中国中药杂志

2020, v.45(16) 3931-3937

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藁本内酯对缺糖缺氧/复氧所致PC12细胞线粒体分裂的影响
Effect of ligustilide on oxygen and glucose deprivation/reperfusion-induced mitochondria fission in PC12 cells

吴倩;汪宁;刘娇;茆志国;王彦翔;
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;Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Chinese Medicine;

摘要(Abstract):

该文旨在探讨川芎中主要活性成分藁本内酯对缺糖缺氧/复氧(oxygen and glucose deprivation/reperfusion,OGD/R)致PC12细胞损伤时线粒体分裂的影响及机制。通过体外建立OGD/R模型,经藁本内酯预处理PC12细胞3 h后,采用CCK-8法检测细胞活力;倒置显微镜下观察藁本内酯不同浓度组对OGD/R损伤后PC12细胞形态的影响;透射电镜下观察OGD/R损伤后PC12细胞线粒体分裂的情况。DCFH-DA免疫荧光染色法检测细胞内活性氧含量(reactive oxygen species,ROS)变化;流式细胞术检测线粒体膜电位(mitochondria membrane potential,MMP)变化,Hochest 33258观察PC12细胞凋亡情况;Western blot法检测线粒体和胞质中细胞色素C(cytochrome C,Cyt C)的含量变化,及线粒体分裂相关蛋白Drp 1及Fis 1的表达。结果显示,与模型组相比,藁本内酯可以增加PC12细胞的存活率,细胞数目明显增多,细胞形态较正常,呈簇状生长。进一步实验表明,藁本内酯可以抑制OGD/R损伤后PC12细胞内ROS的释放及线粒体膜电位的下降,减少Cyt C自线粒体至胞浆的释放,并促进线粒体分裂蛋白Drp 1和Fis 1的表达,促进了线粒体分裂。回复性实验表明,当给予线粒体分裂抑制剂mdivi-1后,抑制了线粒体分裂,同时细胞存活率显著下降,表明藁本内酯可以通过促进线粒体分裂抑制细胞损伤而发挥神经保护作用。初步证明藁本内酯抗缺血性脑损伤的作用机制可能与促进线粒体分裂,维持自身稳态有关。
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.

关键词(KeyWords): 藁本内酯;缺糖缺氧/复氧;PC12细胞;线粒体分裂
ligustilide;oxygen and glucose deprivation/reperfusion(OGD/R);PC12 cell;mitochondria fission

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81903954,81773933);; 安徽省高校学科(专业)拔尖人才学术项目(gxbjZD15)

作者(Author): 吴倩;汪宁;刘娇;茆志国;王彦翔;
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;Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Chinese Medicine;

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