姜黄素通过增强Rab7的表达促进N2a/APP695swe细胞中的自噬流

Curcumin stimulates autophagy flux via enhancing the expression of Rab7 in N2a/APP695swe cells

摘要:

目的： 观察姜黄素（curcumin）对稳定表达APP695swe的Neuro-2a小鼠脑神经瘤细胞（N2a/APP695swe）中自噬流的作用；再进一步深入探讨其作用于N2a/APP695swe细胞中自噬流的可能机制。方法： 首先将细胞分成4组：野生型对照组（WT组）、空白对照组（Control组）、溶剂对照组（Sham组）、姜黄素组（Curcumin组）。透射电镜观察各组中自噬体形态特点，Western blot检测p62和LC3Ⅱ的表达；并分别运用Western blot及免疫荧光检测Rab7蛋白表达，实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测Rab7 mRNA表达。在N2a/APP695swe细胞中分别过表达及沉默Rab7后，将细胞分成5组：Control组、pcDNA3.1组、pcDNA3.1-Rab7组、NC-siRNA组和Rab7-siRNA组。再运用透射电镜观察各组自噬体形态特点，Western blot检测p62和LC3Ⅱ蛋白表达。结果： 经姜黄素处理后，透射电镜观察到细胞中的自噬体主要为单层膜结构的自噬溶酶体，同时，自噬活性关键性蛋白LC3Ⅱ的表达增加（P=0.000），而自噬底物蛋白p62的表达减少（P=0.000）；Rab7在蛋白水平及mRNA水平均明显升高（P=0.000、0.000）。过表达Rab7后，自噬体主要为单层膜结构的自噬溶酶体，p62蛋白表达明显减少（P=0.000），LC3Ⅱ蛋白表达无明显变化（P=0.669）。沉默Rab7后，自噬体主要为双层膜结构的早期自噬体形态，p62蛋白表达明显增加（P=0.000），LC3Ⅱ蛋白表达无明显变化（P=0.622）。结论： 姜黄素促进N2a/APP695swe细胞中的自噬流，其作用机制可能与增强Rab7表达有关。

Abstract:

Objective: To observe the effect of curcumin on autophagic flux in Neuro-2a mouse brain neuroma cells of expressing APP695swe stably (N2a/APP695swe) and explore the potential mechanism. Methods: Firstly, cells were divided into 4 groups: wild type group (WT), blank control group (Control), solvent control group (Sham) and curcumin group (Curcumin). Transmission electron microscopy (TEM) was performed to observe the morphological characteristics of autophagosomes in each group, Western blot was used to test the expressions of p62 and LC3Ⅱproteins. Meanwhile, Western blot and immunofluorescence staining were performed to detect the expression of Rab7 protein, and quantitative real-time PCR (qRT-PCR) was carried out to analyze the mRNA level of Rab7. Then cells were divided into 5 groups after transfection of pcDNA3.1, pcDNA3.1-Rab7, NC-siRNA and Rab7-siRNA respectively: Control group, pcDNA3.1 group, pcDNA3.1-Rab7 group, NC-siRNA group and Rab7-siRNA group. The morphological characteristics of autophagosomes were observed by transmission electron microscopy, and the levels of p62 and LC3Ⅱproteins were detected by Western blot, too. Results: After treatment of curcumin, autolysosomes with monolayer were observed by TEM. And the expression of LC3Ⅱprotein was increased (P=0.000), while the expression of p62 was decreased (P=0.000). Meanwhile, the expressions of Rab7 were increased at both protein and mRNA levels (P=0.000, 0.000). After over-expression of Rab7, more autolysosomes were observed, and the expression of p62 protein was reduced (P=0.000), while the expression of LC3Ⅱprotein did not change significantly (P=0.669). After transfection of Rab7-siRNA in N2a/APP695swe cells, autophagosomes with double membranes were observed visibly. The expression of p62 protein was elevated (P=0.000), while the expression of LC3Ⅱprotein did not change prominently (P=0.622). Conclusion: Curcumin stimulates autophagy flux in N2a/APP695swe cells, and the mechanism may be related to the enhancement of Rab7.