| Deoxynivalenol?DON?is a common trichothecene mycotoxin and can be found throughout the world.DON widely contaminates crops and feed products.DON can cause neurotoxicity,immunotoxicity,reproductive toxicity,carcinogenesis,teratogenesis and mutagenesis in human and animals.Because of the complexity of nervous system,the understanding of people to the neurotoxicity of DON is quite incomplete and is still exploratory stage.In addition,the study of the neurotoxicity of DON in vitro has been reported rarely,especially the specific signal transduction mechanisms of apoptosis in nerve cells induced by mitochondria-mediate signal pathway.PC12 cell is used extensively as a cell line model to study neural mechanism.So,PC12 cells were used in this study to determine the molecular mechanisms of apoptosis induced by DON exposure through the mitochondria-mediate signal pathway.After cells were treated with different concentrations of DON(0,125,250,500,1000,and 2000 ng·mL-1)for 24 h,changes in cellular growth and morphology were observed by an inverted microscopy and a transmission electron microscopy,the situation nuclear chromatin of nucleus was observed after dyeing with hoechst33258.Cellular apoptosis rates were measured using an annexin V-FITC/PI apoptosis detection kit.Mitochondrial membrane potentials were measured by flow cytometry?FCM?.The mRNA levels of apoptois-related genes were determined by real-time polymerase chain reaction?RT-PCR?,and the expression levels of apoptosis-related proteins were determined by western-blot.The activity of caspase3 was detected by immunofluorescene,and the transcriptional activities of P53 and AIF were evaluated by electrophoretic mobility shift assay?EMSA?.The results were as follows:1.After PC12 cells were treated with different concentrations of DON(0,125,250,500,1000,2000 ng·mL-1)for 24 h,cellular morphology in control group was long and spindle-like,and cells were densely packed.While the number of PC12 cells in treatment groups were generally decreased with the increasing DON concentrations.Furthermore,cell bulge disappeared,cell volume was reduced,and cells became round.Regular nuclear,normal organelles,regular chromatin distribution,and clear nucleolus in control group were showed by transmission electron microscopy,but cells in treatment groups were showed typical ultrastructure characteristics of apoptotic cells,including nuclear shrinking,disappearance of nucleoli,chromatin gathering to the nuclear membrane,mitochondrial swelling,and vacuolar degeneration.There was uniform blue in nucleus of control group observed by dyeing with hoechst33258.However,the nucleus of treatment groups were showed bright blue,and the proportion of bright blue and intensity increased in various degrees with the increasing DON concentrations.2.Compared with the control group,the cellular viabilities of PC12 cells in treatment groups were significantly reduced with increasing DON concentrations?P <0.01?.The release of lactate dehydrogenase?LDH?was increased slightly when DON concentration was 125-1000 ng·mL-1,and there was a dose-effect relationship between release of LDH and DON concentrations.Whereas the release of LDH was significantly increased compared with the control group when DON concentration was 2000 ng·mL-1?P <0.01?.3.This study showed that PC12 cells can be induced to apoptosis by DON.Compared with the control group,the apoptosis rates of PC12 cells in treatment groups were significantly increased?P <0.01?.When DON concentration was 125-1000 ng·mL-1,the apoptosis rate was increased in a dose-dependent manner,while the apoptosis rate of 2000 ng·mL-1 DON treatment group was lower than1000 ng·mL-1DON.Therefore,1000 ng·mL-1 DON was used as the optimal concentration in subsequent trial for adding the Caspase8 inhibitor?FMK?.4.Compared with the control group,the mitochondrial membrane potential?MMP?of PC12 cells in treatment group decreased significantly with the DON concentrations increasing?P <0.01?.Compared with 1000 ng·mL-1 DON treatment group,MMP was significantly increased in 1000 ng·mL-1 DON + FMK treatment group?P <0.01?.5.Compared with the control group,the Bax mRNA expression levels and Bax/Bcl-2 ratio of PC12 cells in treatment group increased with increasing concentrations of DON?P <0.01?,peaking at 1000 ng·mL-1.However,the Bcl-2 mRNA expression levels were decreased with increased DON concentrations greater than 500 ng·mL-1?P <0.01?and reached a minimum at 1000 ng·mL-1.Bid mRNA expression levels were significantly increased with increased DON concentrations greater than 250 ng·mL-1?P <0.01?,peaking at 1000 ng·mL-1.Additionally,compared with the 1000ng·mL-1 DON treatment group,the Bax and Bid mRNA expression levels and Bax/Bcl-2 ratio were significantly decreased in PC12 cells treated with 1000 ng·mL-1 DON + FMK?P <0.01?,whereas Bcl-2 mRNA expression levels were significantly increased?P <0.01?.6.Compared with the control group,the expression levels of cleavedCaspase9 was significantly increased with the increasing DON concentrations?P <0.01?,peaking at 1000 ng·mL-1.The expression levels of cleaved-Caspase3 were significantly increased with increased DON concentrations greater than 500 ng·mL-1?P<0.01?,peaking at 1000ng·mL-1.Treatment with increasing concentrations of DON resulted in significant reduction of AIF and Cyt C in mitochondria and reached a minimum at 1000 ng·mL-1.Moreover,compared with 1000 ng·mL-1 DON treatment group,the relative expression levels of cleaved-Caspase9 and cleeaved-Caspase3 in PC12 cell treated with1000 ng·mL-1 DON + FMK were significantly higher compared to those treated with 1000 ng·mL-1 DON alone?P <0.01?.The expression levels of AIF at 1000 ng·mL-1DON + FMK group were significantly increased?P<0.01?,the expression levels of Cyt C at 1000 ng·mL-1 DON + FMK group were increased relative to 1000 ng·mL-1 DON,but the difference was not significant.7.The activation of caspase 3 was observed by using a laser scanning confocal microscopy.It was showed that the cells of control group emitted little red fluorescence,while treatment groups emitted red fluorescence in various degrees.And with the increasing DON concentrations,the red fluorescence of cells enhanced and reached strongest degree at 1000 ng·mL-1 DON.In addition,compared with 1000 ng·mL-1 DON group,red fluorescence was significantly decreased in the treatment groups added 1000 ng·mL-1DON + FMK.8.Compared with the control group,the transcriptional activities of the transcription factor AIF and P53 in treatment groups were significantly increased with increasing concentrations of DON.Compared with 1000ng·mL-1 DON group,the transcriptional activities of the transcription factor AIF and P53 were significantly reduced in the treatment groups applied 1000 ng·mL-1DON + FMK.In conclusion,DON induces apoptosis of PC12 cells by altering the morphology of PC12 cells,damaging cellular membrane and nucleus,and decreasing the mitochondrial membrane potential,increasing the expression levels of mitochondrial related pro-apoptosis genes?Bax and Bid?and decreasing the expression levels of mitochondrial related anti-apoptotic gene?Bcl-2?.In addition,the expression levels cleaved-Caspase3 and cleaved-Caspase9 and the release of Cyt C and AIF from mitochondrial into cytoplasm were promoted by DON.Meanwhile,the transcriptional activities of AIF and P53 also were enhanced.These experimental results suggest that DON can induce apoptosis in PC12 cells via the mitochondrial-dependent signal transduction pathway. |
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