姜黄素对α-突触核蛋白基因突变或过表达诱导的寡聚体形成及线粒体ATP敏感性钾离子通道的影响

Effect of curcumin on oligomer formation and mitochondrial ATP-sensitive potassium channels induced by overexpression or mutation of α-synuclein

目的探讨姜黄素对r突触核蛋白（a-synuclein）寡聚体形成、线粒体膜电位及线粒体ATP敏感性钾通道（mitochondrial ATP-sensitive potassium channels，mitoKATP）的影响。方法构建野生型、A53T突变型{1-synuclein真核表达质粒，用脂质体介导转染方式将构建好的质粒转入PCI2细胞，并分别应用姜黄素（20ttmol／L）、5-羟基葵酸盐（5-hydroxydecanoate，5-HD）进行干预；48h后应用免疫印迹、斑点杂交检测细胞a-synuclein寡聚体形成；应用JC-1荧光探针及乳酸脱氢酶（1actatedehydrogenase，LDH）检测线粒体膜电位及细胞膜毒性损伤；并在姜黄素干预处理前15min给予5-HD预处理，免疫印迹检测mitoKATP蛋白的功能亚基Kir6．2的改变，细胞膜片钳观察mitoKATP的改变。结果将构建所得EGFP-α—synuclein—wT、EGFP—α—synuclein—A53T真核表达质粒转染PCI2细胞，48h后免疫印迹及斑点印迹结果显示，α—synuclein基因过表达或突变诱导均可促进α—synuclein寡聚体形成，姜黄素可明显减弱α-synuclein基因过表达或突变诱导的α—synuclein寡聚体形成；JC-1及LDH结果显示，与正常PCI2细胞相比，转染野生型、A53T突变型组细胞LDH水平分别升高35．5％及42．3％（P〈0．05），jc-1红／绿荧光比率与正常PCI2细胞相比下降60．44％、65．22％（P〈0．05），姜黄素干预后，LDH水平分别下降36．3％及23．5％（P〈0．05），红／绿荧光比率上升48．46％、50．33％（P〈0．05）；转染野生型或A53T突变型α—synuclein融合表达载体组Kir6-2蛋白表达增强，早期通道电流有明显增高的趋势，随后降低，应用姜黄素干预后，α—synuclein基因过表达或突变所诱导的Kir6．2蛋白表达下调，但细胞mitoKATP通道电流增加，与转染组比较有统计学意义（P〈0．05）。结论姜黄素可抑制α-synuclein基因过度表达或突变诱导的α—synuclein寡聚体形成；姜黄素可能通过稳定线粒体膜电位阻断了α—synuclein基因过表达或突变所导致的细胞凋亡；mitoKATP通道的开放可能是α—synuclein基因过表达或A53T突变诱导细胞凋亡的自发始动保护机制，姜黄素可能通过开放mitoKATP通道拮抗α—synuclein基因过表达或突变所导致的细胞毒性，mitoKATP通道的开放程度与Kir6.2蛋白的表达不成正相关。

Objective To investigate the effect of curcumin on oligomer formation and mitochondrial ATP-sensitive potassium channels （mitoKATP） induced by overexpression or mutation of α-synuclein. Methods Recombinant plasmids α-synuclein-pEGFP-A53T and α-synuclein-pEGFP-WT were transfected into PC12 cells by lipofectamin method, and intervened by application of curcumin （20 μmol/L） and 5- hydroxydecanoate （5-HD）. Oligomer formation in the cultured cells was identified by Western blotting and Dot blotting. Cytotoxicity and apoptosis of the PC12 cells were measured by lactate dehydrogenase （LDH） and JC-1 assays, mitoKATP were identified by Western blotting and whole cell patch clamp. Results Curcumin has significantly reduced the oligomer formation induced by overexpression or mutation of α-synuclein in the cultured ceils. LDH has decreased by 36.3% and 23.5%, and red / green fluorescence ratio of JC-1 was increased respectively by 48.46% and 50.33% after application of curcumin （P〈0.05）. Protein expression of Kir6.2 has decreased and mitoKATP channel current has significantly increased （P〈 0.05）. Conclusion Curcumin can inhibit a-synuclein gene overexpression or mutation-induced μ-synuclein oligomers [ormation. It may block apoptosis induced by wild-type overexpression or mutation of α-synuclein. By stabilizing mitochondrial membrane potential. Opening of mitoKATP channel may have been the initiating protective mechanism of apoptosis induced by wild-type overexpression or mutation of α-synuclein. Curcumin may antagonize above cytotoxicity through further opening the mitoKATP channel.