The Effect Of P2X4R Receptor Overxpression On Interleukin Expression In The Brain Of Parkinson's Disese Rat Model

Objective: To investigate the effect of overexpression of P2X4 receptor on the expression of Interleukin-1?(IL-1?)and Interleukin-18(IL-18)in substantia nigra in Parkinson's disease(PD)rat model induced by 6-OHDA.To verify the role of P2X4 R and its mediation on expression of IL-1? and IL-18 in the pathogenesis of PD.Methods: Using 200~250 g Wistar male rats,divided into 6 groups according to the principle of complete randomization.Each group of rats was treated with different methods: 1.control group: saline containing 0.02% ascorbic acid were injected into the left substantia nigra of rats 2.6-OHDA group: 6-OHDA were injected into the left substantia nigra of rats 3.RNA-P2X4 group: targeted lentivirus carrying P2X4 R overexpression were injected into the left substantia nigra 4.RNA-P2X4+6-OHDA group: the rats were injected with the target lentivirus expressing P2X4 through the left substantia nigra,after one week,the 6-OHDA were again injected into the left substantia nigra.5.P2X4-NC group: No-load negative virus were injected into the left substantia nigra of rats 6.P2X4-NC+6-OHDA group: the rats were injected with the No-load negative virus expressing P2X4 through the left substantia nigra,after one week,the 6-OHDA were again injected into the left substantia nigra.Two weeks after different treatment,apomorphine was injected intraperitoneally to induce behavior test,screening successful rat model(right rotation rate?7/min)and continuing the experiment.Four weeks after the model was established,each group of rats was anesthetized by 10% chloral hydrate and then decapitated.The brain tissue was removed and stored for using after different processing.And then detect the changes of TH-positive dopaminergic neurons in substantia nigra of rats by using immunofluorescence staining in each group,using fluorescence quantitative PCR to detected the level of IL-1? m RNAin substantia nigra of brain tissue of rats in each group,and Western blot was used to detect the expression of P2X4 R and IL-1? in substantia nigra of each group.Results: 1.Fluorescence quantitative PCR results showed that P2X4 R,IL-1? and IL-18 content in midbrain substantia nigra was significantly increased in 6-OHDA group and P2X4-NC + 6-OHDA group compared with control group(P <0.001),the content of P2X4 R m RNA(P<0.01),IL-1? m RNA(P<0.05)and IL-18 m RNA(P<0.001)in substantia nigra of RNA-P2X4 + 6-OHDA group increased than the P2X4-NC+6-OHDA gopup,the difference was statistically significant.2.The results of immunofluorescence staining showed that the numbers of TH positive dopaminergic neurons in substantia nigra of 6-OHDA group and P2X4-NC + 6-OHDA group were significantly decreased compared with the control group(P <0.001),compared with the P2X4-NC+ 6-OHDA group,the number of TH-positive dopaminergic neurons in the substantia nigra of RNA-P2X4 + 6-OHDA group decreased significantly,the difference was statistically significant(P <0.001).The numbers of TH positive dopaminergic neurons in substantia nigra in P2X4-NC group and RNA-P2X4 group were not significantly different from those in control group.3.Western Blot showed that compared with the control group,the expression of P2X4 R,IL-1 ? and IL-18 protein in the 6-OHDA group and P2X4-NC + 6-OHDA group increased(P <0.001);Compared with P2X4-NC + 6-OHDA group,the expression of P2X4 R protein(P <0.01),IL-1? protein(P<0.001)and IL-18 protein(P<0.05)in substantia nigra of rats increased in RNA-P2X4 6-OHDA group,the difference was statistically significant.Conclusions: Through our study,lentivirus-mediated P2X4 receptor overexpression can upregulate the expression of IL-1? and IL-18 in the substantia nigra,which can damage dopaminergic neurons and affect the pathogenesis and development of PD.