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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。
AIM: To investigate the effects of curcumin on autophagy in N2a / APP695swe cells and to explore the possible mechanism of curcumin in regulating autophagy in N2a / APP695swe cells. Methods: The cells were divided into 4 groups: WT group, blank control group, Sham group, curcumin treated group (5μmol / L). Western blot was used to detect the expression of autophagic marker protein LC3 Ⅱ and substrate protein SQSTM1, and the expression of Atg protein Beclin1, Atg5, Atg7, Atg12 and Atg16L1 respectively. The expression of SQSTM1 protein was further verified by immunofluorescence. After transfection of autophagy indicator probe (GFPLC3) and lysosomal red probe (Lyso Red), autophagic flow was detected by laser scanning confocal microscope. Results: Western blot results showed that the expression level of LC3Ⅱ in Curcumin group was significantly higher than that in control group (0.59 ± 0.03 vs.0.11 ± 0.01, P = 0.000). Meanwhile, the expression of SQSTM1 in Curcumin group was significantly lower than that in control group (0.19 ± 0.04 vs.0.66 ± 0.05, P = 0.000). Immunofluorescence experiments verified this expression trend of SQSTM1 protein again. Confocal microscopy showed that compared with the control group, the number of autophagosomes in the Curcumin group increased (154.67 ± 7.37 vs.89.00 ± 7.21, P = 0.000) and the number of autophagic lysosomes also increased significantly (84.67 ± 12.50 vs.42.33 ± 6.51, P = 0.000). Western blot analysis showed that the expression of Atg protein Beclin1, Atg7 and Atg16L1 in Curcumin group were significantly increased compared with the 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. 0.03 ± 0.02, P = 0.003), while there was no significant difference between Atg5 and Atg12 (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 can enhance the autophagy of N2a / APP695swe cells, which may be related to the increase of autophagosome-related proteins Beclin1, Atg7 and Atg16L1.