模拟肽Gap27抑制缝隙连接蛋白43在帕金森病小鼠模型中的作用

Inhibition connexin 43 by mimetic peptide Gap27 mediates protective effects on 6-hydroxydopamine induced Parkinson’s disease mouse model

目的:探索在6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的帕金森病(Parkinson’s disease,PD)小鼠模型中,应用缝隙连接蛋白43(connexin 43,Cx43)选择性抑制剂模拟肽Gap27能否改善多巴胺神经元死亡以及对Cx43表达的影响。方法:将18只C57BL/6小鼠随机分为对照组、6-OHDA组与6-OHDA+Gap27组,每组6只,进行双侧黑质脑立体定位注射。对照组注射抗坏血酸盐溶液,6-OHDA组注射6-OHDA溶液,6-OHDA+Gap27组注射6-OHDA和Gap27混合溶液,用免疫组织化学法对多巴胺神经元标志物酪氨酸羟化酶(tyrosine hydroxylase,TH)染色检测多巴胺神经元数量,实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测Cx43信使核糖核酸(messenger ribonucleic acid,mRNA)表达,免疫荧光染色检测Cx43蛋白分布,Western blot法检测小鼠中脑Cx43蛋白及Cx43的第368位点丝氨酸磷酸化(Cx43 phosphorylation at serine 368,Cx43-ps368)蛋白含量。结果:注射6-OHDA后,小鼠出现黑质多巴胺神经元大量死亡,6-OHDA组TH阳性神经元数量降为对照组的27.7%±0.02%(P<0.01),模拟肽Gap27的使用减少了多巴胺神经元死亡数量,6-OHDA+Gap27组TH阳性神经元数量为6-OHDA组的(1.64±0.16)倍(P<0.05);此外,6-OHDA引起Cx43蛋白含量增加,Cx43-ps368蛋白含量降低。Gap27减弱了6-OHDA引起的Cx43蛋白与Cx43-ps368蛋白含量变化,6-OHDA组中脑总Cx43蛋白含量为6-OHDA+Gap27组的(1.44±0.07)倍(P<0.05),为对照组的(1.68±0.07)倍(P<0.01),且6-OHDA组Cx43-ps368蛋白含量及占总Cx43蛋白比例显著低于6-OHDA+Gap27组(P<0.05)。结论:模拟肽Gap27在6-OHDA诱导的小鼠模型中可减少黑质多巴胺神经元死亡从而发挥神经保护作用,6-OHDA引起的Cx43蛋白过表达对多巴胺神经元可能存在神经毒性,而降低Cx43蛋白水平及维持Cx43-ps368蛋白水平可能是Gap27发挥保护作用的机制。

Objective:To explore whether the using of mimetic peptide Gap27,a selective inhibitor of connexin 43(Cx43),could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine(6-OHDA)-induced Parkinson’s disease mouse models.Methods:Eighteen C57BL/6 mice were randomly divided into control group,6-OHDA group and 6-OHDA+Gap27 group,with 6 mice in each group.Bilateral substantia nigra stereotactic injection was performed.The control group was injected with ascorbate solution,6-OHDA group was injected with 6-OHDA solution,and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution.Immuno-histochemical staining was used to detect the number of dopamine neurons,quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of Cx43 messenger ribonucleic acid(mRNA),immuno-fluorescence staining was used to detect the distribution of Cx43 protein,the contents of Cx43 protein and Cx43 phosphorylation at serine 368(Cx43-ps368)in mouse midbrain were detected by Western blot.Results:After injection of 6-OHDA,numerous dopamine neurons in substantia nigra died as Cx43 content increased,Cx43-ps368 content decreased.Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA.The number of tyrosine hydroxylase(TH)immunoreactive positive neurons in 6-OHDA group decreased to 27.7%±0.02%of the control group(P<0.01);The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was(1.64±0.16)times higher than that in 6-OHDA group(P<0.05);The content of total Cx43 protein in 6-OHDA group was(1.44±0.07)times higher than that in 6-OHDA+Gap27 group(P<0.05)while(1.68±0.07)times higher than that in control group(P<0.01).In 6-OHDA group,the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group(P<0.05).Conclusion:In 6-OHDA mouse models,mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons,which was induced by 6-OHDA.The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron.Meanwhile,decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.