八角枫碱通过ERK信号通路抑制神经细胞生长和分化诱导神经毒性的研究

Inhibition of Neuronal Cell Growth and Differentiation by Anabasine via the ERK Signaling Pathway and Its Induction on Neurotoxicity

目的:研究八角枫中的主要活性成分八角枫碱对神经细胞增殖、分化及细胞外信号调节激酶(ERK)通路的影响,探讨其神经毒性及毒性机制。方法:采用人神经母细胞瘤SH-SY5Y和人神经胶质瘤U251细胞作为体外评价模型,通过CCK-8和中性红摄取试验测定细胞毒性;Hoechst 33342核酸染色法检测染色质凝集情况;MitoTracker Red CMXRos线粒体红色荧光探针染色法检测线粒体膜电位的变化;分光光度法检测半胱氨酸蛋白酶Caspase-3、Caspase-9的活力;Western blot法检测胶质纤维酸性蛋白(GFAP)、波形蛋白(VIMENTIN)及BCL2相关X蛋白(BAX)、B淋巴细胞瘤-2(BCL2)、聚腺苷酸二磷酸核糖聚合酶(PARP)、磷酸化的细胞外信号调节激酶(P-ERK)和细胞外信号调节激酶(ERK)的蛋白表达;采用维甲酸诱导神经元分化,检测分化的SH-SY5Y细胞神经突生长和退化的情况。结果:与空白对照组相比,50μg/mL~2000μg/mL八角枫碱能明显抑制U251和SH-SY5Y的细胞生长(P<0.05或P<0.01),作用呈剂量和时间依赖性;八角枫碱作用于U251细胞24 h后,显著抑制胶质细胞标志物GFAP、VIMENTIN蛋白的表达;八角枫碱处理24 h后,U251细胞和SH-SY5Y细胞出现明显凋亡形态学改变,表现为细胞核或细胞质内可见浓染致密的颗粒块状荧光及线粒体膜电位降低,线粒体活性减弱;八角枫碱可剂量依赖性抑制分化的SH-SY5Y神经突的生长及诱导其神经突退化,表现为神经突数量减少,神经突的长度变短;与空白对照组相比,八角枫碱能剂量依赖性明显增加Caspase-3和Caspase-9的酶活力(P<0.05或P<0.01),上调BAX表达,下调BCL2表达,促进PARP切割,明显抑制ERK蛋白的磷酸化水平。结论:八角枫碱能抑制神经细胞及神经胶质细胞的增殖,并抑制神经元轴突的生长。其作用机制可能通过抑制ERK通路,上调BAX表达,下调BCL2表达,促进PARP切割,增加Caspase-3、Caspase-9活力,从而促进线粒体依赖性细胞凋亡。

Objective:To investigate the inhibitory effect of anabasine,the main active component of Alangium chinense(Lour.)Harms,on the cell proliferation,cell differentiation,and Extracellular signal-regulated kinase(ERK)pathway in neural cell lines,and explore its neurotoxicity and underlying mechanisms.Methods:Human neuroblastoma SH-SY5 Y and human glioma U251 cells were used as in vitro models.The cytotoxicity was determined by the cell counting kit-8(CCK-8)assay and neutral red uptake assay.The chromatic agglutination was detected by Hoechst 33342 nucleic acid staining.The MitoTracker Red CMXRos was used to measure changes in mitochondrial membrane potential.The expression levels of caspase-3 and caspase-9 were detected by spectrophotometry.The protein expression levels of glial fibrillary acidic protein(GFAP),vimentin,B-cell lymphoma-2(BCL2),Bcl-2 associated X protein(BAX),poly ADP-ribose polymerase(PARP),Extracellular signal-regulated kinase(ERK),and phosphorylated-ERK(p-ERK)were determined by Western blot.The neuronal differentiation was induced by retinoic acid treatment,and the neurite growth and degeneration of differentiated SH-SY5 Y cells were detected.Results:Compared with the control group,50μg/mL~2000μg/mL anabasine significantly inhibited the cell growth of U251 and SH-SY5 Y(P<0.05 or P<0.01),and the effect was in dose and time-dependent manners.Anabasine significantly inhibited the protein expression levels of glial cell markers GFAP and vimentin in U251 cells after 24 h treatment.After treatment with anabasine for 24 h,obvious apoptotic morphological changes were observed in U251 cells and SH-SY5 Y cells,which were characterized by densely stained granμLar clumps of fluorescence in the nucleus or cytoplasm,decreased mitochondrial membrane potential,and reduced mitochondrial activity.Anabasine inhibited differentiated SH-SY5 Y neurites and induced neurite degeneration in a dose-dependent manner,as evidenced by decreasing the number of neurites and shortening the length of neurites.Compared with the control group,Anabasine increased the activities of caspase-9 and caspase-3 in a dose-dependent manner(P<0.05 or P<0.01).Anabasine also up-regulated the expression level of BAX and down-regulated that of BCL2.In addition,it promoted PARP cleavage and significantly suppressed ERK phosphorylation.Conclusion:Anabasine can inhibit the proliferation of neuron cells and glial cells,as well as inhibit neurite growth.Its mechanism may be related to the inhibition of the ERK pathway,down-regulation of the BCL2 expression,up-regulation of the BAX expression,promotion of PARP cleavage,and increasing of the activities of caspase-9 and caspase-3,thereby promoting the mitochondrial-dependent-cell apoptosis.