Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, Wenzhou Medical University, Wenzhou, China.
CNS Neurosci Ther. 2020 Sep;26(9):952-961. doi: 10.1111/cns.13400. Epub 2020 May 27.
To investigate the protective effect of VX-765 on human umbilical mesenchymal stem cells (HUMSCs) in stroke and its mechanism.
Mouse models of ischemic stroke were established using the distal middle cerebral artery occlusion (dMCAO) method. The dMCAO mice were accordingly transplanted with HUMSCs, VX-765-treated HUMSCs, or VX-765 + MHY185-treated HUMSCs. The HUMSCs were inserted with green fluorescent protein (GFP) for measurement of transplantation efficiency which was determined by immunofluorescence assay. Oxygen-glucose deprivation (OGD) was applied to mimic ischemic environment in vitro experiments, and the HUMSCs herein were transfected with AMPK inhibitor Compound C or autophagy inhibitor 3-MA. MTT assay was used to test the toxicity of VX-765. TUNEL staining and ELISA were applied to measure the levels of apoptosis and inflammatory cytokines (IL-1β, IL-6, and IL-10), respectively. The expressions of autophagy-associated proteins, AMPK, and mTOR were detected by Western blotting. TTC staining was applied to reveal the infarct lesions in the brain of dMCAO mice.
The pro-inflammatory cytokines, TUNEL-positive cells, and p-mTOR were decreased while the anti-inflammatory cytokine, autophagy-related proteins, and p-AMPK were increased in HUMSCs treated with VX-765 under OGD condition. Different expression patterns were found with the above factors after transfection of 3-MA or Compound C. The pro-inflammatory cytokines, TUNEL-positive cells, and infarct sections were decreased while the anti-inflammatory cytokine and autophagy-related proteins were increased in dMCAO mice transplanted with VX-765-treated HUMSCs compared to those transplanted with HUMSCs only. The autophagy was inhibited while p-mTOR was up-regulated after transfection of MHY.
VX-765 protects HUMSCs against stroke-induced apoptosis and inflammatory responses by activating autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.
本研究旨在探讨 VX-765 对人脐带来源间充质干细胞（HUMSCs）在脑卒中的保护作用及其机制。
采用大脑中动脉远端阻塞（dMCAO）法建立缺血性脑卒中模型。将 HUMSCs、VX-765 处理的 HUMSCs 或 VX-765+MHY185 处理的 HUMSCs 移植至 dMCAO 小鼠体内。为检测移植效率，将 GFP 插入 HUMSCs 中，通过免疫荧光法进行测定。在体外实验中采用氧葡萄糖剥夺（OGD）模拟缺血环境，将 AMPK 抑制剂 Compound C 或自噬抑制剂 3-MA 转染至 HUMSCs 中。MTT 法检测 VX-765 的毒性，TUNEL 染色法和 ELISA 法分别检测细胞凋亡和炎症因子（IL-1β、IL-6 和 IL-10）水平，Western blot 法检测自噬相关蛋白、AMPK 和 mTOR 的表达。TTC 染色法检测 dMCAO 小鼠脑梗死灶。
在 OGD 条件下，VX-765 处理的 HUMSCs 中促炎细胞因子、TUNEL 阳性细胞和 p-mTOR 减少，抗炎细胞因子、自噬相关蛋白和 p-AMPK 增加。转染 3-MA 或 Compound C 后，上述因子的表达模式发生了变化。与单独移植 HUMSCs 的小鼠相比，移植 VX-765 处理的 HUMSCs 的小鼠促炎细胞因子、TUNEL 阳性细胞和梗死节段减少，抗炎细胞因子和自噬相关蛋白增加。MHY 转染后，自噬受到抑制，p-mTOR 上调。
在体内和体外，VX-765 通过激活 AMPK/mTOR 信号通路诱导自噬，从而保护 HUMSCs 免受脑卒中诱导的细胞凋亡和炎症反应。
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