红藻氨酸诱导PC12细胞凋亡及阿魏酸对神经元的保护作用

Ferulic acid protects against apoptosis of PC12 cells induced by kainic acid

目的:探讨阿魏酸(ferulic acid,FA)对红藻氨酸(kainic acid,KA)诱导的PC12细胞凋亡的作用及其机制。方法:采用50μmol/L KA诱导PC12细胞凋亡建立阿尔茨海默病神经细胞模型,然后将处理后的PC12细胞分为KA模型组和KA+FA(25、50和100μmol/L)处理的低、中、高剂量组,同时设立正常对照组。采用MTT比色法检测PC12细胞的存活率;采用免疫细胞化学法观察PC12细胞中凋亡蛋白Bcl-2、Bax和细胞色素C(Cyt C)的表达;annexinⅤ+PI双染流式细胞术检测PC12的细胞凋亡率;蛋白免疫印记技术检测PC12细胞中Bcl-2、Bax和Cyt C的表达水平。结果:MTT法和免疫细胞化学检测显示,与正常组相比,模型组PC12细胞的存活率明显下降,且细胞中Bcl-2表达减少(P<0.01),而Bax和Cyt C表达升高,Bcl-2/Bax比值下降(P<0.01),流式细胞术检测细胞的凋亡率显示,模型组细胞的凋亡率显著上升(P<0.01)。蛋白印迹术检测显示,模型组细胞中Bcl-2表达量减少,Bax和Cyt C表达量升高,与正常组比较差异显著(均P<0.01)。当采用FA干预后,与模型组相比,25、50和100μmol/L组细胞的存活率明显上升,细胞凋亡率减少,而且能增加Bcl-2阳性百分率和表达水平,明显减少Bax和Cyt C阳性百分率和表达水平,使Bcl-2/Bax比值增加(P<0.05或P<0.01)。结论:KA在50μmol/L时可明显诱导PC12发生凋亡,FA在25～100μmol/L时能显著抑制KA诱导的PC12细胞凋亡,其神经保护机制可能是通过抑制Bax和Cyt C的表达,升高Bcl-2表达和Bcl-2/Bax比值,从而阻断内源性细胞凋亡通路而提高神经细胞的存活率。

AIM： To investigate the effect of ferulic acid（FA） on the apoptosis of PC12 cells induced by kainic acid（KA） in vitro.METHODS： In order to establish an Alzheimer disease neuronal cell model,the rat pheochromocytoma cell line PC12 was treated with KA at a concentration of 50 μmol / L.These model neurons were divided into KA model group and 3 groups treated with FA at doses of 25,50 and 100 μmol / L,respectively.At the same time,normal group was established without KA pretreatment.The viability of the PC12 cells was detected by MTT assay.The expression of Bcl-2,Bax and cytochrome C（Cyt C） was determined by immunocytochemical method.Apoptotic rate of the PC12 cells was measured by flow cytometry with annexin V / PI double staining.The protein levels of Bcl-2,Bax and Cyt C were analyzed by Western blotting.RESULTS： The cell survival rate,the expression of Bcl-2 and the ratio of Bcl-2 to Bax in KA model group were significantly decreased（P 0.01）,while the expression of Bax and Cyt C was obviously increased compared with normal control group（P 0.01）.The apoptotic rate in KA model group was obviously increased compared with normal control group（P 0.01） After the intervention of FA,the cell survival rates were increased and the apoptotic rates were decreased.Furthermore,the positive rate and expression of Bcl-2,and the ratio of Bcl-2 to Bax in each dose of FA treatment group were significantly increased,while the expression of Bax and Cyt C in each dose group was significantly reduced as compared with KA model group（P 0.05 or P 0.01）.CONCLUSION： KA obviously induces apoptosis of PC12 cells.FA had obvious protective effect on PC12 cells against the toxicity of KA.FA blocks endogenous apoptic pathway through inhibiting the expression of Bax and Cyt C and increasing the expression of Bcl-2 and the ratio of Bcl-2 / Bax,thus improving the survival rate of PC12 cells.