β-细辛醚对Aβ_(1-42)诱导星形胶质细胞活化所致PC12细胞损伤的保护作用研究

Protective effect of β-asarone on PC12 cells injury induced by Aβ_(1-42) astrocytic activation

该文探讨β-细辛醚对Aβ1-42诱导星形胶质细胞活化所致PC12细胞损伤的保护作用及其机制,为β-细辛醚在阿尔兹海默病(Alzheimer’s diease,AD)防治中的应用提供实验依据。首先,采用实时无标记动态细胞分析技术(Real time cell analysis,RTCA)构建RA-h/PC12共培养体系,实时动态观察Aβ1-42诱导星形胶质细胞损伤后对共培养体系中PC12细胞存活率的影响,根据系统自动生成的存活率曲线和参数选出最佳的药物干预时间,采用不同浓度的β-细辛醚对星形胶质细胞进行干预,观察共培养体系中PC12细胞存活率的改变;其次,采用MTT法检测Aβ1-42作用于RA-h对PC12细胞存活率的影响以及β-细辛醚的干预效应,验证RTCA的检测结果;进一步采用ELISA法检测下室培养液中IL-1β,TNF-α,BDNF的水平;Western blot法检测PC12细胞NF-κB的活性和ERK,p38,JNK磷酸化水平。结果显示,β-细辛醚(55.5 mg·L-1)能显著减缓Aβ1-42诱导的RA-h活化引起的PC12细胞存活率下降(P<0.01),显著降低下室培养液中IL-1β,TNF-α的水平和PC12细胞ERK,p38,JNK磷酸化水平(P<0.01);β-细辛醚(166.7 mg·L-1)能促进下室培养液中BDNF的释放(P<0.05)。以上结果表明,Aβ1-42诱导RA-h活化并释放IL-1β,TNF-α等炎症因子加剧PC12细胞的损伤;β-细辛醚能降低IL-1β,TNF-α和促进BDNF的释放,抑制PC12细胞NF-κB的活性和ERK,p38,JNK磷酸化水平。

This study was aimed to investigate the protective effect and mechanism of β-asarone on PC12 cells injury induced by Aβ1-42 activated astrocytes,and provide experimental basis for β-asarone application in the prevention and control of Alzheimer＇s disease（ AD）. Firstly,RA-h and PC12 cells were co-cultured in the special transwell chamber,and the Real time cell analysis（ RTCA）system was used to real-time observe its effect on PC12 cells survival rate in the co-culture system after astrocytes injury induced by Aβ1-42. The best intervention time of β-asarone was selected according to the survival curve and parameters generated automatically.β-asarone with different concentrations was used for intervention on astrocytes,then the changes of PC12 cells survival rate in the coculture system were observed. Secondly,MTT assay was used to detect the effect of Aβ1-42 on PC12 cells survival rate as well as the intervention effect of β-asarone,and verify the testing results of RTCA. The levels of IL-1β,TNF-α and BDNF in culture media of the lower chamber were detected by ELISA. The NF-κB activity and phosphorylation levels of ERK,p38 and JNK were detected by Western blot. Results showed that β-asarone（ 55. 5 mg·L- 1） could significantly slowdown the decline of PC12 cells survival rate caused by Aβ1-42-induced RA-h activation（ P 0. 01）,significantly reduce the levels of IL-1β,TNF-α and the phosphorylation levels of ERK,p38 and JNK in culture media of the lower chamber（ P 0. 01）. β-asarone（ 166. 7 mg·L- 1） could promote the release of BDNF in culture media of the lower chamber（ P 0. 05）. These results indicated that Aβ1-42 could induce RA-h activation and its release of IL-1β,TNF-α and other inflammatory factors to aggravate the PC12 cells injury; β-asarone could reduce the levels of IL-1β,TNF-α,promote the release of BDNF,and inhibit the NF-κB activity as well as phosphorylation levels of ERK,p38 and JNK protein in PC12 cells.