Öz Ahmi, Çelik Ömer
a Department of Biophysics , School of Medicine, Süleyman Demirel University , Isparta , Turkey.
b Neuroscience Research Center , Süleyman Demirel University , Isparta , Turkey.
Mol Membr Biol. 2016 May-Aug;33(3-5):76-88. doi: 10.1080/09687688.2017.1318224. Epub 2017 Jun 1.
Transient Receptor Potential (TRP) channels are mostly Ca permeable cation channels. Transient Receptor Potential Melastatin-like 2 (TRPM2) is expressed in neurological tissues such as brain, dorsal root ganglia (DRG) neurons, hippocampus and also liver, heart and kidney. The SH-SY5Y cells are mostly used as a cellular model of neurodegenerative diseases, Alzheimer's and Parkinson's diseases. Curcumin, shows phenolic structure, synthesized by Curcuma longa L. (turmeric), has powerful non-enzymatically antioxidant effects compared with Vitamin E. Hence, we aimed to investigate that effects of curcumin on TRPM2 cation channel currents using the whole-cell Patch-Clamp method, Ca signaling, apoptosis and cell viability (MTT) assays, reactive oxygen species (ROS) production, mitochondrial membrane potential levels, caspase 3 and caspase 9 activities in TRPM2 transfected SH-SY5Y neuroblastoma cells. For this aim, we designed four experimental groups named; control, curcumin, transfected and transfected + curcumin groups. Cytosolic free calcium concentrations were higher in transfected group compared with curcumin and transfected + curcumin group. Moreover, these data examined with whole-cell Patch-Clamp recordings of single cells in all groups. ROS levels were significantly higher in transfected group than in transfected + curcumin group. Apoptosis levels in transfected + curcumin group were lower than in transfected group. Procaspase 9 and procaspase 3 levels measured by western blotting and caspase 3 and caspase 9 levels by spectrophotometric methods show that TRPM2 transfected cells are more tended to apoptosis. In conclusion, curcumin strongly induces modulator effects on TRPM2-mediated Ca influx caused by ROS and caspase 3 and 9 processes in SH-SY5Y neuroblastoma cells.
瞬时受体电位(TRP)通道大多是钙离子通透的阳离子通道。瞬时受体电位褪黑素样2(TRPM2)在脑、背根神经节(DRG)神经元、海马体等神经组织以及肝脏、心脏和肾脏中均有表达。SH-SY5Y细胞常被用作神经退行性疾病、阿尔茨海默病和帕金森病的细胞模型。姜黄素具有酚类结构,由姜黄(Curcuma longa L.)合成,与维生素E相比具有强大的非酶抗氧化作用。因此,我们旨在使用全细胞膜片钳方法、钙信号、凋亡和细胞活力(MTT)测定、活性氧(ROS)产生、线粒体膜电位水平、caspase 3和caspase 9活性,研究姜黄素对TRPM2转染的SH-SY5Y神经母细胞瘤细胞中TRPM2阳离子通道电流的影响。为此,我们设计了四个实验组,分别为:对照组、姜黄素组、转染组和转染+姜黄素组。与姜黄素组和转染+姜黄素组相比,转染组的胞质游离钙浓度更高。此外,所有组均通过全细胞膜片钳记录单个细胞来检测这些数据。转染组的ROS水平显著高于转染+姜黄素组。转染+姜黄素组的凋亡水平低于转染组。通过蛋白质印迹法测定的原半胱天冬酶-9和原半胱天冬酶-3水平以及通过分光光度法测定的半胱天冬酶-3和半胱天冬酶-9水平表明,TRPM2转染细胞更易发生凋亡。总之,姜黄素强烈诱导对SH-SY5Y神经母细胞瘤细胞中由ROS以及caspase 3和9过程介导的TRPM2介导的钙内流的调节作用。