Wu Qian, Wang Ning, Liu Jiao, Mao Zhi-Guo, Wang Yan-Xiang
Key Laboratory of Chinese Medicinal Formula of Anhui Province, Anhui University of Chinese Medicine Hefei 230012, China Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Chinese Medicine Hefei 230012, China.
Key Laboratory of Chinese Medicinal Formula of Anhui Province, Anhui University of Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2020 Aug;45(16):3931-3937. doi: 10.19540/j.cnki.cjcmm.20200316.401.
This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.
本研究旨在探讨川芎主要活性成分藁本内酯对氧糖剥夺/复灌注（OGD/R）诱导的PC12细胞损伤后线粒体分裂的影响及其机制。实验中，体外建立OGD/R模型，PC12细胞用藁本内酯预处理3小时，然后用CCK-8法检测细胞活力。在倒置显微镜下观察不同浓度藁本内酯对OGD/R损伤后PC12细胞形态的影响。采用透射电子显微镜观察OGD/R损伤后PC12细胞的线粒体分裂情况。采用DCFH-DA免疫荧光染色法检测细胞内活性氧（ROS）的变化。通过流式细胞术检测线粒体膜电位（MMP）的变化。用Hochest 33258观察PC12细胞的凋亡情况。采用蛋白质免疫印迹法检测线粒体和细胞质中细胞色素C（Cyt C）含量以及线粒体分裂相关蛋白Drp 1和Fis 1的变化。所有结果显示，与模型组相比，藁本内酯显著提高了PC12细胞的存活率和细胞数量。进一步实验表明，藁本内酯抑制OGD/R损伤后PC12细胞中ROS的释放和线粒体膜电位的下降。此外，藁本内酯减少了Cyt C的释放，并促进了线粒体分裂蛋白Drp1和Fis1的表达。验证实验表明，线粒体分裂抑制剂mdivi-1降低了细胞存活率并抑制了分裂。结果表明，藁本内酯通过促进线粒体分裂和减少细胞损伤发挥神经保护作用。初步证明藁本内酯对缺血性脑损伤的作用机制可能与促进线粒体分裂和维持细胞稳态有关。
