State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, New drug screening center, Jiangsu Center for Pharmacodynamics Research and Evaluation, Institute of Pharmaceutical Sciences, China Pharmaceutical University, Nanjing 210009, PR China; College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 311402, PR China; Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, PR China; School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Yantai University, Yantai 264005, PR China.
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, New drug screening center, Jiangsu Center for Pharmacodynamics Research and Evaluation, Institute of Pharmaceutical Sciences, China Pharmaceutical University, Nanjing 210009, PR China.
Pharmacol Res. 2021 Jul;169:105640. doi: 10.1016/j.phrs.2021.105640. Epub 2021 Apr 27.
Brain microvascular endothelial cells (BMVECs), as the important structure of blood-brain barrier (BBB), play a vital role in ischemic stroke. Pyroptosis of different cells in the brain may aggravate cerebral ischemic injury, and PGC-1α plays a major role in pyroptosis. However, it is not known whether BMVECs undergo pyroptosis after ischemic stroke and whether PGC-1α activator Medioresinol (MDN) we discovered may be useful against pyroptosis of endothelial cells and ischemic brain injury.
For in vitro experiments, the bEnd.3 cells and BMVECs under oxygen and glucose-deprivation (OGD) were treated with or without MDN, and the LDH release, tight junction protein degradation, GSDMD-NT membrane location and pyroptosis-associated proteins were evaluated. For in vivo experiments, mice underwent transient middle cerebral artery occlusion (tMCAO) for ischemia model, and the neuroprotective effects of MDN were measured by infarct volume, the permeability of BBB and pyroptosis of BMVECs. For mechanistic study, effects of MDN on the accumulation of phenylalanine, mitochondrial reactive oxygen species (mtROS) were tested by untargeted metabolomics and MitoSOX Red probe, respectively.
BMVECs underwent pyroptosis after ischemia. MDN dose-dependently activated PGC-1α, significantly reduced pyroptosis, mtROS and the expressions of pyroptosis-associated proteins (NLRP3, ASC, cleaved caspase-1, IL-1β, GSDMD-NT), and increased ZO-1 and Occludin protein expressions in BMVECs. In tMCAO mice, MDN remarkably reduced brain infarct volume and the permeability of BBB, inhibited pyroptosis of BMVECs, and promoted long-term neurobehavioral functional recovery. Mechanistically, MDN promoted the interaction of PGC-1α with PPARα to increase PPARα nuclear translocation and transcription activity, further increased the expression of GOT1 and PAH, resulting in enhanced phenylalanine metabolism to reduce the ischemia-caused phenylalanine accumulation and mtROS and further ameliorate pyroptosis of BMVECs.
In this study, we for the first time discovered that pyroptosis of BMVECs was involved in the pathogenesis of ischemic stroke and MDN as a novel PGC-1α activator could ameliorate the pyroptosis of endothelial cells and ischemic brain injury, which might attribute to reduction of mtROS through PPARα/GOT1 axis in BMVECs. Taken together, targeting endothelial pyroptosis by MDN may provide alternative therapeutics for brain ischemic stroke.
脑微血管内皮细胞（BMVECs）作为血脑屏障（BBB）的重要结构，在缺血性卒中中发挥重要作用。不同细胞的细胞焦亡可能加重脑缺血损伤，而过氧化物酶体增殖物激活受体γ共激活因子 1α（PGC-1α）在细胞焦亡中起主要作用。然而，目前尚不清楚缺血性卒中后 BMVEC 是否发生细胞焦亡，以及我们发现的 PGC-1α 激活剂 Medioresinol（MDN）是否对内皮细胞焦亡和缺血性脑损伤有治疗作用。
在体外实验中，用或不用 MDN 处理氧葡萄糖剥夺（OGD）下的 bEnd.3 细胞和 BMVECs，评估乳酸脱氢酶（LDH）释放、紧密连接蛋白降解、Gasdermin D-N 端（GSDMD-NT）膜定位和细胞焦亡相关蛋白。在体内实验中，小鼠接受短暂性大脑中动脉闭塞（tMCAO）缺血模型，通过梗死体积、BBB 通透性和 BMVEC 细胞焦亡评估 MDN 的神经保护作用。为了进行机制研究，分别通过靶向代谢组学和 MitoSOX Red 探针测试了 MDN 对苯丙氨酸积累、线粒体活性氧（mtROS）的影响。
缺血后 BMVEC 发生细胞焦亡。MDN 剂量依赖性地激活 PGC-1α，显著减少细胞焦亡、mtROS 和细胞焦亡相关蛋白（NLRP3、ASC、cleaved caspase-1、IL-1β、GSDMD-NT）的表达，增加 BMVECs 中紧密连接蛋白 ZO-1 和 Occludin 的表达。在 tMCAO 小鼠中，MDN 显著减少脑梗死体积和 BBB 通透性，抑制 BMVEC 细胞焦亡，促进长期神经行为功能恢复。机制上，MDN 促进 PGC-1α 与过氧化物酶体增殖物激活受体α（PPARα）相互作用，增加 PPARα 核转位和转录活性，进一步增加谷草转氨酶 1（GOT1）和苯丙氨酸羟化酶（PAH）的表达，导致苯丙氨酸代谢增强，减轻缺血引起的苯丙氨酸积累和 mtROS，进一步改善 BMVEC 细胞焦亡。
本研究首次发现 BMVEC 细胞焦亡参与缺血性卒中的发病机制，新型 PGC-1α 激活剂 MDN 可改善内皮细胞焦亡和缺血性脑损伤，这可能归因于通过 BMVECs 中的过氧化物酶体增殖物激活受体α/GOT1 轴减少 mtROS。总之，通过 MDN 靶向内皮细胞焦亡可能为脑缺血性卒中提供新的治疗策略。
