14,15-EET Maintains Mitochondrial Homeostasis to Inhibit Neuronal Pyroptosis After Ischemic Stroke
14,15-EET 通过维持线粒体稳态抑制缺血性卒中后的神经元焦亡

BACKGROUND: Neuronal pyroptosis is involved in neuronal cell death and neurological damage after cerebral ischemiareperfusion. 14,15-Epoxyeicosatrienoic acid (14,15-EET) can reduce neuronal loss induced by cerebral ischemia-reperfusion by regulating mitochondrial biological processes. However, it remains unclear how 14,15-EET regulates mitochondrial homeostasis, inhibits neuronal pyroptosis, and promotes neurological functional recovery after cerebral ischemia-reperfusion.
背景：神经元焦亡参与了脑缺血再灌注后的神经元细胞死亡和神经损伤。14,15-环氧二十碳三烯酸（14,15-EET）可以通过调节线粒体生物学过程来减少脑缺血再灌注引起的神经元丢失。然而，14,15-EET 如何调节线粒体稳态、抑制神经元焦亡以及促进脑缺血再灌注后的神经功能恢复尚不清楚。

METHODS: Mice with middle cerebral artery occlusion and reperfusion were used as an animal model to study the cerebral ischemia-reperfusion disease. The neurological function of mice was performed at 1,3 , and 5 days to test the therapeutic effects of 14,15-EET. Transmission electron microscope imaging and Nissl staining were used to analyze neuronal morphological structure, mitophagy, and neuronal pyroptosis. Western blot and transcriptome were used to detect the levels of mitophagy and neuronal pyroptosis signaling pathway-related molecules. HT22 cells were used in in vitro studies to detect the mechanism by which 14,15-EET reduces neuronal pyroptosis after oxygen-glucose deprivation/reoxygenation treatment.
方法：采用中动脉闭塞再灌注的小鼠模型研究脑缺血再灌注疾病。在 1、3 和 5 天时对小鼠进行神经功能评估，以测试 14,15-EET 的治疗效果。使用透射电子显微镜成像和尼氏染色分析神经元形态结构、线粒体自噬和神经元焦亡。通过蛋白质印迹和转录组检测线粒体自噬和神经元焦亡信号通路相关分子的水平。在体外研究中使用 HT22 细胞检测 14,15-EET 在氧糖剥夺/复氧处理减少神经元焦亡的机制。

RESULTS: 14,15-EET treatment reduced cerebral infarct volumes and improved neurological functional recovery in mice after cerebral ischemia-reperfusion. 14,15-EET treatment maintained the morphological structure of neurons in the ischemic penumbra area as well as the dendritic spine density in mice after cerebral ischemia-reperfusion. The upregulation of NLRP1 (NOD-like receptor thermal protein domain associated protein 1), IL (interleukin)- $1\beta$$1\beta$1beta1 \beta, caspase- 1 , and GSDMD (gasdermin D) induced by cerebral ischemia-reperfusion was inhibited, and the expression of mitophagy proteins Parkin and LC3B was increased by 14,15-EET treatment. Transcriptome profiling found that 14,15-EET exerts a neuroprotection role in promoting neural function recovery by activating the WNT (wingless-type MMTV integration site family) signaling pathway. We found that 14,15-EET upregulated the WNT pathway proteins such as WNT1, WNT3A, $\beta$$\beta$beta\beta-catenin, and p-GSK-3 $\beta$$\beta$beta\beta (phosphorylation of glycogen synthase kinase $3\beta$$3\beta$3beta3 \beta ) in vivo and in vitro. The WNT signaling pathway inhibitor XAV-939 reduced the expression of mitophagy protein Parkin and upregulated the expression of caspase-1 and GSDMD in HT22 cells with oxygen-glucose deprivation/reoxygenation and 14,15-EET treatment.
结果：14,15-EET 处理减少了脑缺血再灌注后小鼠的脑梗死体积，并改善了神经功能恢复。14,15-EET 处理维持了脑缺血再灌注后小鼠缺血半暗带区域神经元的形态结构以及树突棘密度。脑缺血再灌注上调的 NLRP1（NOD 样受体热蛋白结构域相关蛋白 1）、IL（白介素）- $1\beta$$1\beta$1beta1 \beta 、caspase-1 和 GSDMD（气孔蛋白 D）被抑制，而 14,15-EET 处理增加了线粒体自噬蛋白 Parkin 和 LC3B 的表达。转录组分析发现，14,15-EET 通过激活 WNT（无翅型 MMTV 整合位点家族）信号通路，在促进神经功能恢复中发挥神经保护作用。我们发现 14,15-EET 在体内和体外上调了 WNT 通路蛋白，如 WNT1、WNT3A、 $\beta$$\beta$beta\beta -catenin 和 p-GSK-3 $\beta$$\beta$beta\beta （糖原合成酶激酶 3 的磷酸化）。 WNT 信号通路抑制剂 XAV-939 降低了缺氧/复氧和 14,15-EET 处理下 HT22 细胞中线粒体自噬蛋白 Parkin 的表达，并上调了 caspase-1 和 GSDMD 的表达。

CONCLUSIONS: 14,15-EET regulates mitochondrial homeostasis to inhibit neuronal pyroptosis, thereby promoting the recovery of neurological function in mice after cerebral ischemia-reperfusion. These results provide new ideas for maintaining mitochondrial homeostasis and inhibiting neuronal pyroptosis after cerebral ischemia-reperfusion.
结论：14,15-EET 通过调节线粒体稳态抑制神经元焦亡，从而促进脑缺血再灌注后小鼠神经功能的恢复。这些结果为脑缺血再灌注后维持线粒体稳态和抑制神经元焦亡提供了新思路。

GRAPHIC ABSTRACT: A graphic abstract is available for this article.
图示摘要：本文提供图示摘要。

Key Words: homeostasis mischemia mitophagy moptosis m reperfusion
关键词：稳态 缺血 线粒体自噬 焦亡 再灌注

Nonstandard Abbreviations and Acronyms
非标准缩写和首字母缩略词

Stroke is an acute cerebrovascular disease characterized by high morbidity and mortality. Globally, $\approx 5$$\approx 5$~~5\approx 5 million people die from stroke each year. ${}^1$${}^1$^(1){ }^{1} Stroke is divided into ischemic, hemorrhagic, and transient ischemic. Among them, ischemic stroke has the highest incidence rate, accounting for $\approx 80\mathrm{\%}$$\approx 80\mathrm{\%}$~~80%\approx 80 \% of patients with stroke. The current main treatment strategies are intravenous thrombolysis, endovascular intervention, and the use of antiplatelet drugs. Intravenous thrombolysis is the main measure to restore blood flow, r-tPA (recombinant tissue-type plasminogen activator) is often used as a thrombolytic drug. However, thrombolytic therapy has a strict and limited time window ( $<4.5$$<4.5$< 4.5<4.5 hours) and adverse reactions, and blood reflow after thrombolysis can easily lead to secondary damage to neurons. Therefore, it is of great practical significance to explore treatment strategies and methods for neuronal protection and neurological function recovery after cerebral ischemia-reperfusion.
中风是一种以高发病率和死亡率为特征的急性脑血管疾病。全球每年有 $\approx 5$$\approx 5$~~5\approx 5 万人死于中风。 ${}^1$${}^1$^(1){ }^{1} 中风分为缺血性、出血性和短暂性缺血性。其中，缺血性中风发病率最高，占中风患者的 $\approx 80\mathrm{\%}$$\approx 80\mathrm{\%}$~~80%\approx 80 \% 。目前主要的治疗策略包括静脉溶栓、血管内介入和抗血小板药物的使用。静脉溶栓是恢复血流的主要措施，r-tPA（重组组织型纤溶酶原激活剂）通常用作溶栓药物。然而，溶栓治疗有严格且有限的时间窗（ $<4.5$$<4.5$< 4.5<4.5 小时）和不良反应，溶栓后的血流再通容易导致神经元继发性损伤。因此，探索脑缺血再灌注后神经元保护和神经功能恢复的治疗策略和方法具有重要的实际意义。

Recent studies have shown that pyroptosis is involved in the pathological process of neuronal apoptosis and neurological damage after cerebral ischemiareperfusion. ${}^2$${}^2$^(2){ }^{2} Cerebral ischemia-reperfusion causes neuronal mitochondrial membrane structures to rupture and swell, mitochondrial cristae disappearance, and mitochondrial contents release into the cytoplasm. ${}^3$${}^3$^(3){ }^{3} Mitochondrial DNA, ATP, and other contents can be recognized by PRRs (pattern recognition receptors) as DAMPs (damage-associated molecular patterns) of the cells, which activate caspase-1, cleave GSDMD (gasdermin D), and lead to cell pyroptosis. ${}^4$${}^4$^(4){ }^{4} Mitochondrial structural destruction and functional damage,
近期研究表明，焦亡参与了脑缺血再灌注后神经元凋亡及神经损伤的病理过程。 ${}^2$${}^2$^(2){ }^{2} 脑缺血再灌注会导致神经元线粒体膜结构破裂、肿胀，线粒体内嵴消失，线粒体内容物释放到细胞质中。 ${}^3$${}^3$^(3){ }^{3} 线粒体 DNA、ATP 等内容物可被 PRRs（模式识别受体）识别为细胞的 DAMPs（损伤相关分子模式），从而激活 caspase-1，切割 GSDMD（气孔蛋白 D），导致细胞焦亡。 ${}^4$${}^4$^(4){ }^{4} 线粒体结构破坏和功能损伤，
neuroinflammatory response caused by the release of contents (mtDNA, ATP), and oxidative stress caused by the production of reactive oxygen species are involved in the neuropathological process induced by cerebral ischemia-reperfusion. Mitophagy is an important mechanism for mitochondrial homeostasis by clearing mitochondria with structural and functional damages. ${}^5$${}^5$^(5){ }^{5} Several studies have shown that promoting mitophagy reduces neuronal apoptosis induced by cerebral ischemia-reperfusion. ${}^6$${}^6$^(6){ }^{6}
由内容释放（mtDNA、ATP）引起的神经炎症反应，以及由活性氧产生引起的氧化应激，都参与了脑缺血再灌注诱导的神经病理过程。线粒体自噬是维持线粒体稳态的重要机制，通过清除结构和功能受损的线粒体。 ${}^5$${}^5$^(5){ }^{5} 多项研究表明，促进线粒体自噬可以减少脑缺血再灌注诱导的神经元凋亡。 ${}^6$${}^6$^(6){ }^{6}

Epoxyeicosatrienoic acids (EETs) play important physiological and pathological functions in the body, such as promoting vasodilation, increasing angiogenesis, reducing pain, alleviating inflammation. ${}^7$${}^7$^(7){ }^{7} There are 4 isomers of EETs, including 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET, and are metabolized by sEH (soluble epoxide hydrolase) into dihydroxyeicosatrieonic acids without biological activity. Studies have shown that the level of 14,15-dihydroxyeicosatrieonic acid in the brain tissue of sEH gene knockout mice is much lower than that of control mice. The study results suggest that sEH has the strongest affinity for 14,15-EET, and gene knockout mainly inhibits the metabolism of 14,15-EET and correspondingly increases the devel of 14,15-EET. ${}^8$${}^8$^(8){ }^{8}
二十碳五烯酸（EETs）在人体中具有重要的生理和病理功能，例如促进血管舒张、增加血管生成、减轻疼痛、缓解炎症。 ${}^7$${}^7$^(7){ }^{7} EETs 有 4 种异构体，包括 5,6-EET、8,9-EET、11,12-EET 和 14,15-EET，它们通过可溶性环氧水解酶（sEH）代谢为无生物活性的二羟基二十碳五烯酸。研究表明，sEH 基因敲除小鼠脑组织中的 14,15-二羟基二十碳五烯酸水平远低于对照组小鼠。研究结果提示 sEH 对 14,15-EET 具有最强的亲和力，基因敲除主要抑制了 14,15-EET 的代谢，相应地增加了 14,15-EET 的发育。 ${}^8$${}^8$^(8){ }^{8}

We have previously reported that after cerebral ischemia-reperfusion, mitochondrial dynamics disorder in the mouse brain aggravates neuronal apoptosis and neurological damage. 14,15-EET treatment upregulates the mitochondrial fusion proteins by regulating the AMPK/ SIRT1/p-FoxO1 signaling pathway, maintains mitochondrial dynamic balance, reduces neuronal apoptosis, promotes dendritic spine formation, and alleviates neurological damage, ${}^{3,9}$${}^{3,9}$^(3,9){ }^{3,9} Because-pyroptosis is involved in mitochondrial structure and function disorders, the purpose of this study was to elucidate the potential mechanism of 14,15-EET in maintaining mitochondrial homeostasis and promoting neurological recovery through inhibiting neuronal pyroptosis in cerebral ischemia-reperfusion mice.
我们之前报道过，在脑缺血再灌注后，小鼠脑中的线粒体动力学紊乱会加剧神经元凋亡和神经损伤。14,15-EET 治疗通过调节 AMPK/SIRT1/p-FoxO1 信号通路上调线粒体融合蛋白，维持线粒体动态平衡，减少神经元凋亡，促进树突棘形成，并减轻神经损伤。 ${}^{3,9}$${}^{3,9}$^(3,9){ }^{3,9} 由于炎性细胞死亡与线粒体结构和功能紊乱有关，本研究旨在阐明 14,15-EET 通过抑制脑缺血再灌注小鼠的神经元炎性细胞死亡来维持线粒体稳态和促进神经恢复的潜在机制。

Data supporting the findings of the study are available from the corresponding authors upon reasonable request. A detailed methods section is available in the Supplemental Material.
支持本研究结果的数据可在合理请求下向通讯作者获取。详细的实验方法部分可在补充材料中找到。

The original contributions presented in the study are included within the article and further inquiries can be directed to the corresponding authors.
研究中所呈现的原创性贡献已包含在文章中，进一步咨询可致相应作者。

The animal study was reviewed and approved by the Committee of Medical Ethics and Welfare for Experimental Animals, Henan University School of Medicine (Approval No. HUSOM-2018-333).
动物研究已由河南大学医学院实验动物医学伦理与福利委员会审查批准（批准编号 HUSOM-2018-333）。

Healthy 5- to 6-week-old male C57BL/6N mice, weighing 19 to 21 g , were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd and GemPharmatech Co, Ltd. The mice ( 5 mice per cage) were housed in an SPF-level animal facility with a room temperature of ${20}^{\circ }\mathrm{C}$${20}^{\circ }\mathrm{C}$20^(@)C20^{\circ} \mathrm{C} to ${25}^{\circ }\mathrm{C}$${25}^{\circ }\mathrm{C}$25^(@)C25^{\circ} \mathrm{C} and a humidity of $40\mathrm{\%}$$40\mathrm{\%}$40%40 \% to $60\mathrm{\%}$$60\mathrm{\%}$60%60 \%, a 12 -hour light/dark cycle, and free access to water and food. The mice were environmentally adapted for 2 days and were used to establish middle cerebral artery occlusion (MCAO) model. All animals were randomly divided into groups, in accordance with the ARRIVE guidelines ${}^{10}$${}^{10}$^(10){ }^{10} and the guidelines for experimental animals, and experiments were conducted with the permission of the Henan University School of Medicine and Research Ethics Committee. The HT22 cell, a mouse hippocampal neuron cell, was used for in vitro experiments.
健康 5 至 6 周龄的雄性 C57BL/6N 小鼠，体重 19 至 21 克，购自北京维科生物技术公司有限公司和 GemPharmatech 公司。小鼠（每笼 5 只）饲养在 SPF 级动物设施中，室温为 ${20}^{\circ }\mathrm{C}$${20}^{\circ }\mathrm{C}$20^(@)C20^{\circ} \mathrm{C} 至 ${25}^{\circ }\mathrm{C}$${25}^{\circ }\mathrm{C}$25^(@)C25^{\circ} \mathrm{C} ，湿度为 $40\mathrm{\%}$$40\mathrm{\%}$40%40 \% 至 $60\mathrm{\%}$$60\mathrm{\%}$60%60 \% ，12 小时光照/黑暗循环，自由获取水和食物。小鼠进行环境适应 2 天，用于建立中动脉闭塞（MCAO）模型。所有动物根据 ARRIVE 指南 ${}^{10}$${}^{10}$^(10){ }^{10} 和实验动物指南随机分组，实验在河南大学医学院和科研伦理委员会的许可下进行。HT22 细胞，一种小鼠海马神经元细胞，用于体外实验。

The MCAO mice model was established by the endovascular suture-occluded method according to previous methods. ${}^3$${}^3$^(3){ }^{3} In brief, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, their neck hair was shaved, and their neck skin was cut open after disinfection. The internal carotid artery, external carotid artery, and common carotid artery were separated under a stereomicroscope, and the 3 blood vessels were ligated respectively. The external carotid artery was cut into a small incision by ophthalmic scissors. A thread plug was inserted into the external carotid artery and the thread plug was fixed with a thread. The slipknot of the internal carotid artery was untied. The thread plug was turned over and slowly inserted into the internal carotid artery. Insertion was stopped after the middle artery was blocked. After surgery, the mice were placed on a constant temperature incubator to monitor vital signs, and the ischemia duration was set to 2 hours. During reperfusion, the thread plug was slowly pulled out, the slipknot was tied, blood flow to the common carotid artery was restored, and the skin was sutured.
采用血管缝合阻塞法根据先前方法建立 MCAO 小鼠模型。 ${}^3$${}^3$^(3){ }^{3} 简而言之，通过腹腔注射 1%戊巴比妥麻醉小鼠，剃掉颈部毛发，消毒后切开颈部皮肤。在立体显微镜下分离颈内动脉、颈外动脉和颈总动脉，分别结扎这三条血管。用眼科剪刀在颈外动脉上切一个小口，插入线塞并用线固定。解开颈内动脉的滑结。翻转线塞，缓慢插入颈内动脉。阻塞中动脉后停止插入。手术后，将小鼠放置在恒温培养箱上监测生命体征，缺血时间设置为 2 小时。在再灌注期间，缓慢拉出线塞，系上滑结，恢复颈总动脉的血流，并缝合皮肤。

Cerebral blood flow was constantly measured with a laser Doppler blood flow monitor (PeriFlux 5000). The MCAO model was considered successful when cerebral blood flow was reduced by $80\mathrm{\%}$$80\mathrm{\%}$80%80 \%. During reperfusion, $10\mu \mathrm{M}$$10\mu \mathrm{M}$10 muM10 \mu \mathrm{M} 14,15-EET was injected into the tail vein, followed by tail vein injection every 12 hours. 14,15-EET (No. 50651) was purchased from Cayman Chemical Company. It contained no additives except the solvent ethanol, and its purity was $\ge 98\mathrm{\%}$$\ge 98\mathrm{\%}$>= 98%\geq 98 \%. It was sealed and stored in a low-temperature refrigerator and taken out when used. Except that no thread plug was inserted into the blood vessel, the other steps in the sham group were the same as those in the MCAO group.
脑血流量通过激光多普勒血流监测仪（PeriFlux 5000）持续测量。当脑血流量减少至 $80\mathrm{\%}$$80\mathrm{\%}$80%80 \% 时，认为 MCAO 模型建立成功。在再灌注期间，向尾静脉注射 $10\mu \mathrm{M}$$10\mu \mathrm{M}$10 muM10 \mu \mathrm{M} 14,15-EET，随后每 12 小时进行一次尾静脉注射。14,15-EET（编号 50651）购自 Cayman Chemical Company。除溶剂乙醇外，其不含任何添加剂，纯度为 $\ge 98\mathrm{\%}$$\ge 98\mathrm{\%}$>= 98%\geq 98 \% 。将其密封后存放在低温冰箱中，使用时取出。除了未在血管中插入栓子外，假手术组的其他步骤与 MCAO 组相同。

The mouse hippocampal cell line HT22 were seeded into cell plates with DMEM containing 10% FBS and placed in a cell culture incubator for culture. Passage the cells in the culture dish when the growth density is $80\mathrm{\%}$$80\mathrm{\%}$80%80 \% to $90\mathrm{\%}$$90\mathrm{\%}$90%90 \% and conduct subsequent experiments after the cells are fully activated for 3 passages. Cells were randomly divided into 4 groups: sham, oxygen-glucose deprivation/reoxygenation (OGD/R), OGD/ R+14,15-EET, OGD/R+14,15-EET+XAV-939.
将小鼠海马细胞系 HT22 接种于含 10% FBS 的 DMEM 培养基中，置于细胞培养箱中培养。当生长密度达到 $80\mathrm{\%}$$80\mathrm{\%}$80%80 \% 至 $90\mathrm{\%}$$90\mathrm{\%}$90%90 \% 时，在培养皿中传代细胞，并在细胞完全激活 3 代后进行后续实验。将细胞随机分为 4 组：假手术组、缺氧/复氧（OGD/R）、OGD/R+14,15-EET、OGD/R+14,15-EET+XAV-939。

Sterilize the anaerobic tank with UV irradiation for 1 hour, and then place it at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} to preheat. The cells were washed twice with PBS solution, sugar-free DMEM medium was added, and placed in an anaerobic tank. The air in the anaerobic tank is replaced with $95\mathrm{\%}{\mathrm{N}}_2$$95\mathrm{\%}{\mathrm{N}}_2$95%N_(2)95 \% \mathrm{~N}_{2} and $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2}. The cells were placed in a ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} incubator for 2 hours to complete oxygen and sugar deprivation. In the sham group, a complete culture medium was added and cultured at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} and $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2} for 2 hours. After OGD, the cells were taken out of the anaerobic tank and replaced with normal DMEM medium, DMEM medium+14,15EET ( $10\mathrm{nmol}/\mathrm{L}$$10\mathrm{nmol}/\mathrm{L}$10nmol//L10 \mathrm{nmol} / \mathrm{L} ), and DMEM medium+14,15-EET ( $10\mathrm{nmol}/$$10\mathrm{nmol}/$10nmol//10 \mathrm{nmol} / L)+XAV-939 ( $10\mu \mathrm{M}$$10\mu \mathrm{M}$10 muM10 \mu \mathrm{M} ).
用紫外线照射对厌氧罐进行 1 小时的灭菌，然后将其放置于 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 处预热。细胞用 PBS 溶液清洗两次，加入无糖 DMEM 培养基，置于厌氧罐中。厌氧罐中的空气用 $95\mathrm{\%}{\mathrm{N}}_2$$95\mathrm{\%}{\mathrm{N}}_2$95%N_(2)95 \% \mathrm{~N}_{2} 和 $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2} 替换。细胞置于 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 培养箱中 2 小时以完成缺氧和糖剥夺。在假手术组中，加入完全培养基，并在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 和 $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2} 处培养 2 小时。OGD 后，将细胞从厌氧罐中取出，更换为正常 DMEM 培养基、DMEM 培养基+14,15EET ( $10\mathrm{nmol}/\mathrm{L}$$10\mathrm{nmol}/\mathrm{L}$10nmol//L10 \mathrm{nmol} / \mathrm{L} ) 和 DMEM 培养基+14,15-EET ( $10\mathrm{nmol}/$$10\mathrm{nmol}/$10nmol//10 \mathrm{nmol} / L)+XAV-939 ( $10\mu \mathrm{M}$$10\mu \mathrm{M}$10 muM10 \mu \mathrm{M} )。

The brain sections were taken, proteinase K working solution was added to cover them, incubated at room temperature for 20 minutes, and washed with PBS $3\times ,5$$3\times ,5$3xx,53 \times, 5 minutes each time. A $0.1\mathrm{\%}$$0.1\mathrm{\%}$0.1%0.1 \% Triton X-100 solution was added to the tissue, which was incubated at room temperature for 20 minutes to permeabilize the cell membranes, followed by washing with PBS $3\times ,5$$3\times ,5$3xx,53 \times, 5 minutes each time. The TUNEL assay kit manufacturer’s instructions were strictly followed then, $50\mu \mathrm{L}$$50\mu \mathrm{L}$50 muL50 \mu \mathrm{~L} of equilibration buffer was added to each section and incubated at room temperature for 20 minutes. Thereafter, $56\mu \mathrm{L}$$56\mu \mathrm{L}$56 muL56 \mu \mathrm{~L} of TdT incubation buffer was added and incubated at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} for 2 hours. Five percent BSA was added dropwise to cover the tissue and blocked at room temperature for 30 minutes. The blocking solution was removed, rabbit anti-NeuN primary antibody ( $1:500$$1:500$1:5001: 500 ) was added to the slices, and incubation at $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} was performed overnight. The next day, the sections were washed with PBST $3\times ,5$$3\times ,5$3xx,53 \times, 5 minutes each time, secondary antibody was added, and incubated at room temperature for 60 minutes in the dark. 4’,6-Diamidino-2-phenylindole (DAPI) dye was added dropwise and incubated at room temperature in the dark for 10 minutes. The slides were sealed and taken picture with a fluorescence microscope.
脑组织切片被取出，加入蛋白酶 K 工作液覆盖，室温孵育 20 分钟，每次用 PBS 清洗。加入 $0.1\mathrm{\%}$$0.1\mathrm{\%}$0.1%0.1 \% Triton X-100 溶液至组织中，室温孵育 20 分钟以通透细胞膜，每次用 PBS 清洗。随后严格遵循 TUNEL 检测试剂盒说明书，向每张切片加入 $50\mu \mathrm{L}$$50\mu \mathrm{L}$50 muL50 \mu \mathrm{~L} 平衡缓冲液，室温孵育 20 分钟。接着加入 $56\mu \mathrm{L}$$56\mu \mathrm{L}$56 muL56 \mu \mathrm{~L} TdT 孵育缓冲液，孵育 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 2 小时。滴加 5% BSA 覆盖组织，室温封闭 30 分钟。封闭液被移除，向切片中加入兔抗 NeuN 一抗（ $1:500$$1:500$1:5001: 500 ）， $4^{\circ }\mathrm{C}$$4^{\circ }\mathrm{C}$4^(@)C4^{\circ} \mathrm{C} 孵育过夜。第二天，用 PBST 清洗切片 $3\times ,5$$3\times ,5$3xx,53 \times, 5 分钟，加入二抗，避光室温孵育 60 分钟。滴加 4’,6-二脒基-2-苯基吲哚（DAPI）染料，避光室温孵育 10 分钟。 幻灯片被密封并用荧光显微镜拍摄照片。

Total RNA was extracted from the brain tissues of mice in different groups, and RNA sequences were provided by GENEWIZ Biotechnology Co, Ltd (Suzhou). Raw reads were aligned to the ENSEMBL mouse reference genome using Hisat2, v2.0.1 alignment software. Use R language, v4.2.2 and DESeq2 software package to perform data analysis and screen differential genes. The difference fold is $>2,P<0.05$$>2,P<0.05$> 2,P < 0.05>2, P<0.05. Use the ClusterProfiler software package to perform Gene Ontology enrichment and KEGG pathway analysis on differential genes. The obtained $P$$P$PP value was corrected using the Benjamini-Hochberg correction method.
从不同组的小鼠脑组织中提取总 RNA，RNA 序列由 GENEWIZ 生物技术有限公司（苏州）提供。使用 Hisat2，v2.0.1 对齐软件将原始读数与 ENSEMBL 小鼠参考基因组进行比对。使用 R 语言，v4.2.2 和 DESeq2 软件包进行数据分析和筛选差异基因。差异倍数是 $>2,P<0.05$$>2,P<0.05$> 2,P < 0.05>2, P<0.05 。使用 ClusterProfiler 软件包对差异基因进行 Gene Ontology 富集和 KEGG 通路分析。获得的 $P$$P$PP 值使用 Benjamini-Hochberg 校正方法进行校正。

Mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and PBS was perfused through the heart until the flushing fluid was clear. The brain was quickly removed on ice, and samples were cut in the penumbral area and immediately immersed in 2.5% glutaraldehyde solution for fixation, overnight
小鼠通过腹腔注射 1%戊巴比妥进行麻醉，并使 PBS 通过心脏灌流直至冲洗液变清澈。大脑迅速在冰上取出，并在梗死边缘区域切取样本，立即浸入 2.5%戊二醛溶液中进行固定，持续过夜。