Effect Of P2X4 Signal Axis On Iron Metabolism In Parkinson’s Disease Animal Model

Objective: To elucidate the effect of P2X4 signal axis on iron metabolism in substantia nigra(SN)of male rats with Parkinson’s disease(PD)induced by 6-hydroxydopamine(6-OHDA).Methods: 120 male rats were randomly divided into control group,6-OHDA group(PD group),P2X4 gene carrying virus(P2X4-Positive Intervention,P2X4-PI)group,P2X4 gene unloaded virus(P2X4-Negative Control,P2X4-NC)group,P2X4-PI+6-OHDA group and P2X4-NC+6-OHDA group.There were 6 groups with 20 rats in each group.The normal saline containing 0.02% ascorbic acid,6-OHDA,carrying P2X4 gene virus,no-load virus was locally injected into the left SN of rats,and the formation was induced:control group,PD group,P2X4-PI group and P2X4-NC group.In P2X4-PI+6-OHDA group and P2X4-NC+6-OHDA group,P2X4 gene virus and unloaded virus were injected into the left SN of rats,respectively,and then 6-OHDA was injected into the left SN after2 weeks.Two weeks after the completion of the model,the behavior test was carried out,and the qualified rat models were selected.And the initiation and balance ability of each group of rats were further evaluated by a balance bar experiment.After 4 weeks of modeling,the brain of the qualified rat models were decapitated,the brain tissue was taken away and preserved after related treatment.Immunofluorescence staining and Western blot method were used to detect the number of(TH)positive dopaminergic neurons in the left SN of the 6 groups,and the expression levels of protein in P2X4 purinergic receptor(P2X4R),Divalent metal-ion transporter-1(DMT1)and Ferroportin1(FPN1).Results: 1.The result of immunofluorescence staining showed that the number of TH positive dopaminergic neurons in the left SN of PD group(4724.0±261.1,t = 13.17,P<0.01)and P2X4-NC+6-OHDA group(4470.0±228.9,t = 14.21,P < 0.001)were significantly lower than that of the control group(7942.0±461.6),the difference was of statistical significance.Compared with P2X4-NC+6-OHDA group,the number of TH positive dopaminergic neurons in the left SN of P2X4-PI+6-OHDA group(2493.0±371.6,t = 8.092,P<0.01)were significantly lower than that of P2X4-NC+6-OHDA group,and the difference was statistically significant.Compared with the control group,the number of TH positive dopaminergic neurons in the left SN of P2X4-PI group(7878.0±356.7)and P2X4-NC group(7785.0±342.6)were similar,and there was no significant difference between the two groups.2.The result of Western blot suggested that compared with the control group(1.723±0.146、1.369±0.107、1.02±0.059),the expression of P2X4 R,DMT1in the left SN of PD group(2.107±0.070,t = 4.368,P<0.05;1.733±0.117,t = 4.245,P<0.05;0.783±0.042,t = 5.795,P<0.01)and P2X4-NC+6-OHDA group(2.104±0.110,t = 4.326;1.737±0.073,t = 4.291;0.804±0.037,t = 5.282;P<0.05)were increased,FPN1 decreased,and the differences were statistically significant.Compared with P2X4-NC+6-OHDA group,the expression of P2X4 R,DMT1 in left SN of P2X4-PI+6-OHDA group(2.875±0.170,t = 8.770;2.845±0.180,t = 12.92;0.550±0.040,t = 6.216;P<0.01)increased and FPN1 decreased,the differences were statistically significant.Compared with the control group,the expression of DMT1,FPN1 protein in the left SN of P2X4-PI group(2.099±0.108、1.367±0.084、0.985±0.006)and P2X4-NC group(1.675±0.119 、 1.316±0.136 、 1.007±0.089)were similar,and there were no significant differences between the two groups.Conclusion: The overexpression of P2X4 gene can up-regulate the expression of DMT1 and down-regulate the expression of FPN1 in the SN of rats,which leads to the deposition of iron in the SN of the midbrain,and then may cause damage to dopaminergic neurons,and finally has an effect on the occurrence and development of PD.