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.
