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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.
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Objective To investigate the effect and mechanism of interfering the nicotinamide mononucleotide adenylyltransferase 1(NMNAT1)gene in Parkinson's disease(PD)mouse models. Methods Thirty mice were randomly assigned to three groups: the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)group, the small interfering nicotinamide mononucleotide adenylyltransferase 1 (siNMNAT1)+MPTP group, and the control group, with 10 mice in each group.After injecting siRNA-green fluorescent protein(GFP)lentivirals directly into substantia nigra(SN),mice received intraperitoneal injections of MPTP, which was the siNMNAT1 +MPTP group.While the MPTP group was only with injections of MPTP,and the control group was with neither siRNA nor MPTP.Then we assessed the motor coordination ability firstly.To observe the variation of nigrostriatal pathway, the counts of dopamanergic neurons in SN were measured by tyrosine hydroxylase(TH)immunofluorescence staining.And the expression of TH in striatum, which was used to estimate the dopaminergic neurons axonal variation, was analyzed by RT-PCR.Then the expression of TH, SOD1, Bcl2, Bax, Bcl2/Bax in SN was estimated through Western blotting.Results Compared with the control group,the siNMNAT1+MPTP group and the MPTP group decreased significantly in motor coordination ability(creep down time: siNMNAT1 +MPTP group(62.8 ±15.7)s,MPTP group(77.9 ±13.5)s, control group(122.0 ±25.2)s), dopamanergic neuron counts(siNMNAT1 +MPTP group 45.0 ±6.7, MPTP group 68.0 ±11.3, control group 93.0 ± 12.8)and the striatal TH expression(Creep down time: t=-6.291, P=0.000; t=-4.865, P=0.000.Dopamanergic neuron counts:t=-10.482,P=0.000;t=-4.624, P=0.000.TH expression:t=-9.117,P=0.000;t=-5.716, P=0.000).Although the siNMNAT1+MPTP group showed lower coordination ability than the MPTP group, there was no statistically significant difference.Whereas the counts of dopamanergic neurons in SN(t=-5.487, P=0.000), the expression of TH in striatum(t=-5.146,P=0.003),SOD1(t=-4.143, P=0.001)and Bcl2/Bax(t=-6.303, P=0.000)were obviously decreased in the siNMNAT1+MPTP group,in which Bax increased significantly(t=3.550,P=0.002).Conclusions Interfering the expression of NMNAT1 aggravated the neurodegeneration in PD, and the mechanism might be related to oxidative stress and programmed cell death.
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Objective To investigate the effect of overexpression of P2X4 gene on cell apoptosis in 6-OHDA (6-hydroxydopamine, 6-OHDA) induced PD rat model. Methods One hundred twenty male Wistar rats were randomly divided into 6 groups: 6-OHDA group, control group, objective RNA-P2X4 + 6-OHDA group, target gene RNA-P2X4 group, P2X4-NC + 6-OHDA group and negative virus P2X4-NC only group. Lentivirus or negative virus carrying the target gene and or 6-OHDA or the same amount of physiological saline were injected into the left substantia nigra of rats according to the groups. The number of dopaminergic neurons in substantia nigra was measured by immunofluorescence method.The expression levels of P2X4R,caspase-1 and NLRP3 in substantia nigra were detected by Western blot assay. Results Compared with P2X4-NC + 6-OHDA group, the expression of protein P2X4R (1.099 ± 0.05569 vs. 0.7821 ± 0.02008, P=0.0003 ), NLRP3 (0.9875 ± 0.01932 vs. 0.6645 ± 0.01747. P<0.0001), caspase-1 (0.9948 ± 0.01788 vs. 0.8276 ± 0.04543, P<0.0001) increased significantly in RNA-P2X4+6-OHDA group. Conclusion overexpression of P2X4 gene can increase the expression of NLPR3 and caspase-1 protein and promote apoptosis in PD rats.
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Objective To investigate the effects of gene silencing of Fas-associated death domain (FADD) with synthetic small interfering RNA (siRNA) on apomorphine-induced contralateral rotation,and the expression of Fas and caspase-8 in rat models of parkinsonism. Methods Sprague-Dawley rats were randomly divided into 5 groups:control group,Parkinson' s disease (PD) group,FADD siRNA group,FADD siRNA positive control group and FADD siRNA negative control group. Synthetic FADD siRNA sequences,siRNA positive sequences or siRNA negative sequences were infused into right substantianigra of midbrain using RNA interference and stereotactic techniques before parkinsonian rat model establishment.Apomorphine-induced contralateral rotations of the rats were observed after the injection.The protein and mRNA expression levels of FADD,Fas and caspase-8 were measured by Western blot and RT-PCR.Results In the control group,no rotation was observed after injecting apomorphine; however,in the rest groups,the number of rats respectively was 12 (12/14),3 (3/13),4 (4/15) and 11 ( 11/14 ) in apomorphine-induced contralateral rotation,which had significant statistical differences ( x2 ＝ 18.56,P =0.000).In parkinsonian substantia nigra,marked increases in the protein and mRNA levels of FADD,Fas and caspase-8 were observed,compared with control group.Furthermore,compared with PD group,FADD protein and mRNA levels were strongly suppressed by administration of FADD siRNA in FADD siRNA group. FADD siRNA administration also resulted in great attenuation of 6-hydroxydopamine-induced increases in expression and activation of caspase-8.However,no decrease in expression of Fas was observed in FADD siRNA group and FADD siRNA positive control group,compared with PD group.Conclusion Our results suggest that death receptor signaling pathway plays a critical role in the pathogenesis of PD.FADD siRNA is effective against this pathway and it may,at least in part,provide a potential neuroprotective effect.