The Role Of ATP-P2X4 Receptor Signaling In Regulation Of Apoptotic Factors In Parkinson’s Disease

Objective: To investigate the expression of P2X4 receptor and apoptotic protein caspase-3 in 6-OHDA-induced Parkinson’s rat model,and to find out the effect of the regulation of ATP-P2X4 receptor signaling on caspase-3 expression.Methods: Male Wistar rats were randomly divided into 8 groups with 20 rats in each group.After anesthetizing rats and accurately locating the left substantia nigra,rats in each group were treated as follows: control group and 6-OHDA group were injected with saline containing 0.3% ascorbic acid into substantia nigra;control group with no-load si-RNA lentivirus and 6-OHDA group with no-load si-RNA lentivirus were injected with no-load si-RNA lentivirus;control group with gene-overexpression si-RNA lentivirus and6-OHDA group with gene-overexpression si-RNA lentivirus were injected with P2X4 R gene overexpression si-RNA lentivirus;control group with gene-inhibited si-RNA lentivirus and 6-OHDA group with gene-inhibited si-RNA lentivirus were injected with P2X4 R gene-inhibited si-RNA lentivirus.One week after adapting to the experimental environment,the Parkinson’s disease model were built.0.3% ascorbic acid saline were injected into the substantia nigra of the above control groups rats,while in the above6-OHDA group rats,6-OHDA solution was injected into the same area.Apomorphine-inducted rotation tests were carried out one week after model establishment,and successful PD models with positive test were screened.After killed the rats,the brain tissue was frozen and stained with immunofluorescence to count the number of TH positive dopaminergic neurons.Total RNA and protein in substantia nigra of rats were isolated and extracted and the relative expression of P2X4 receptor and caspase-3 was detected by real-time fluorescence quantitative PCR and Western blotting.Results: 1.Immunofluorescence detecting the number of TH-positive dopaminergic neurons: Compared with the control group,the number of TH-positive dopaminergic neurons in the 6-OHDA group was significantly reduced(P < 0.01),and there was no significant difference in the number of cells in the control group with no-load si-RNA lentivirus.Compared with the control group with no-load si-RNA lentivirus,the number of cells in the 6-OHDA group with gene-overexpression si-RNA lentivirus decreased significantly,while that in the 6-OHDA group with gnen-inhibited si-RNA lentivirus increased.And the differences were with statistical significance(P < 0.01).2.PCR detecting the relative expression of caspase-3 in substantia nigra of rats: Compared with the expression of caspase-3 in substantia nigra of rats in control group,the expression in6-OHDA group was significantly increased(P < 0.01).And the expression of caspase-3 in the 6-OHDA group with gene-overexpression si-RNA lentivirus was higher than that in the 6-OHDA group with no-load of si-RNA lentivirus(P < 0.05),while caspase-3expressed lower in the 6-OHDA group with gene-inhibited si-RNA lentivirus than that the6-OHDA group with no-load of si-RNA lentivirus(P < 0.05).3.Western bloting was used to detect the relative expression of caspase-3 protein in substantia nigra of rats: Compared with the control group,the expression of target protein increased in 6-OHDA group(P <0.01).The expression of caspase-3 protein was significantly higher in the 6-OHDA group with gene-overexpression si-RNA lentivirus than in the 6-OHDA group with no-load si-RNA lentivirus(P < 0.01),while the protein in the substantia nigra of the 6-OHDA group with gene-inhibited si-RNA lentivirus was significantly lower than that in the6-OHDA group with no-load si-RNA lentivirus(P < 0.01).Conclusion: When P2X4 receptor gene is overexpressed,caspase-3 gene and protein expression are increased,resulting in increased loss of functional cells in substantia nigra.When the expression of P2X4 receptor gene was inhibited,the expression of caspase-3gene and protein was lower than that of PD rats with no-load virus,which accordingly reduced the loss of dopaminergic neurons in substantia nigra.ATP-P2X4 receptor signaling can participate in the pathogenesis of Parkinson’s disease by regulating the expression of caspase-3.