| Objective:To investigate the role of P2X4 receptor in the pathogenesis of Parkinson’s disease and the protective effect of 5-BDBD on the damage of dopaminergic neurons caused by 6-OHDA and find the target of PD protection intervention therapy by testing the effect of P2X4 receptor antagonist 5-BDBD on the expression of Interleukin-1β(IL-1β)and Interleukin-18(IL-18)in Parkinson’s disease(PD)rat model induced by 6-OHDA.Methods:Wistar male rats were divided into 4 groups(n=30):(1)control group: one week after normal saline pretreatment,0.02% ascorbic acid was stereotactic injected into the substantia nigra;(2)6-OHDA group: one week after normal saline pretreatment,6-OHDA was stereotactic injected into the substantia nigra;(3)5-BDBD group: one week after 5-BDBD pretreatment,0.02% ascorbic acid was stereotactic injected into the substantia nigra;(4)5-BDBD+6-OHDA group: one week after 5-BDBD pretreatment,6-OHDA was stereotactic injected into the substantia nigra.14 d after the operation,the rats in each group were injected with APO(0.5mg/kg)intraperitoneally.The rotation behavior of the rats was induced and the number of rotations to the right of the rats in the 30 min was counted.The speed >420r/h was seen as the success of the PD model.After 4 weeks,10 rats in each group were anaesthetized by intraperitoneal anesthesia,and 4% polyoxymethylene was injected into the heart.The brain was taken out of the head.Then after the sucrose was dehydrated,the frozen section was frozen.TH-positive neurons in PD rats substantia nigra were detected by immunohistofluorescence stain.The brain of 10 rats in each group which were taken from each group was extracted from the head after abdominal anesthesia,and the left substantia nigra tissue was quickly separated.Then the mRNA was extracted and the concentration of RNA was measured.The expression of P2X4 R,IL-1 beta and IL-18 m RNA in substantia nigra was detected by RT-PCR.The relative expression of the target gene was calculated by 2-DeltaDelta Ct formula.The brain of 10 rats in each group which were taken from each group was extracted from the head after abdominal anesthesia,and the left substantia nigra tissue was quickly separated.The the tissue were lysised and protein were extracted quickly.The relative expression of P2X4 R,IL-1 beta and IL-18 protein in the substantia nigra of each group was detected by Western-blot.GraphPad Prism6 statistical software was used to analyze the data and the measurement data was expressed in s.The two factor variance analysis(two-way ANOVA)was used for the interaction analysis.The P>0.05 showed no interaction,and the single factor analysis of variance(one-way ANOVA)was used,and inter-group comparison used the LSD method.P<0.05 indicated that the difference was statistically significant.Results:Compared with the control group and 5-BDBD group,the TH-positive neurons were decreased in 6-OHDA group and 5-BDBD+6-OHDA group(P<0.01);the mRNA level of P2X4 R,IL-1βand IL-18 were increased in 6-OHDA group and 5-BDBD+6-OHDA group(P<0.01);the protein level of P2X4 R,IL-1β and IL-18 were increased in 6-OHDA group and 5-BDBD+6-OHDA group(P<0.01).Compared with 6-OHDA group,the TH-positive neurons were increased in 5-BDBD+6-OHDA group(P<0.01);the mRNA level of P2X4 R,IL-1βand IL-18 were decreased in 5-BDBD+6-OHDA group(P<0.01);the protein level of P2X4 R,IL-1β and IL-18 were decreased in 5-BDBD+6-OHDA group(P<0.01).Conclusion:By detecting the changes in the number of TH positive neurons,P2X4 R,IL-1 beta and IL-18 protein,this study confirmed the role of P2X4 R in the pathogenesis of PD.Inhibiting the expression of P2X4 R can reduce the degeneration of dopaminergic neurons in the substantia nigra of PD model rats,and inhibit the up regulation of the expression of IL-1β and IL-18 which induced by 6-OHDA.The P2X4 R inhibitor 5-BDBD maybe has the potential neuroprotective effect,which may provides a new direction for the treatment of PD. |
PDF Full Text Request