姜黄素通过提高Rab家族蛋白的表达增强N2a/APP695swe细胞的自噬作用

Curcumin enhances the autophagy of N2a/APP695swe cells by up-regulating expression of the Rab family proteins

摘要:

目的：研究姜黄素（curcumin）对N2a/APP695swe细胞中自噬和Rab家族蛋白的影响，并且探讨姜黄素调节N2a/APP695swe细胞自噬的可能机制。方法：采用N2a/WT和N2a/APP695swe细胞，并将其分为4个组：N2a/WT组（WT组）、N2a/APP695swe组（APP组）、DMSO溶剂对照组（DMSO组，5 μmol/L，24 h）、curcumin处理组（curcumin组，5 μmol/L，24 h）。Western blot分别检测各组自噬标志性蛋白LC3BⅡ和底物蛋白SQSTM1的表达，以及Rab家族蛋白Rab1、Rab5、Rab7、Rab9、Rab24和Rab33B的表达；共聚焦显微镜观察免疫荧光进一步验证各组LC3BⅡ与SQSTM1蛋白的表达情况。结果：Western blot结果表示，curcumin组LC3BⅡ蛋白表达量明显高于APP组和DMSO组（0.54±0.02 vs. 0.29±0.09，0.54±0.02 vs. 0.29±0.09，P=0.000）；同时，curcumin组SQSTM1蛋白表达量则明显低于APP组（0.67±0.01 vs. 1.02±0.02，P=0.000）。共聚焦显微镜观察免疫荧光实验再次验证了LC3B和SQSTM1蛋白的这种表达趋势。Western blot进一步检测发现，与APP组相比，curcumin组Rab家族蛋白Rab1、Rab5、Rab7和Rab33B的蛋白表达量均明显增加（1.00±0.02 vs. 0.63±0.02，P=0.000；0.94±0.03 vs. 0.79±0.03，P=0.000；0.75±0.00 vs. 0.56±0.01，P=0.000；0.88±0.04 vs. 0.59±0.03，P=0.000），而Rab9和Rab24的改变差异无统计学意义（1.04±0.02 vs. 1.06±0.02，P=0.578；0.79±0.00 vs. 0.80±0.00，P=0.067）。结论：姜黄素提高N2a/APP695swe细胞的自噬水平，其机制可能与增强Rab家族蛋白Rab1、Rab5、Rab7和Rab33B的表达有关。

Abstract:

Objective：To investigate the effect of curcumin the autophagy of N2a/APP695swe cells and its possible mechanism. Methods：N2a/WT cells and N2a/APP695swe cells were selected as experimental cells. They were divided into four groups：N2a/WT group（WT group），N2a/APP695swe group（APP group），dimethylsulfoxide（DMSO） solvent control group（DMSO group，5 μmol/L，24 h），and cur－cumin treatment group（curcumin group，5 μmol/L，24 h）. Western blot was used to determine the expression of autophagy marker — microtubule-associated protein light chain 3B-Ⅱ（LC3BⅡ），substrate protein-sequestosome 1（SQSTM1），and Rab family proteins（Rab1，Rab5，Rab7，Rab9，Rab24，and Rab33B）. Immunofluorescence assay was performed using a confocal microscope to verify the expression of LC3BⅡ and SQSTM1. Results：Western blot results demonstrated that the curcumin group had significantly higher ex－pression of LC3BⅡ compared with the APP group and the DMSO group（0.54±0.02 vs. 0.29±0.09，P=0.000；0.54±0.02 vs. 0.29±0.09，P=0.000）. However，the curcumin group had significantly lower expression of SQSTM1 than the APP group（0.67±0.01 vs. 1.02±0.02，P=0.000）. And the immunofluorescence assay of LC3BⅡ and SQSTM1 confirmed the above results. Western blot results also showed that the curcumin group had significantly increased expression of Rab1，Rab5，Rab7，and Rab33B compared with the APP group（1.00±0.02 vs. 0.63±0.02，P=0.000；0.94±0.03 vs. 0.79±0.03，P=0.000；0.75±0.00 vs. 0.56±0.01，P=0.000；0.88±0.04 vs. 0.59±0.03，P=0.000）. However，there were no significant differences in the expression of Rab9 and Rab24 between the APP group and the curcumin group（1.04±0.02 vs. 1.06±0.02，P=0.578；0.79±0.00 vs. 0.80±0.00，P=0.067）. Conclusion：Curcumin can enhance the autophagy of N2a/APP695swe cells，probably by increasing the expression of Rab1，Rab5，Rab7，and Rab33B.