ABSTRACT
Objective:To elucidate the effect of P2X4 signal axis on iron metabolism in the substantia nigra (SN) of male rats with Parkinson′s disease (PD) successfully induced by 6-hydroxydopamine (6-OHDA).Methods:A total of 120 male rats were randomly divided into control group, 6-OHDA group (PD group), P2X4-gene virus (P2X4-positive intervention, P2X4-PI) group, P2X4-gene unloaded virus (P2X4-negative control, P2X4-NC) group, P2X4-PI+6-OHDA group (inject P2X4 gene virus first, then 6-OHDA two weeks later) and P2X4-NC+6-OHDA group (inject no-load gene virus first, then two weeks later with 6-OHDA) using a completely random numbers method, with 20 rats in each group. Brain stereotactic instrument was used to inject the corresponding grouped drugs into the left SN of rats. A behavioral test was performed two weeks after the modeling was completed to select the qualified rat models, and the initiation and balance ability of each group of rats were further evaluated by a balance bar experiment. The brains of the qualified rat models were decapitated, and the brain tissue was taken away and preserved after related treatment. Immunofluorescence staining and Western blotting methods were used to detect the number of tyrosine hydroxylase (TH) positive dopaminergic neurons, and the expression levels of protein in P2X4 purinergic receptor (P2X4R), divalent metal-ion transporter-1 (DMT1) and ferroportin 1 (FPN1).Results:The results of immunofluorescence staining showed that the number of TH positive dopaminergic neurons in the left SN of the PD group (4 724.0±261.1, t=13.17, P<0.01) and the P2X4-NC+6-OHDA group (4 470.0±228.9, t=14.21, P<0.001) was significantly lower than that of the control group (7 942.0±461.6). The number of TH positive dopaminergic neurons of the P2X4-PI+6-OHDA group (2 493.0±371.6, t=8.092, P<0.01) was significantly lower than that of the P2X4-NC+6-OHDA group. The results of Western blotting suggested that compared with the control group (1.723±0.146, 1.369±0.107, 1.020±0.059), the expression of P2X4R, DMT1 was increased, whereas FPN1 was decreased in the left SN of the 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 the 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). Compared with the P2X4-NC+6-OHDA group, the expression of P2X4R, DMT1 was increased and FPN1 was decreased in the left SN of the 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). Conclusion:The overexpression of P2X4 gene can up-regulate the expression of DMT1 and down-regulate the expression of FPN1 in the SN, 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.