姜黄素影响N2a/APP695swe细胞中ABCA1表达的机制研究

Curcumin up-regulates ABCA1 expression through inhibiting the activity of CaN in N2a/APP695swe cells

摘要:

目的：研究姜黄素（Curcumin）对N2a/APP695swe细胞钙调神经磷酸酶（Calcineurin，CaN）活性和三磷酸腺苷结合转运子A1（ATP-binding cassette transporter A1，ABCA1）表达的影响；探讨Curcumin影响N2a/APP695swe细胞中ABCA1表达的可能机制。方法：MTT法分别检测1、5、10、20、40 μmol/L Curcumin或0.1、0.5、1、2、4 μmol/L CaN活性抑制剂环孢素A（Cyclospo－rinA，CsA）对N2a/APP695swe细胞存活率的影响；依据MTT筛选结果，分为正常对照组、模型组、Curcumin组：5 μmol/L Cur－cumin处理24 h的N2a/APP695swe细胞、CsA组：0.5 μmol/L CsA处理48 h的N2a/APP695swe细胞。酶底物显色法检测CaN活性，real-time PCR、Western blot检测ABCA1表达水平的变化。结果：与未处理组比较，5 μmol/L Curcumin组细胞存活率[（110.495±4.005）%]最高（P=0.023）；0.1、0.5、1、2、4 μmol/L CsA组细胞存活率依次为（105.532±4.292）%、（115.211±4.826）%、（76.317±4.475）%、（69.177±5.267）%、（54.687±3.912）%，与未处理组比较，0.5、1、2、4 μmol/L CsA组差异均有统计学意义（P值依次为0.001、0.000、0.000、0.000）；酶底物显色法显示与正常对照组[（18.519±1.664） nmol/mg]比较，模型组CaN活性[（24.488±3.041） nmol/mg]增加（P=0.007），Curcumin组[（16.717±2.055） nmol/mg]、CsA组[（4.928±1.232）nmol/mg] CaN活性明显受到抑制（P值依次为0.002、0.000）；real-time PCR和Western blot显示Curcumin组ABCA1表达量（real-time PCR：1.988±0.355；West－ern blot：0.294±0.015）高于模型组（real-time PCR：1.000±0.000；Western blot：0.175±0.008），差异有统计学意义（real-time PCR：P=0.009；Western blot：P=0.000）；与模型组比较，CsA组（real-time PCR：4.000±0.464；Western blot：0.470±0.016）的ABCA1表达水平增加（real-time PCR：P=0.000；Western blot：P=0.000）。结论：Curcumin能上调N2a/APP695swe细胞中ABCA1的表达，其机理可能与抑制CaN活性有关。

Abstract:

Objective：To investigate the effect of Curcumin on the activity of Calcineurin（CaN） and the expression of ATP-binding cas－sette transporter A1（ABCA1） in N2a/APP695swe cells，and to explore the mechanism of the regulation of Curcumin on the expression of ABCA1 in N2a/APP695swe cells. Methods：MTT assay was used to detect N2a/APP695swe cell viabilities in different concentrations of Curcumin or the CaN activity inhabitor cyclosporinA（CsA） treatment. According to the results determined by MTT，N2a/APP695swe cells were co-cultured with 5 μmol/L Curcumin for 24 h，or 0.5 μmol/L CsA for 48 h，then，the CaN activities were measured by enzyme substrate color metric methods. Western blot and real-time PCR were performed to evaluate the expression of ABCA1. Results：Cell survival rate was （110.495±4.005）%，the highest in 5 μmol/L Curcumin group（P=0.023），followed by （105.532±4.292）%，（115.211±4.826）%，（76.317±4.475）%，（69.177±5.267）%，（54.687±3.912）%，respectively in 0.1，0.5，1，2，4 μmol/L CsA groups. There were significant differences in cell survival rate between untreated group and 0.5，1，2，4 μmol/L CsA groups（P=0.001，0.000，0.000，0.000）. Compared with that（18.519±1.664） nmol/mg of normal group，the CaN activity was increased in N2a/APP695swe cells（24.488±3.041） nmol/mg（P=0.007），CaN activities were distinctly decreased in Curcumin group（16.717±2.055） nmol/mg（P=0.002） and CsA group（4.928±1.232）nmol/mg（P=0.000）. Furthermore，Western blot and real-time PCR showed that the expression of ABCA1 was higher in Curcumin group（real-time PCR：1.988±0.355；Western blot：0.294±0.015） than in the model group（real-time PCR：1.000；Western blot：0.175±0.008）（P=0.009 in real-time PCR and P=0.000 in Western blot）. Compared with that of model group，the expression of ABCA1 in CsA group（real-time PCR：4.000±0.464；Western blot：0.470±0.016） was increased（both P=0.000 in real-time PCR and Western blot）. Conclusion：Curcumin may up-regulate the expression of ABCA1 in N2a/APP695swe cells via inhibiting the CaN activity.