Protective Effect And Its Signal Transduction Mechanism Of Autophagy On Apoptosis In PC12 Cells Induced By Deoxynivalenol

The trichothecene mycotoxin deoxynivalenol(DON)is produced in wheat,barley and corn following infestation by the fungus Fusarium in the field as well as during storage.Colloquially it is known as “vomitoxin”.Recently,the research on DON nerve injury is mainly focused on neuronal apoptosis and tissue necrosis.However,whether autophagy is involved in DON induced neurotoxicity as well as the role of autophagy has not been reported.In this experiment,PC12 cells were selected as an object of in vitro study.PC12 cells were exposed to DON at different concentrations(0,125,250,500,1 000 and 2 000 ng/mL)for 24 h.The changes of cell morphology and cell ultrastructure were determined by inverted microscopy,transmission electron microscopy.Autophagic vacuoles were determined by acridine orange(AO),monodansylcadaverin(MDC)under fluorescence microscope,Cellular apoptosis rates were measured using an annexin V-FITC/PI apoptosis detection kit by flow cytometry(FCM).Formation of GFP–LC3 dots,the presence of LC3 puncta were determined by immunofluorescence.Autophagy-related genes class ? PI3 K,Beclin-1,Bcl-2,P62 and LC3 mRNA expression as well as autophagy-related protein class ? PI3K?Beclin-1?Bcl-2?P62?LC3 expression were determined by,quantitative real time polymerase chain reaction(qRT-PCR)and Western-blot(WB)respectively.Chloroquine(an autophagic inhibitor)and rapamycin(an autophagic inducer)were used to explore the relationship between autophagy and apoptosis.The results obtained are as follows:1.PC12 cells were treated with different concentrations of DON for 24 h.In the control group,the cells were full and the links were dense.During treatment,the numbers of PC12 cells decreased with increasing concentration of DON.Cell bulge disappeared,their volume decreased followed by cells shrinkage as well as turned smaller round.PC12 cells in the control group showed regular chromatin distribution compared with the vacuoles in the cells cytoplasm of DON treated.The autophagosome and late autophagic lysosomes with double or multilayer membrane are typically features of autophagosome.The cells in control group after AO staining displayed green fluorescence,basically no red fluorescence in cytoplasm and nucleolus but displayed considerable red fluorescent dots in cytoplasm of DON treated cells.The control cells have aweak fluorescence intensity.As the DON concentration increased,fluorescence intensity signal becomes strengthened gradually and fluorimetric dots began to bank up.GFP-LC3 are randomly sprinkled throughout cells without autophagy;most of GFP-LC3 appeared as spot with autophagy induced.2.As the DON concentration increased,it will increase the rate of apoptosis.The percentage of apoptotic cells in all groups treated with DON was significantly higher than control PC12 cells(P <0.01),While at 2000 ng/mL DON have declined from their peaks of 1000 ng/mL DON.3.The distribution of endogenous LC3 in cells before and after DON treatment for 24 h was monitored by indirect immunofluorescence staining.Specific punctatedistribution of endogenous LC3? was observed in PC12,which shows more dots appeared in DON-treated(500,1 000,2 000 ng/mL DON)cells than in control cells(P <0.01).4.The expression level of experimental group class ? PI3 K mRNA was significantly higher than that of the control group(P <0.01),and the expression level of class ? PI3 K mRNA increased gradually at 125~1 000 ng/mL DON,reached peak at 1000 ng/mL DON concentration,and then gradually decreased.The expression level of Beclin-1 mRNA increased significantly with DON treatment(P <0.01),and reached the maximum at 1000 ng/mL DON concentration.Bcl-2 mRNA expression levels were significantly decreased with increased DON concentrations greater than 250 ng/mL,at1000 ng/mL DON fell to its lowest point.Treatment with increasing concentrations of DON resulted in significant difference(P <0.05)or highly significant(P <0.01)induction of LC3 mRNA expression(P <0.01),peaking at 1000 ng/mL.P62 mRNA expression levels were significantly different(P <0.05)or highly significant(P <0.01)decreased with increased DON concentrations,but at1000 ng/mL DON fell to its lowest point.In addition,compared with the 1000 ng/mL DON treatment group and 1000 ng/mL DON+PI3K inhibitors(LY294002)treatment group,the expression levels of class ? PI3K?Beclin-1?LC3 mRNA were significantly decreased(P <0.01),while the expression levels of Bcl-2 and P62 mRNA were significantly increased.5.The expression levels of class ? PI3 K was significantly(P <0.05)or highly significantly(P <0.01)increased with increasing DON concentration,and reached a peak level at 1000 ng/mL.The expression levels of Beclin-1 was significantly increased(P <0.01)and also topping at 1000 ng/mL.The expression of LC3 was significantly increased(P <0.05)at the 250 ng/mL and highly significantly increased(P <0.01)with increasing DON concentration,reached a maximum at 1000 ng/mL.Treatment with increasing concentrations of DON resulted in significant reduction of Bcl-2 and P62 in mitochondria and reached a minimum at 1000 ng/mL(P <0.01).Moreover,compared with 1000 ng/mL DON group,the expression levels of class ? PI3K?Beclin-1?LC3 at1000 ng/mL DON+LY294002 group were significantly decreased(P <0.01).However,the relative expression levels of Bcl-2 and P62 in PC12 cells treated with 1000 ng/mL DON + LY294002 were significantly higher compared to those treated with 1000 ng/mL DON alone(P <0.01).6.Compared with the 1000 ng/mL DON treatment group,the expression of LC3? protein in 1000 ng/mL DON+CQ was significantly increased(P <0.01).The relative expression levels of LC3? in PC12 cells treated with 125 ng/mL DON + RAP was significantly higher compared to those treated with 125 ng/mL DON alone(P <0.01).Compared with the control group,the apoptotic rate of each treatment group was significantly(P <0.05)or highly significantly(P <0.01)increased.7.During Comparison with the control group,the apoptosis rates of PC12 cells in treatment groups were significantly increased(P <0.01).Compared with the 125 ng/mL DON treatment group,the apoptosis rates of PC12 cells in 125 ng/mL DON+RAP was significantly decreased(P <0.01).Compared with the 1000 ng/mL DON treatment group,the apoptosis rates of PC12 cells in 1000 ng/mL DON+CQ was significantly increased(P <0.01).In conclusion,DON can induce apoptosis and autophagy in PC12 cells.Autophagy related genes and protein class ? PI3 K,Beclin-1,Bcl-2,P62 and LC3 participate in the regulation of DON induced autophagy.CQ inhibits DON induced autophagy in PC12 cells,and RAP can increase the level of autophagy expression.The apoptosis rate showed that inhibition of autophagy could increase the apoptosis rate of PC12 cells.