姜黄素通过提高自噬体形成相关蛋白的表达增强N2a/APP695swe细胞的自噬作用  

Curcumin improves autophagy in N2a/APP695swe cells via elevating the expressions of Atg proteins

作　　者：康清梅 梁洁[1] 张雄[2] 周凡琳[1] 李丹[1] 高敏娜[1] 李昱[1] Kang Qingmei Liang Jie Zhang Xiong Zhou Fanlin Li Dan Gao Minna Li Yu(Teaching and Research Section of Pathology,College of Basic Medicine Institute of Neuroscience,Chongqing Medical University)

机构地区：[1]重庆医科大学基础医学院病理学教研室,重庆400016 [2]重庆医科大学神经科学研究中心,重庆400016

出　　处：《重庆医科大学学报》2017年第5期481-486,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81671261)

摘　　要：目的:全面研究姜黄素(curcumin)对N2a/APP695swe细胞自噬作用的影响,进一步探讨姜黄素调节N2a/APP695swe细胞自噬的可能机制。方法:将细胞分为4组:野生型对照组(WT组)、空白对照组(Control组)、溶剂对照组(Sham组)、姜黄素处理组(Curcumin组,5μmol/L)。Western blot分别检测自噬标志性蛋白LC3Ⅱ和底物蛋白SQSTM1的表达,以及Atg蛋白Beclin1、Atg5、Atg7、Atg12、Atg16L1的表达;免疫荧光法进一步验证SQSTM1蛋白的表达。各组分别转染细胞自噬体指示探针(GFPLC3)和溶酶体红色探针(Lyso Red)后,激光共聚焦显微镜观察自噬体和自噬溶酶体的数量检测自噬流。结果:Western blot结果表明,Curcumin组LC3Ⅱ蛋白表达量较对照组明显增高(0.59±0.03 vs.0.11±0.01,P=0.000);同时,Curcumin组SQSTM1蛋白表达量较对照组明显降低(0.19±0.04 vs.0.66±0.05,P=0.000)。免疫荧光实验再次验证了SQSTM1蛋白的这个表达趋势。共聚焦显微镜观察发现,与对照组相比,Curcumin组细胞内自噬体数量增加(154.67±7.37 vs.89.00±7.21,P=0.000),同时自噬溶酶体数量也显著增加(84.67±12.50 vs.42.33±6.51,P=0.000)。Western blot进一步检测发现,与对照组相比,Curcumin组Atg蛋白Beclin1、Atg7、Atg16L1的表达均明显增加(0.55±0.09 vs.0.10±0.02,P=0.000;0.89±0.12 vs.0.39±0.07,P=0.000;0.36±0.07vs.0.13±0.02,P=0.003),而Atg5和Atg12改变差异无统计学意义(0.79±0.12 vs.0.79±0.10,P=0.470;0.94±0.13 vs.0.87±0.16,P=0.587)。结论:姜黄素增强N2a/APP695swe细胞的自噬水平,其机制可能与提高自噬体形成相关蛋白Beclin1、Atg7、Atg16L1的表达有关。Objective：To explore the effects of curcumin on autophagy of N2a/APP695 swe cells and the underlying mechanisms.Methods：The N2a/WT and N2a/APP695 swe cells were involved,and were divided into four groups：wild type（WT）group,control group,sham group and curcumin group（5 μmol/L）. Western blot was carried out to detect the expressions of LC3Ⅱ,SQSTM1,and the autophagy-related genes（Atg）including Beclin1,Atg5,Atg7,Atg12,and Atg16L1 proteins. Immunofluorescence was used to test the expression of SQSTM1 protein,too. After transfection of p SELECT-GFP-LC3 and Lyso Red into the cells of four groups,respectively,confocal microscope was performed to observe the difference of the number of autophagosomes and autolysosomes in each group.Results：Western blot demonstrated that compared with those of control group,the expressions of LC3 Ⅱ protein were significantly increased in curcumin group（0.59±0.03 vs. 0.11±0.01,P=0.000）. But the expression of SQSTM1 protein was attenuated in curcumin group than that in control group,simultaneously（0.19±0.04 vs. 0.66±0.05,P=0.000）. And immunofluorescence also verified this trend of SQSTM1 protein. Then confocal microscope showed that the number of autophagosomes and the quantity of autolysosomes were improved in curcumin group,compared with those in control group（154.67±7.37 vs. 89.00±7.21,P=0.000;84.67±12.50 vs. 42.33±6.51,P =0.000）. At last,Western blot further found that the expressions of Beclin1,Atg7,and Atg16L1 at protein levels were increased in curcumin group when compared with those of control group（0.55±0.09 vs. 0.10±0.02,P=0.000;0.89±0.12 vs. 0.39±0.07,P=0.000;0.36±0.07 vs. 0.13±0.02,P=0.003）. However,there was no significant difference in the two groups at the levels of Atg5 and Atg12 proteins（0.79±0.12 vs. 0.79±0.10,P=0.470;0.94±0.13 vs. 0.87±0.16,P=0.587）. Conclusion：Curcumin plays a key role in elevating autophagy in N2a/APP695 swe cells and the potential mechanisms is associated with Atg proteins including Beclin

关 键 词：姜黄素 自噬 阿尔茨海默病 BECLIN1 Atg7 Atg16L1 

分 类 号：R741.02[医药卫生—神经病学与精神病学]