Aim

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.

Methods

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.

Results

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.

Conclusion

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.

中文翻译：

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Medioresinol 作为一种新型 PGC-1α 激活剂通过 PPARα-GOT1 轴防止缺血性卒中内皮细胞焦亡

目标

脑微血管内皮细胞（BMVECs）作为血脑屏障（BBB）的重要结构，在缺血性脑卒中中发挥着至关重要的作用。脑内不同细胞的焦亡可能加重脑缺血性损伤，PGC-1α在焦亡中起主要作用。然而，尚不清楚 BMVECs 在缺血性中风后是否会发生细胞焦亡，以及我们发现的 PGC-1α 激活剂 Medioresinol (MDN) 是否可能对内皮细胞焦亡和缺血性脑损伤有用。

方法

对于体外实验，在有或没有 MDN 的情况下，在氧和葡萄糖剥夺 (OGD) 下处理 bEnd.3 细胞和 BMVEC，并评估 LDH 释放、紧密连接蛋白降解、GSDMD-NT 膜定位和焦亡相关蛋白. 对于体内实验，小鼠接受短暂大脑中动脉闭塞（tMCAO）的缺血模型，并通过梗死体积、BBB 通透性和 BMVECs 焦亡测量 MDN 的神经保护作用。对于机理研究，分别通过非靶向代谢组学和 MitoSOX Red 探针测试了 MDN 对苯丙氨酸、线粒体活性氧 (mtROS) 积累的影响。

结果

BMVECs 在缺血后发生焦亡。MDN 剂量依赖性地激活 PGC-1α，显着降低细胞焦亡、mtROS 和细胞焦亡相关蛋白（NLRP3、ASC、cleaved caspase-1、IL-1β、GSDMD-NT）的表达，并增加 ZO-1 和 Occludin 蛋白的表达在 BMVEC 中。在 tMCAO 小鼠中，MDN 显着减少脑梗死体积和 BBB 通透性，抑制 BMVEC 焦亡，促进长期神经行为功能恢复。机制上，MDN促进PGC-1α与PPARα相互作用，增加PPARα核转位和转录活性，进一步增加GOT1和PAH的表达，导致苯丙氨酸代谢增强，减少缺血引起的苯丙氨酸积累和mtROS，进一步改善细胞焦亡。 BMVEC。

结论

在这项研究中，我们首次发现 BMVECs 的焦亡参与了缺血性卒中的发病机制，MDN 作为一种新型 PGC-1α 激活剂可以改善内皮细胞焦亡和缺血性脑损伤，这可能归因于 mtROS 的减少通过 BMVEC 中的 PPARα/GOT1 轴。总之，通过 MDN 靶向内皮细胞焦亡可能为脑缺血性中风提供替代疗法。