藁本内酯介导的线粒体自噬减轻HT22细胞的缺糖缺氧/复氧损伤  

Mitophagy mediated by ligustilide relieves OGD/R-induced injury in HT22 cells

作　　者：吴倩 刘娇[1] 田丽宇 汪宁 WU Qian;LIU Jiao;TIAN Li-yu;WANG Ning(College of Pharmacy y Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Province Key Laboratory of Research&Development of Chinese Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Institute for Pharnuicodynamics and Safety Evaluation of Chinese Medicine,Anhui Academy of Traditional Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽中医药大学中药复方安徽省重点实验室,安徽合肥230012 [3]安徽省中医药科学院中药药效与安全评价研究所,安徽合肥230012

出　　处：《中国中药杂志》2022年第7期1897-1903,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81903954,81773933);安徽高校自然科学研究重点项目(KJ2020A0384,KJ2020A0410);安徽省高校学科(专业)拔尖人才学术项目(gxbjZD15);安徽省第十三批115产业创新团队“针药结合防治脑病产业创新团队”项目。

摘　　要：线粒体作为细胞能量供应的主要场所,是决定缺血后神经细胞生存和死亡的关键靶区。缺血性脑卒中发生时,及时清除受损线粒体对于改善线粒体功能修复神经损伤至关重要。该文旨在研究中药活性成分藁本内酯(ligustilide, LIG)对缺糖缺氧/复氧(oxygen and glucose deprivation/reperfusion, OGD/R)致HT22细胞损伤时线粒体功能及线粒体自噬的影响。通过体外建立OGD/R模型,经藁本内酯预处理HT22细胞3 h后,采用CCK-8法检测细胞活力;免疫荧光法及流式细胞术检测线粒体功能相关指标如线粒体膜电位、钙离子超载情况、活性氧(reactive oxygen species, ROS)含量变化;Western blot法检测线粒体分裂蛋白Drp1及凋亡执行蛋白cleaved caspase-3表达;免疫荧光观察线粒体标志蛋白TOMM20和自噬发生标志物——溶酶体关联膜蛋白2的共定位情况。结果显示,与模型组相比,藁本内酯可以增加HT22细胞的存活率。进一步实验表明,藁本内酯可以抑制OGD/R损伤后HT22细胞内ROS的释放、钙离子超载及线粒体膜电位的下降,同时促进线粒体分裂蛋白Drp1的表达,增加线粒体与溶酶体关联膜蛋白2的共定位。以上结果表明藁本内酯可以改善OGD/R损伤后的线粒体功能,促进线粒体自噬的发生。当给予线粒体自噬抑制剂mdivi-1后,凋亡蛋白表达升高,表明藁本内酯发挥的神经保护作用可能与促进线粒体自噬有关。Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.

关 键 词：藁本内酯 线粒体自噬 缺糖缺氧/复氧 HT22细胞 

分 类 号：R285[医药卫生—中药学]