摘要
目的:通过制作谷氨酸诱导的PC-12细胞损伤模型,进一步探讨川芎嗪影响凋亡的作用机制。方法:应用细胞毒性试验,确定细胞模型中谷氨酸浓度;进而,应用不同浓度的川芎嗪同时处理PC-12细胞,采用荧光燃料Hoechst-33258染色直接观察细胞核的改变,采用流式细胞术检测细胞凋亡和Caspase-3,Caspase-8和Caspase-9活性,最后应用实时荧光定量RT-PCR检测Bax和Bcl2转录水平。结果:谷氨酸具有细胞毒性,其半数抑制浓度(ID50)是21.72mmol/L;川芎嗪呈剂量依赖性的提高细胞存活率。谷氨酸诱导的PC-12细胞以中晚期凋亡为主,但川芎嗪处理后,PC-12细胞凋亡率显著降低(P<0.05),且以早期凋亡为主。川芎嗪处理谷氨酸细胞模型后,Caspase-8活性未见显著改变(P>0.05),但在1mmol/L和10mmol/L川芎嗪干预组,Caspase-3和Caspase-9活性显著降低,同时伴随Bax/Bcl2比值显著降低(P<0.05)。结论:谷氨酸通过Caspase9而不是Caspase8途经活化Caspase3诱导凋亡;川芎嗪通过干预Bax/Bcl2平衡调控Caspase9途径抑制谷氨酸诱导的PC-12细胞凋亡。
Objective： In previous studies we have discovered that its effective component Tertrramethylpyrazine （TMPz） can inhibit the apoptosis of brain cells. In this paper, a glutamate-induced-injury modal is proposed for further discussion of the TMPz-influencedapoptosis mechanism. Methods： First determine the glutamate concentration in the cell model by cytotoxicity tests. Then synchronously treat PC- 12 cells with TMPz at different concentrations, and directly observe changes of nuclei with fluorescent dyes. Flow cytometry is adopted to detect apoptosis and the activity of Caspase- 3, Caspase- 8 and Caspase- 9. Finally a real-time fluorescent quantitative RT-PCR technique is used to measure the transcriptional level of Bax and Bcl2. Results： Glutamic acid is cytotoxic with an inhibitory concentration 50 （ID 50） of 21.72 mmol/L. TMPz dose-dependently enhances cell survival rate. For the case of glutamate-induced PC- 12 cells, advanced apoptosis take the leading place. After TMPz treatment, apoptmis rate of PC- 12 cells decreases remarkably （P〈 0.05 ） and early apoptosis become dominating. After treating glutamate-call modal with TMPz, there is no obvious change in Caspase- 8 activity （P 〉 0.05）, while in 1 mmol/L and 10 mmol/L TMPz-treat groups the activities of Caspase- 3 and Caspase- 9 exhibit tremendous decline, coupled with significant decrease of Bax/Bcl2 ratio （P〈0.05）. Conclusion： Glutamic acid activates Caspase3, inducing apoptosis through Caspase- 9 rather than Caspase- 8. TMPz inhibit the glutamate-induced-apoptosis of PC- 12 through controlling caspase9 by adjusting the ratio of Bax/Bcl2.
出处
《河南中医学院学报》
2008年第2期24-27,共4页
Journal of Henan University of Chinese Medicine
基金
江苏省研究生培养创新工程资助项目(编号:CX07B-222Z)
