Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via the AMPK/mTOR signaling pathway
Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via the AMPK/mTOR signaling pathway
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Mu-Xiang-You-Fang 通过 AMPK/mTOR 信号通路保护 PC12 细胞免受 OGD/R 诱导的自噬
DOI：
10.1016/j.jep.2020.112583
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发表时间：
2020-04-24
影响因子：
5.4
通讯作者：
Zhao, Qi-peng
中科院分区：
文献类型：
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作者：
Ma, Hui-xia;Hou, Fan;Zhao, Qi-peng
民族药理学相关性：木香攸方是回族医药的经典方剂。它由五种草药组成，多年来一直用于治疗缺血性中风。然而，MXYF的潜在药理学机制仍不清楚。本研究旨在探讨MXYF对体外脑缺血再灌注损伤模型的保护作用及其可能机制。材料与方法：建立PC12细胞氧糖剥夺再灌注（OGD/R）模型。使用细胞计数试剂盒（CCK-8）测定来确定MXYF对OGD/R损伤后的细胞活力的影响。采用比色法测定乳酸脱氢酶（LDH）漏出率。采用化学荧光法测定细胞内钙离子浓度，流式细胞仪测定线粒体膜电位。采用单丹酰尸胺（MDC）染色和电镜观察PC12细胞缺氧缺糖再灌注后的自噬现象。结果：MXYF（1、2、4 μ g/mL）能显著提高PC12细胞的存活率和线粒体膜电位，降低细胞内钙离子浓度和LDH释放速率。PC12细胞OGD/R损伤后，自噬体和自噬溶酶体数量显著增加。MXYF（4 μ g/mL）抑制OGD/R诱导的自噬，并抑制LC 3、beclinl、p-AMPK和ULK 1的表达。结论：MXYF通过AMPK-mTOR途径抑制OGD/R诱导的PC12细胞损伤后的自噬。因此，MXYF可能具有治疗缺血性中风的潜力。
Ethnopharmacological relevance: Mu-Xiang-You-Fang (MXYF) is a classic prescription of Hui medicine. It is composed of five herbs and has been used to treat ischemic stroke for many years. However, the potential pharmacological mechanisms of MXYF remain unclear. The present research is aimed to investigate the protective effect and possible mechanisms of MXYF treatment in an in vitro model of cerebral ischemia-reperfusion injury.Materials and methods: An oxygen-glucose deprivation and reperfusion (OGD/R) model of PC12 cells was established. The effect of MXYF on the cell viability after OGD/R injury was determined using a cell counting kit (CCK-8) assay. The colorimetric method was used to determine the lactate dehydrogenase (LDH) leakage rate. The calcium concentration was determined by the chemical fluorescence method, and mitochondrial membrane potential was determined using flow cytometry. Monodansylcadaverine (MDC) staining and electron microscopic analysis were then conducted to detect autophagy after oxygen-glucose deprivation and reperfusion in PC12 cells. Immunofluorescence and western blot analyses were used to detect the expression of proteins associated with autophagy.Results: It was found that MXYF (1, 2, 4 mu g/mL) could significantly increase cell viability and mitochondrial membrane potential and decrease the calcium concentration and LDH release rate in PC12 cells. After OGD/R injury in PC12 cells, the number of autophagosomes and autophagolysosome significantly increased. MXYF (4 mu g/mL) inhibited the autophagy induced by OGD/R and inhibited the expression of LC3, beclinl, p-AMPK, and ULK1. In contrast, the expression of p-mTOR, p-p70s6k, and p62 was significantly enhanced.Conclusions: These findings suggest that MXYF inhibits autophagy after OGD/R-induced PC12 cell injury through the AMPK-mTOR pathway. Thus, MXYF might have therapeutic potential in treating ischemic stroke.