: A total of 40 SPF-grade female Sprague-Dewley(SD)rats weighing 180~220 g was purchased from experimental animal center of Disease Control and Prevention Center of Hubei.These rats were randomly divided into two groups after adaptive feeding for one week: control group [sodium fluoride(NaF)concentration lower than 1 mg/L in drinking water] and NaF(30 rats with NaF concentration at 100 mg/L in drinking water)group.After 4 months of fluoride treatment,rats were further divided into four groups: control group,NaF group,NaF+ resveratrol(RSV,200 mg/kg)group,and NaF+ nicotinamide(NIC,100 mg/kg)group.RSV and NIC gavage solution were prepared with 1.5% carboxymethyl cellulose sodium solution,and the dosage of gavage solution was 0.01 ml/g bw/day according to rats' weights.After 2 months,Morris water maze experiment was used to observe learning and memory abilities of rats.Hippocampal tissues of rats were extracted and neuronal apoptosis and pathological changes were detected by TUNEL staining and Nissl staining;ultrastructural changes of mitochondria in hippocampal tissues were observed by transmission electron microscope(TEM);Western blot and immunohistochemical methods were used to detect protein expression levels of SIRT1,mitochondrial fusion/fission proteins including mitofusion 1(Mfn1),mitofusion 2(Mfn2),dynamic related protein 1(Drp1)and fission protein(fission 1,Fis1)as well as their localization and distribution in hippocampal tissues.(2)In vitro experiments: SH-SY5 Y cells were transfected with adenovirus overexpressing SIRT1(Ad-SIRT1)and control Vector adenovirus for 24 h and treated with NaF(60 mg/L)for 24 h.SIRT1 inhibitor NIC(3m M)was combined with NaF to treat SH-SY5 Y cells for 24 h.Western blot was used to detect protein expressions of SIRT1,mitochondrial fusion/fission proteins,pro-apoptotic proteins cytochrome C(Cyto C)and apoptotic proteins cleaved cysteinyl aspartate specific proteinase 3(cleaved caspase 3)and poly ADP-ribose polymerase(PARP);Flow cytometry and laser scanning confocal microscope(LSCM)were used to detect mitochondrial membrane potential(MMP)and mitochondrial morphological changes.Cell viability was determined by CCK-8.Results:(1)In vivo experiments: Compared with control group,the number of times crossing the platform,ratio of residence time and distance in the target quadrant to the total time and distance rats spent were all reduced in NaF group(P < 0.05).Compared with NaF group,the number of times crossing the platform and ratio of residence time to the total time rats spent in the target quadrant in NaF+RSV group were increased(P < 0.05).However,there was no difference as to the ratio of distance in the target quadrant to the total distance in rats of NaF+RSV group.The above indexes did not change significantly in NaF+NIC group.Results of morphological changes of hippocampus in rats showed that,TUNEL positive cells and Nissl bodies were significantly increased and pathological changes such as swelling,vacuolization and cristae disappearance occurred in mitochondria in NaF group.Compared with NaF group,TUNEL positive cells and Nissl bodies in NaF+RSV group were decreased and mitochondrial abnormal morphology was alleviated.However,TUNEL positive cells and Nissl bodies in NaF+NIC group were further increased and mitochondrial abnormal morphology were more severe.Compared with control group,protein expressions of Mfn1 and Mfn2 in NaF group were increased,while protein expressions of Drp1 and Fis1 were significantly decreased(P < 0.05).Compared with NaF group,protein expressions of Mfn1,Mfn2 in NaF+RSV group were decreased,while protein expressions of Drp1 and Fis1 were significantly increased(P < 0.05).However,protein expressions of Mfn1 and Mfn2 in NaF+NIC group were further increased,while protein expressions of Fis1 were further significantly decreased(P < 0.05).Immunohistochemical results of SIRT1,Mfn2 and Drp1 in CA1 region of rat hippocampal tissues were similar to that of protein expressions.(2)In vitro experiments: Results of mitochondrial indexes showed that,compared with control group,protein expressions of SIRT1 and mitochondrial fusion were increased with mitochondrial fission protein levels decreased in NaF group(P < 0.05).In addition,MMP levels were decreased and mitochondria showed such abnormal morphologies as excessive elongation.Compared with NaF group,SIRT1 protein expressions were further increased and mitochondrial fusion protein levels were further augmented in Ad-SIRT1+NaF group(P < 0.05).Furthermore,expression levels of mitochondrial fission proteins were further decreased and MMP level were more decreased along with more aggravated mitochondrial abnormal morphologies.Results of cellular outcome showed that,compared with control group,protein expressions of Cyto C,PARP and cleaved caspase 3 were increased and cell viability was decreased in NaF group(P < 0.05).Compared with NaF group,expressions of apoptosis-associated proteins were decreased and cell viability was increased in Ad-SIRT1+NaF group(P < 0.05),whereas expression of apoptosis-associated proteins were further increased and cell viability was further decreased in NaF+NIC group(P < 0.05).Conclusions: NaF promoted mitochondrial fusion and inhibited mitochondrial fission,caused mitochondrial abnormal morphologies and dysfunction,resulting in mitochondria-mediated apoptosis and subsequent neurotoxicity.RSV/Ad-SIRT1 alleviated fluoride neurotoxicity and NIC aggravated fluoride neurotoxicity.Above all,SIRT1 promotion alleviated fluoride-induced mitochondrial fusion/fission imbalance,improved mitochondrial abnormal morphologies and dysfunction caused by fluoride,reduced mitochondria-mediated apoptosis and improved cell survival rates,thus antagonizing neurotoxic effect of fluoride.Part 2.Roles of SIRT1 in fluoride neurotoxicity by regulating PGC-1?Objective: to investigate roles of PGC-1? in SIRT1-mediated fluoride neurotoxicity.Methods: The targeted binding of SIRT1 and PGC-1? in cells was detected by Chromatin immunoprecipitation-PCR(Ch IP-PCR).Proteins from hippocampal tissues and SH-SY5 Y cells in Part 1 were extracted.Protein expression of PGC-1? was detected by western blot.After transfection of PGC-1?(Ad-PGC-1?)and Vector into SH-SY5 Y cells for 24 h,NaF and NIC co-treated cells for 24 h.Western blot was used to detect protein expressions of PGC-1?,mitochondrial fusion/fission proteins,pro-apoptotic protein Cyto C and apoptotic proteins cleaved caspase 3 and PARP.Mitochondrial morphology was observed by LSCM.Results: Ch IP-PCR results showed that SIRT1 could target PGC-1? in SH-SY5 Y cells.PGC-1? protein expression was significantly decreased in NaF group compared with the control group(P < 0.05).Compared with NaF group,PGC-1? protein expression was further increased in NaF+RSV group in vivo and NaF+Ad-SIRT1 group in vitro(P < 0.05),which was further decreased in NaF+NIC group both in vivo and in vitro(P < 0.05).In addition,compared with Vector+NaF group,expression levels of PGC-1? were decreased,Mfn1 and Mfn2 protein levels were increased,Drp1 and Fis1 expression levels were decreased in in Vector+NaF+NIC group(P < 0.05).Also mitochondrial abnormal morphologies were aggravated and protein expressions of Cyto C,cleaved caspase 3 and PARP were increased(P < 0.05).Compared with Vector+NaF+NIC group,PGC-1? and mitochondrial fission protein expressions were increased(P < 0.05),mitochondrial abnormal morphologies were alleviated and expressions of apoptosis-associated proteins were evidently decreased(P < 0.05)in Ad-PGC-1?+NaF+NIC group.However,fusion protein expression levels did not change significantly.Conclusions: Fluoride causes mitochondrial fusion/fission imbalance,abnormal mitochondrial morphology and induced mitochondria-mediated apoptosis,which are significantly aggravated by NIC.Ad-PGC-1? blocks the hazardous role of NIC on fluoride neurotoxicity.Taken together,SIRT1 up-regulates PGC-1? expression and inversed mitochondrial fission decrease caused by fluoride in SH-SY5 Y cells,alleviated abnormal mitochondrial morphology and mitochondria-mediated apoptosis induced by fluoride,thus preventing fluoride neurotoxicity.Part 3.Roles of miR-708-3p targeting SIRT1 in fluoride neurotoxicityObjective: to investigate upstream miR-708-3p targeting SIRT1 and its roles in fluoride neurotoxicity.Methods:(1)RNAhybrid(v2.1.2),Miranda(v3.3a)and Target Scan(v7.0)were used to predict miRNA targeting SIRT1 expression(highly homologous between human and rat).The target miRNA was screened by real-time PCR(q RT-PCR).(2)Mi RNA mimic and inhibitor were designed and transfected into SH-SY5 Y cells for 48 h.The miRNA expression and protein expressions of SIRT1 were detected by q RT-PCR and western blot.The specific binding of selected miRNA to SIRT1 was detected by double luciferase reporter assay.(3)Mi RNA mimic and inhibitor were transfected into cells for 24 h,followed by further fluoride treatment for 24 h.Expression of SIRT1,pro-apoptotic protein Cyto C and apoptotic proteins cleaved caspase 3 and PARP were detected by western blot.(4)miRNA mimic and Ad-SIRT1,miRNA inhibitor and NIC treated SH SY5 Y cells with fluoride exposure.Western blot was used to detect protein expressions of SIRT1,mitochondria fusion/fission proteins and Cyto C,cleaved caspase 3;Morphology of mitochondria was observed by LSCM.Results:(1)A total of 287 miRNA that could regulate SIRT1 were predicted by miRNA software.In combination with miRNA in the transcriptome of hippocampal tissues of rats in our laboratory,miR-708-3p,miR-29c-3p,miR-3085 and miR-194-5p were chosen.q RT-PCR results showed that compared with control group,miR-708-3p expression in both fluorine-infected rats and cell group was decreased(P < 0.05),while expressions of the other three were unstable.Therefore,we selected miR-708-3p for subsequent studies.(2)miR-708-3p expression was increased and SIRT1 protein expression was decreased in miR-708-3p mimic group compared with control group(P < 0.05).However,miR-708-3p expression was decreased and SIRT1 protein expression was increased in miR-708-3p inhibitor(P < 0.05).Results of dual luciferase reporter gene showed that miR-708-3p mimic could specifically bind to SIRT1.(3)SIRT1 protein expression was elevated and protein expression of Cyto C,cleaved caspase 3,and PARP were elevated in NaF group compared with control group(P < 0.05).Compared with NaF group,SIRT1 protein expression in NaF+miR-708-3p mimic group was decreased,and expression levels of apoptosis-associated proteins were increased(P < 0.05).Conversely,expression levels of SIRT1 protein in NaF+miR-708-3p inhibitor group was further increased(P < 0.05),while expression levels of apoptosis-associated proteins were decreased(P < 0.05).(4)Compared with NaF+miR-708-3p mimic group,SIRT1 protein expression was increased,protein expression of Cyto C,cleaved caspase 3 were decreased(P < 0.05),mitochondrial fusion protein expressions were decreased whereas fission protein expressions were increased(P < 0.05),mitochondrial abnormal morphologies was alleviated in NaF+miR-708-3p mimic+Ad-SIRT1 group.Whereas,compared with NaF+miR-708-3p inhibitor group,SIRT1 protein expression was decreased,protein expression of Cyto C,cleaved caspase 3 were increased(P < 0.05),mitochondrial fusion protein expressions were increased whereas fission protein expressions were decreased(P < 0.05),mitochondrial abnormal morphologies was aggravated in NaF+miR-708-3p inhibitor+NIC group.Conclusions: NaF decreases miR-708-3p expression in neuronal cells.In SH-SY5 Y cells,miR-708-3p targeted SIRT1.Mi R-708-3p mimic inhibited SIRT1 protein expression,aggravated mitochondrial fusion/fission imbalance and abnormal morphologies induced by NaF,thus triggering mitochondria-mediated apoptosis.The aggravated roles of miR-708-3p mimic in neurotoxicity induced by NaF were resisted by Ad-SIRT1.On the other hand,Mi R-708-3p inhibitor increased SIRT1 protein expression,alleviated mitochondrial fusion/fission imbalance and abnormal morphologies induced by NaF,thus alleviating mitochondria-mediated apoptosis.The protective roles of miR-708-3p inhibitor in neurotoxicity induced by NaF were blocked by NIC.In conclusion,decreased miR-708-3p expression can lead to SIRT1 upregulation,improves mitochondrial fusion/fission imbalance caused by fluoride,alleviates abnormal mitochondrial morphology and mitochondria-mediated apoptosis,thus antagonizing fluoride neurotoxicity. |
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