川芎嗪拼合物对PC12细胞损伤保护作用的研究

Neuroprotective Effect of Ligustrazine Derivative T-CA against Apoptosis on CoCl2-Induced PC12 Cells

目的:探讨川芎嗪拼合物T-CA对CoCl_2拟缺血模型PC12细胞损伤的神经保护作用及其机制。方法:体外培养NGF诱导分化的拟缺血损伤PC12细胞,将其随机分为Normal组、Model组、T-CA组及Positive组。采用MTT比色法检测各组细胞OD值的变化,应用AO/EB荧光染色及DAPI染色法观察各组细胞的凋亡情况,应用HE染色法观察各组细胞形态学的变化情况,并采用免疫细胞化学方法检测各组细胞中Bad和Bcl-xl阳性颗粒的表达,最后应用Western Blot法检测各组细胞中NF-κB/P65和Caspase-8蛋白的表达情况。结果:与Normal组及Positive组相比,经T-CA处理后的CoCl_2拟缺血模型PC12细胞的活性明显增强(P<0.05或P<0.01);给药组中细胞数量增多,细胞形态较好,细胞突起生长正常、密集,坏死及凋亡的细胞减少;Bad阳性颗粒的表达明显减弱,而Bcl-xl阳性颗粒的表达明显增多;NF-κB/P65的表达量变化不明显,T-CA组Caspase-8的表达水平稍有升高,但差异没有统计学意义(P>0.05);Positive组Caspase-8的表达略有降低,但差异不具有统计学意义(P>0.05)。结论:化合物T-CA可能通过作用于细胞凋亡的非死亡受体途径,上调Bcl-xl蛋白表达和下调Bad蛋白表达来抑制PC12细胞凋亡,发挥神经保护作用。

Objective： To investigate the neuroprotective mechanisms of T - CA on CoCl2 - induceddifferentiated PC12 cell damage. Methods： PC12 cell differentiated by NGF and cultured in vitro, and thenwere randomly divided into the normal group, the model group, T - CA group and the positive group. Thechanges of optical density（OD） of PC12 cells were detected by methyl thiazolyl tetrazolium（MTT） assay. Theapoptosis of the cells were confirmed using Acridine Orange / Ethidium Bromide（AO/ EB） double staining and DAPI staining. The morphological changes of PC12 cells induced by T - CA were observed via HE staining.The expression of Bad and Bcl - xl positive granules in PC12 cells were detected by using mmunohistochemistry technique. The expressions of NF - κB / P6 and Caspase - 8 in PC12 cells were detected by Western blotmethod. Results： Compared to the normal group and the positive group, the proliferation of PC12 cells intervened with T - CA were promoted significantly（P 〈 0. 05, P 〈 0. 01）. In the medication groups, there weremore live cells with an decreased number of apoptotic cells, the cells grew exuberant with morphology intact,and showed more and longer nerve protrusions. The expression of Bad positive granules decreased significantly and the expression of Bcl - xl increased. The expression of NF - κB / P65 was not changed evidently, and grayscale analysis was not statistically significant（P 〉 0. 05）. Cells treated with T - CA showed an increase of Capase - 8, but with no significant difference（P 〉 0. 05）, and the positive group made a bit reduction in theexpression with no significant difference（P 〉 0. 05）. Conclusion： T - CA can possibly up - regulate the expression of Bcl - xl and down - regulate Bad expression to inhibit apoptosis of PC12, playing the role of neuroprotective effect.