The Effect Of P2X4 On The Expression Of ?-synuclien In The Substantia Nigra Of PD Rat Model

Objective To investigate the effect of P2X4 on the expression of ?-synuclein in Parkinson's disease(PD)rat model which induced by 6-hydroxydopamine(6-OHDA).Methods The male Wistar rats were randomly divided into 6 groups,including control group,6-OHDA group,P2X4 negative control lentiviral vectors(P2X4-NC)group,lentivirus vectors with small interference RNA for P2X4(si RNA-P2X4)group,P2X4-NC+6-OHDA group,and si RNA-P2X4+6-OHDA group,with 30 rats each group.The 6-OHDA group and control group were given 6-OHDA or equivalent amount of normal saline containing 0.02% ascorbic acid for substantia nigra positioning injection alone.The P2X4-NC group and si RNA-P2X4 group were given positioning injection of empty negative virus or virus carrying the target lentivirus that suppressed P2X4 expression into the rat left substantia nigra using the cerebral stereotaxic apparatus,respectively.The P2X4-NC+6-OHDA group and si RNA-P2X4+6-OHDA group were given positioning injection of empty negative virus or virus carrying the target lentivirus that suppressed P2X4 expression through the substantia nigra,respectively;followed by positioning injection of 6-OHDA in the left substantia nigra 1 weeks later.Apomorphine was injected intraperitoneally to induce behavior test,after 2 weeks.To be induced once a week.4 weeks later,the rats were decapitated to collect the brains,the left substantia nigra tissues in rats were collected,and the protein expression levels of P2X4,?-synuclein,and caspase-1 in the left substantia nigra were detected using Western blotting.Meanwhile,the m RNA expression levels of P2X4,?-synuclein,and caspase-1 in the left substantia nigra were detected through fluorescence quantitative PCR.Changes in the TH positive neuron number in the left substantia nigra in each group were detected by immunofluorescence staining.Results The immunofluorescence staining results of dopaminergic neurons in substantia nigra suggested that,compared with the control group,the numbers TH positive dopaminergic neurons in the substantia nigra of 6-OHDA group and P2X4-NC+6-OHDA group were markedly reduced,and the differences were statistically significant(P<0.01).Compared with P2X4-NC+6-OHDA group,the numbers TH positive dopaminergic neurons in the substantia nigra of P2X4-NC group and si RNA-P2X4+6-OHDA group were notably increased,and the differences were statistically significant(P<0.01).Compared with control group,the numbers TH positive dopaminergic neurons in the substantia nigra of P2X4-NC group and si RNA-P2X4 group showed no statistical significance compared with that in control group.Western blotting results indicated that,compared with the control group,the expression quantities of P2X4,?-synuclein and caspase-1 in the substantia nigra of 6-OHDA group and P2X4-NC+6-OHDA group were dramatically up-regulated,and the differences were statistically significant(P<0.01).Compared with the P2X4-NC+6-OHDA group,the P2X4 expression quantities in the substantia nigra of P2X4-NC group and si RNA-P2X4+6-OHDA group were notably down-regulated,with difference being of statistical significance(P<0.01).The ?-synuclein and caspase-1 expression quantities in si RNA-P2X4+6-OHDA group were lower than those in P2X4-NC+6-OHDA group,with statistically significant difference(P<0.01).Fluorescence quantitative PCR results revealed that,compared with the control group,the P2X4 expression in the substantia nigra of 6-OHDA group and P2X4-NC+6-OHDA group was markedly up-regulated,and the differences were statistically significant(P<0.01).P2X4 expression in P2X4-NC group and si RNA-P2X4+6-OHDA group was apparently down-regulated compared with that in P2X4-NC+6-OHDA group,with statistically significant differences(P<0.01).Compared with the control group,?-synuclein expression in 6-OHDA group was distinctly up-regulated,and the difference was statistically significant(P<0.01).?-synuclein expression in si RNA-P2X4+6-OHDA group was outstandingly down-regulated compared with that in P2X4-NC+6-OHDA group,and the difference was statistically significant(P<0.01).Compared with the control group,the caspase-1 expression in 6-OHDA group was notably increased,and the difference was statistically significant(P<0.001).Compared with the P2X4-NC+6-OHDA group,the caspase-1 expression in si RNA-P2X4+6-OHDA was markedly down-regulated,and the difference was statistically significant(P<0.001).Conclusion P2X4 plays a certain role in the pathogenesis of PD model,and down-regulation of P2X4 can affect ?-synuclein expression.Its mechanism may be potentially achieved through suppressing caspase-1 expression.Therefore,down-regulation of P2X4 may protect the dopaminergic neurons.