| Parkinson’s disease(PD)is the second most common neurodegenerative disease in middle-aged and elderly people after Alzheimer’s.The main pathological features of PD are the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the depletion of dopamine(DA)in the striatum.However,the etiology of PD has yet to be fully uncovered.Many factors such as oxidative stress,abnormal accumulation of iron in the substantia nigra,mitochondrial dysfunction,apoptosis,and inflammation are thought to be involved in the process of neurodegeneration.The multiple risk factors make the prevention and treatment of PD extremely complicated.In addition,many side-effects occur after prolonged pharmacological treatment.Therefore,it is of great practical significance to find effective drugs for the treatment of PD with low side effects.4-aminopyridine(4-AP)is a pyridine derivative with a molecular weight of 94.12 Da.It has good lipid solubility and easily penetrates the blood-brain barrier.It is a classic blocker of A-type potassium channel,which can improve the excitability of neurons and increase the release of neurotransmitters.4-AP plays an important role in the treatment of a variety of neurological diseases such as multiple sclerosis,spinal cord injury,cerebellar ataxia,nystagmus and PD.Clinical reports showed that the oral administration of 4-AP improved the movement symptoms of PD patients,such as stride frequency and stride length.In a PD rat model prepared by neurotoxin 6-hydroxydopamine(6-OHDA),4-AP can reduce the rotation times induced by apomorphine.In our previous study,we also observed that 4-AP can improve the motor speed and motor coordination ability of mice in the PD mouse model prepared by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).These studies suggest that 4-AP may regulate the function of the substantia nigra striatum-system,and thus improve the motor behavior of the body.However,whether 4-AP has a direct protective effect on DA neurons is not completely clear.To investigate this issue,this research used DA cell line MES23.5 cells and primary cultured ventral mesencephalon(VM)neurons,with 1-methyl-4-phenyl pyridine ion(MPP~+)preparation of PD cell model.The survival rate of MES23.5 cells was detected by the Cell Counting Kit-8(CCK-8),and the release level of lactate dehydrogenase(LDH)in the culture medium of VM neurons was detected by the LDH kit.The levels of mitochondrial transmembrane potential(△Ψm)and reactive oxygen species(ROS)were detected by flow cytometry,and the protein expressions of tyrosine hydroxylase(TH),Caspase-3,Bcl-2 and Bax were detected by western blotting.Based on the above,this study aims to explore the protective effect and mechanism of 4-AP on SN DA neurons during the pathogenesis of PD.The experimental results are as follows:1.The survival rate of MES23.5 cells treated with different concentrations of 4-AP for 24h was detected by CCK-8 kit.The results showed that compared with the control group,0.01,0.1,0.5 and 1 m M 4-AP did not affect the cell survival,while 5 m M and 10 m M4-AP reduced the cell survival rate of MES23.5 cells to 64.69%and 42.24%of that in control group(P<0.001).2.The survival rate of MES23.5 cells treated with MPP~+(200μM)for 24 h decreased to77.64%of that in control group(P<0.001).Compared with MPP~+group,MES23.5 cells pretreated with 4-AP(0.01,0.1,0.5 and 1 m M)for 4 h could significantly improve the survival rate of cells(P<0.001).3.MES23.5 cells were treatd with MPP~+(200μM)for 24 h.Western blotting was used to detect the expression of TH protein in MES23.5 cells.The results showed that TH protein expression was decreased compared with the control group(P<0.01).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreated cells for 4 h could antagonize the decrease of TH protein expression in MES23.5 cells induced by MPP~+(P<0.05,P<0.01,P<0.01,P<0.05).4.MES23.5 cells were treated with MPP~+(200μM)for 24 h.The change of△Ψm was detected by flow cytometry.Compared with the control group,the△Ψm in MPP~+treatment group was significantly decreased(P<0.01).Compared with MPP~+group,4-AP(0.1,0.5,and 1 m M)pretreatment cells for 4 h could antagonize the reduction of△Ψm induced by MPP~+(P<0.01,P<0.05,P<0.05).5.MES23.5 cells were treated with MPP~+(200μM)for 24 h.The change of ROS level was detected by flow cytometry.Compared with the control group,the ROS level in MPP~+treatment group was significantly increased(P<0.05).Compared with MPP~+group,4-AP(0.1,0.5,and 1 m M)pretreatment cells for 4 h could antagonize the increase of intracellular ROS level induced by MPP~+(P<0.05).6.VM neurons were treated with MPP~+(100μM)for 24 h.The activity of LDH in the supernatant of the culture medium of primary VM neurons was detected by lactate dehydrogenase(LDH)kit.The results showed that the release of LDH was significantly increased compared with the control group(P<0.001).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreatment of VM neurons for 4 h could inhibit the increase of LDH release induced by MPP~+(P<0.001).7.VM neurons were treatd with MPP~+(100μM)for 24 h.Western blotting was used to detect the expression of TH protein in primary VM neurons.The results showed that TH protein expression was decreased compared with the control group(P<0.01).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreated VM neurons for 4 h could antagonize the decrease of TH protein expression in VM neurons induced by MPP~+(P<0.05).8.VM neurons were treated with MPP~+(100μM)for 6 h.The changes of△Ψm in VM neurons were detected by flow cytometry.The results showed that△Ψm of VM neurons was significantly lower than that of the control group(P<0.01).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreatment of VM neurons for 4 h could antagonize the decrease of△Ψm of VM neurons induced by MPP~+(P<0.05,P<0.05,P<0.05,P<0.01).9.VM neurons were treated with MPP~+(100μM)for 24 h.Western blotting was used to detect the expression of Bcl-2/Bax protein in primary VM neurons.The results showed that Bcl-2/Bax ratio was decreased compared with the control group(P<0.01).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreated VM neurons for 4 h could antagonize the decrease of the ratio(P<0.05,P<0.05,P<0.01,P<0.05).10.VM neurons were treated with MPP~+(100μM)for 24 h.Western blotting was used to detect the expression of cleaved Caspase-3 protein in primary VM neurons.The results showed that Caspase-3 protein expression was increased compared with the control group(P<0.05).Compared with MPP~+group,4-AP(0.01,0.1,0.5 and 1 m M)pretreated VM neurons for 4 h could antagonize the decrease of the cleaved Caspase-3 protein expression in VM neurons induced by MPP~+(P<0.05,P<0.05,P<0.01,P<0.01).These results indicate that 4-AP can improve the cell survival rate,reduce the release of LDH,and increase the expression of TH protein,suggesting that 4-AP had a protective effect on the damage of DA MES23.5 cell lines and VM neurons induced by MPP~+.4-AP can antagonize the decrease of△Ψm and the increase of ROS level induced by MPP~+,and antagonize the decrease of Bcl-2/Bax ratio and the activation of Caspase-3 protein,suggesting that the cell protection mechanism of 4-AP may be related to the protection of mitochondrial function,the anti-apoptosis and the reduction of oxidative stress.The results of this study provide experimental basis for fully understanding the protective mechanism of 4-AP in PD,and provide a new strategy for the prevention and treatment of PD. |
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