姜黄素改善N2a/APP695swe细胞线粒体功能和抑制细胞凋亡的作用

Curcumin improved mitochondrial function of N2a/APP695swe cells and inhibited apoptosis

摘要:

目的：观察姜黄素（curcumin）对N2a/APP695swe细胞线粒体形态和功能及细胞凋亡的影响；探讨其可能的神经保护机制。方法：实验细胞分为4个组：N2a/APP695组（WT）、N2a/APP695swe组（APP）、溶剂对照组（DMSO，5 μmol/L）、姜黄素组（Curcumin，5 μmol/L）。流式细胞术分别检测细胞凋亡、活性氧水平和膜电位水平、免疫细胞化学检测细胞色素C的表达、电镜观察细胞线粒体形态的改变，Western blot检测Caspase3、Caspase9蛋白表达，分光光度检测Caspase3、Caspase9酶活性。结果：流式细胞术结果显示，Curcumin组细胞的凋亡率、活性氧水平较APP组明显降低[（0.05±0.01） vs. （0.18±0.01），P=0.000；（49.67±4.16） vs. （197.67±8.08），P=0.000]；而Curcumin组线粒体膜电位较APP组升高[（1.00±0.03） vs. （0.64±0.03），P=0.000]。电镜结果发现姜黄素可以减轻线粒体的肿胀，改善其形态。同时免疫细胞化学结果显示姜黄素减少了细胞色素C的释放。Western blot检测发现，与APP组相比，Curcumin组Caspase3、Caspase9蛋白的表达均降低[（0.62±0.12） vs. （1.39±0.10），P=0.000；（0.64±0.20） vs. （1.78±0.03），P=0.000]；并且Curcumin组较APP组的Caspase3与Caspase9的活性明显降低[（0.66±0.04） vs. （2.12±0.06）, P=0.000；（0.58±0.04） vs. （0.93±0.03）, P=0.000]。结论：姜黄素能抑制N2a/APP695swe细胞的凋亡，其机制可能与改善线粒体功能有关。

Abstract:

Objective：To observe the effects of curcumin on the mitochondrial morphology and functions，and on the apoptosis in N2a/APP695swe cells，and to explore the underlying mechanisms. Methods：The experimental cells were divided into four groups：N2a/APP695 group（WT），N2a/APP695swe group（APP），dissolvent control group（DMSO，5 μmol/L） and Curcumin group（5 μmol/L）. Flow cytometry（FCM） method was used to investigate apoptosis，the level of cytosolic ROS and the change of mitochondrial membrane potential. Immunocytochemistry technique was performed to analyze changes of cytochrome C in experimental cells. The morphology of mitochondria was observed by electron microscope. Western blot was carried out to detect the expression of caspase3 and caspase9 protein. And the activities of caspase3 and caspase9 were assessed by spectrophotometry. Results：Apoptosis and cytosolic ROS were significantly lower in the curcumin group than in the APP group（0.05±0.01 vs. 0.18±0.01，P=0.000；49.67±4.16 vs. 197.67±8.08，P=0.000），and the mitochondrial membrane potential was higher in the curcumin group than in the APP group（1.00±0.03 vs. 0.64±0.03，P=0.000）. The electron microscopy found that curcumin could reduce the swelling of the mitochondria and improve its shape. Furthermore，curcumin could reduce the release of cytochrome C and the expression of caspase3 and caspase9 protein in the curcumin group than in the APP group（0.62±0.12 vs. 1.39±0.10，P=0.000；0.64±0.20 vs. 1.78±0.03，P=0.000）. Similarly，the activity of caspase3 and caspase9 was significantly lower in the curcumin group than in the APP group（0.66±0.04 vs. 2.12±0.06，P=0.000；0.58±0.04 vs. 0.93±0.03，P=0.000）. Conclusions：Curcumin could reduce the apoptosis of the N2a/APP695swe cell，and its mechanism may be associated with improved mitochondrial function.