| Objective: To explore the role of N-terminal kinase(c-jun) and caspase 8 in cer ebellar granule neurons apoptosis induced by heroin. Methods: Primary cultured ce rebellar granule neurons cells, observing the change of nucleus interved by Heroin in the different dosages and time at same concentration with Hoechst 33258 stainin g.After the interference of virus vector with synthetic miRNA silencing c-jun gene was transfected into cerebellar granule cells 72 h,Heroin has been intervened cerebel lar granule neurons for 24 h with 80 ug/ml dosage.Experiment was divided into thre e groups: control group,heroin group, heroin + virus group.Using flow cytometry to detect cell apoptosis rate in each group, each group detected the expression of c-j un and caspase-8 protein fluorescence intensity by immunofluorescence.Real-time qu antitative polymerase chain reaction(real-time quantitative PCR, qRT-PCR) detected the expression of c-jun and caspase-8 mRNA in each group cells, Western blot was used to detect the expression of p-c-jun, c-jun and caspase-8 protein in three groups. Results: With the increasing of heroin concentrions and time prolonged to intervene cerebellar granule neurons cells, the number of neuron cells apoptosis gr adually increased, and the difference was statistically significant(Pconcentration = 0.007,Ptime = 0.005).After the expression of c-jun gene silenced, heroin has intervented cerebellar granule neurons for 24 h, the apoptosis rate of heroin group(46.50 ± 4.39%) was significantly higher than the heroin + virus group’s apoptotic rate(6.06± 3.31%) and control group’s apoptosis rate(7.93 ± 3.68%), the difference was sta tistically significant(P=0.012, P=0.009)(P <0.05).Immunofluoresce-nce staining fou nd:after the 80ug/ml heroin intervened cerebellar granule neurons for 24 h,the leve l of c-jun protein expression showed higher than control group and the heroin + virus group(P=0.041, P=0.010),The difference was statistically significant(P <0.05).Caspase-8 protein highly expressed in the heroin group was different in the control group and the heroin + virus group(P= 0.027, P= 0.018), the difference was stat istically significant(P <0.05).q RT-PCR detected the results of three groups showed:The m RNA expression level of c-jun and caspase-8 in heroin group was significant ly higher than the c-jun and caspase-8 mRNA expression levels in control group(P=0.007, P=0.036) and heroin + virus group(P=0.001, P=0.017), the difference w as statistically significant(P<0.05), meanwhile the mRNA expression level of c-jun and caspase-8 in heroin + virus group was significantly lower than the control gr oup, the difference was statistically significant(P=0.006), There was no significant difference in the level of caspase-8 mRNA with heroin + virus group and control groups,caspase-8 mRNA expression levels was not statistically significant(P=0.088).Western blot analyzed the results showed: p-c-jun, c-jun and caspase-8 protein e xpression level in heroin group was significantly higher than control group(P=0.018, P=0.013, P=0.018) and heroin + virus group(P=0.002, P=0.002, P=0.004), the difference was statistically significant(P<0.05);The protein expression of p-c-jun, cjun and caspase-8 in heroin + virus group lower than the control group(P=0.005,P=0.007, P=0.027), the difference was statistically significant(P<0.05). Conclusion:with increasing ratio of heroin concentration and action time,Heroin-induced apop tosis rate in cerebellar granule neurons cells rised with concentration and time prop ortional dependencies.There is the closely relationship between c-jun, caspase-8 prot ein and heroin induced apoptosis in cerebellar granule neurons cells, caspase-8 pro tein is one of the important promoting apoptosis candidate target genes in c-jun sig naling pathway. |
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