| ObjectiveSodium azide(NaN3)is classed as a highly toxic substance,Which can be absorbed through respiratory tract,digestive tract and skin to cause poisoning and multi-system damage in the process of production and processing.Among all these toxic tissues and organs in the body,the nervous system consumes a large amount of oxygen and as the major target organ which is the most sensitive to hypoxia,has the least tolerance and the most severe reaction.The studies have shown that acute poisoning of NaN3 can induce pathological changes in the brain,caudate-putamina nucleus,cerebral cortex,hippocampus,cerebellar cortex and thalamus in rats and accompany with the apoptosis of neurons.In the present study,We used the rats as the research objects and PC 12 cell was used to generate a dopamine neuron model,The effects and mechanism of NaN3 on the rat striatum and PC 12 cells were investigated,to identify whether NaN3 apoptosis-inducing effects in the rat striatum and cultured PC 12 cells and the underlying mechanisms.MethodsIn vivo,for the induction of poisoning model,32 male Sprague-Dawley(SD)rats were randomly divided in to the following 4 groups,8 rats in each group:Control group(group C),NaN3-treated with a low dose(group L,1.5mg/kg),NaN3-treated with a moderate dose(group M,3mg/kg),NaN3-treated with a high dose(group H,6mg/kg),Different doses of NaN3 or physiological saline were administered by intraperitoneal injection six daily to the rats for 6 days and every injection interval was 0.5h.on day 7,the blood of all rats were taken from orbital vein and sacrificed by cardiac perfusion and the striatums in the brains were removed.Peroxide levels and antioxidant activities in the blood were measured by assay kits,including superoxide dismutase(SOD),glutathione(GSH)and lactate dehydrogenase(LDH).The neurons in the striatums were detected by HE dying and the expressionof bcl-2,bax,cytochrome c and caspase 3 protein and apoptosis were detected by immunohistochemistry.In vitro,The nerve cell toxic model was induced by various concentration of NaN3(0,5,10,20,40 and 80 mM)for 12,24,48 and 72 h in PC12 cells.The viability of damaged PC 12 cells was determined by cell counting kit-8(CCK-8).After selection of optimal concentration,the damaged PC 12 cells induced by NaN3 were treated with 10,20 and 40mM for 24h.DAPI staining was employed to additionally examine apoptotic cells and their nuclear changes.Production of reactive oxygen species(ROS),mitochondrial membrane potential(??m)and apoptotic rate of PC 12 cells were also assessed by the corresponding staining kit in a flow cytometry(FCM).Cellular ATP content was estimated by firefly luciferase assay.In addition,following extraction of total protein in treated PC 12 cells,the expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cytochrome c(Cyt-c),peroxisome proliferator-activated receptor ? co-activator 1-?(Pgc-1?),nuclear respiratory factor(Nrf-1/2),mitochondrial transcription factor A(Tfam),complex ?(Cox ?),pan-calcineurin A(CaN),phosphorylated(p)-Ca2+/calmodulin-dependent protein kinase(Camk),p-p38 mitogen-activated protein kinase(p38 MAPK)and p-extracellular signal-regulated kinase(Erk)1/2 were determined by western blot analysis.Results?.In vivo experiment(1).With the increase of the number of injections and the extension of the time of exposure in NaN3,all rats in each dose group showed different degrees of symptoms such as:reduced activity,slow reaction,lethargy,reduced diet and so on.In the low-dose group,poisoning occurred on the 4d of exposure in NaN3,with the first occurrence of accelerated respiration rate,following by restlessness and unsteady gait,but soon entered in a state of the inhibitory which showing like lying prostrate or on the side,But the symptoms of poisoning can be restored automatically after a period of time.The above symptoms appeared in the medium dose group on the 3d and the high dose group on the 2d of exposure in NaN3,respectively.And the duration of symptoms was prolonged compared with the low dose group,but the symptoms could still be recovered.In the high-dose group,there was a rat died on day 5 and one on day 6,respectively.(2).Biochemical test results:In group-NaN3 rats,LDH levels were elevated but SOD and GSH activitives were decreases as compared with those in group C rats(p<0.01).(3).HE dying results:In the control group,the striatum nerve cells were normal in shape,abundant in number,evenly distributed and closely arranged.In the NaN3-treated groups,the cells were arranged in a disorderly,loose and irregular pattern,with hyperchromatic cytoplasm,nuclear pyknosis,loose and swollen nerve fiber bundles,and in the severe group,the number of neurons was significantly reduced.(4)Immunohistochemistry(IHC)dying results:With the increase of the number of injections and the extension of the time of exposure in NaN3,The number of remaining Bax,Caspase-3 and Cytochrome c+neurons in the striatum from the medicated group rats was significantly increased as compared with the control group rats(p<0.01),and The number of remaining Bcl-2 neurons in the striatum from the medicated group rats was significantly lower as compared to the control group rats(p<0.01).?.In vitro experiment(1).CCK-8 assay showed that NaN3 had a significant anti-proliferation effect on PC 12 cells in a dose-dependant and time-dependant manner.After treated with 0,5,10,20,40 and 80 mM NaN3 in PC12cells for 12h,24h,48h and 72h,the inhibition rates were decreasing respectively,and PC 12 cell models were successfully induced by 20mM concentration of NaN3 for 24h,which resulted in 24.89±3.33%viability.(2).In DAPI staining,After treated with 0,10,20 and 40 mM NaN3 in PC12cells for 24h,The morphological changes of PC 12 cells were observed by fluorescence microscopy and cell nuclei shrinkage and chromatin condensation were increased slightly with the concentration of NaN3 in PC 12 cells as compared to the control.In these cells,apoptotic cells were smaller and brighter compared with normal cells.Chromatin condensation and nuclear fragmentation were also observed,whereas blue nuclei of viable cells were identified in the control group.In addition,the number of apoptotic cells and the intensity of green fluorescence were increased in cells exposed to NaN3.(3).Apoptotic rate determination by Annexin V-FITC staining.The numbers of apoptotic cells were 8.2±0.14%,43.77±10.28%and 78.67±13.92%,8.20%(P<0.05)in the PC 12 cells following exposure to NaN3 at different concentrations(10,20 and 40 mM)for 24 h as compared with those in the control group(5.03±1.38%,0 mM NaN3),respectively.These results additionally confirmed that NaN3 induced cell apoptosis in a dose-dependent manner.(4).Changes in mitochondrial membrane potential(??m).After treated with 0,10,20 and 40 mM NaN3 in PC12cells for 24h,The ratios of red and green fluorescence were 1.74±0.2,1.66±0.1,0.32±0.1,as compared with those in the control group were 1.84±0.01,(P<0.05).Suggesting that NaN3 can gradually decreased the mitochondria membrane potential in the PC 12 cells.(5).NaN3-induced accumulation of mitochondrial ROS.The result indicates that the fluorescence intensities in control cells and cells exposed to NaN3 at various concentrations(0,10,20 and 40 mM)for 24 h were 3.65±0.28,6.99±1.06,18.63±4.27 and 39.35±6.85.respectively,indicating that NaN3 exposure significantly increased the production of mitochondrial ROS compared with the control group(P<0.05).These results suggested that NaN3-induced apoptosis improved the production of intracellular ROS.(6).NaN3 downregulates the mitochondrial energy production of cellular ATP.To evaluate the ATP content in PC 12 cells exposed to NaN3,the relative luminescence unit(RLU)was quantitatively determined by a luminometer.And after treated with 0,10,20 and 40 mM NaN3 in PC12cells for 24h,The ATP levels in the PC12 cells were 0.21±0.02 nmol/mg,0.11 ±0.02 nmol/mg,0.07±0.01 nmol/mg,0.04±0.008nmol/mg,respectively(P<0.05).The result indicates that NaN3 inhibited mitochondrial ATP production and gradually decreased the cellular ATP level.(7).Western blot analyses demonstratesd that the expression levels of pro-apoptotic proteins Bax and cytochrome c were upregulated,while the expression levels of anti-apoptotic proteins procaspase-3 and Bcl-2 were downregulated.In addition,the phosphorylation of MAPK and Ca2+/calmodulin-dependent protein kinase ?(Camk?)family members including pan-calcineurin A was increased,in particular the ratios of p-p38/p38 and p-Camk?/Camk?.However,the expression levels of Pgc-1? and its associated proteins,including Nrf-1/2,Tfam and p-Erk1/2 were decreased(p<0.05).ConclusionsThese results suggested that NaN3 can damage the striatum in the rat toxic model and induce apoptosis in PC 12 cells in a dose-dependant manner.The effect may be related to the decrease of mitochondrial membrane potential,ATP level and elevated the level of ROS.Multiple factors acting on the activation and regulation of Pgc-1?-associated signaling pathway may be one of its possible molecular mechanisms. |
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