外泌体通过NRF2/SLC7A11/GPX4通路调控铁死亡治疗小鼠缺血性脑卒中

doi:10.12300/j.issn.1674-5817.2025.034

摘要/Abstract

摘要：

目的通过电凝法阻断小鼠大脑中动脉（middle cerebral artery，MCA），构建小鼠大脑中动脉闭塞模型（middle cerebral artery occlusion， MCAO），比较模型小鼠和外泌体（exosome，EXO）干预后的模型小鼠神经细胞损伤程度和铁死亡（ferroptosis）相关分子表达量的差异，以探索MCAO发病过程中，外泌体抑制铁死亡，改善缺血性脑卒中的作用机制。方法将32只6～8周龄SPF级雄性C57BL/6小鼠随机分为4组，每组8只，假手术组（Sham组）、模型组（MCAO组）、模型+生理盐水组（MCAO+NACL组）、模型+外泌体组（MCAO+EXO组）。利用离心和过滤的方法从人羊膜间充质干细胞（human amniotic mesenchymal stem cells，hAMSCs）的上清培养液中提取外泌体，通过电子显微镜对外泌体的粒径进行分析确认。对MCAO+EXO组的小鼠尾静脉注射1 mL的外泌体，MCAO+NACL组的小鼠尾静脉注射1 mL生理盐水。使用电凝法建立大脑中动脉缺血（MCAO）模型，Sham组暴露大脑中动脉但不实施电凝。采用Longa神经功能缺损评分评价各组小鼠神经功能损害程度，然后进行心脏灌注并取脑。通过TTC 染色法评估各组小鼠脑梗死体积百分比差异；通过HE染色评估各组小鼠脑组织神经细胞的形态特点差异；通过微量法检测各组小鼠脑梗死区及其周围组织亚铁离子（Fe²⁺）、丙二醛（MDA）、总谷胱甘肽（total glutathione，Total GSH）、氧化型谷胱甘肽（glutathione oxidized，GSSG）和谷胱甘肽（glutathione， GSH）含量的差异；取适量脑梗死区及周围组织，通过实时RT-PCR方法检测各组小鼠铁死亡相关因子：核因子红细胞相关因子2（NRF2）、溶质载体家族7成员11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）的mRNA表达；通过Western blot检测各组小鼠脑梗死区及其周围组织中NRF2、SLC7A11和GPX4的蛋白质表达。结果相较于MCAO组，MCAO+EXO组的Longa评分显著降低（P<0.01）。TTC染色发现：MCAO组小鼠脑组织出现明显梗死灶，而MCAO+EXO组脑梗死体积百分比显著减少（P<0.001）。HE染色发现：与Sham组相比，MCAO组出现神经细胞胞体空泡变性、细胞核固缩，深染，细胞核结构不清晰，神经细胞排列不整齐。而相较于MCAO组小鼠，MCAO+EXO组小鼠神经细胞结构较完整，细胞核大而规整，位于细胞中央。微量法检测发现：MCAO组脑梗死区及周围组织Fe²⁺、MDA含量较Sham组显著上升（P<0.001），MCAO+EXO组Fe²⁺、MDA含量较MCAO组显著下降（P<0.01）。相较于Sham组，MCAO模型组Total GSH、GSSG、GSH含量显著下降（P<0.01），相较于MCAO组，MCAO+EXO组Total GSH、GSH含量显著上升（P<0.001），GSSG含量无显著差异。实时RT-PCR发现：相较于Sham组，MCAO组NRF2、SLC7A11、GPX4的mRNA表达量均显著下降（P<0.01），相较于MCAO组，MCAO+EXO组NRF2、SLC7A11、GPX4的mRNA表达量显著升高（P<0.05）。Western blot发现：相较于Sham组，MCAO组NRF2、SLC7A11、GPX4的蛋白表达量显著下降（P<0.001）。相较于MCAO组，MCAO+EXO组NRF2、SLC7A11、GPX4的蛋白表达量显著上升（P<0.05）。结论在小鼠MCAO模型中，尾静脉注射人羊膜间充质干细胞来源外泌体可以改善运动功能、减少梗死面积、保护神经细胞形态、降低神经损伤程度；外泌体可能通过NRF2/SLC7A11/GPX4通路，减少MCAO模型小鼠脑神经细胞铁死亡而发挥对神经细胞的保护作用。

关键词: 动物模型, 缺血性脑卒中, 铁死亡, 外泌体, 小鼠

Abstract:

Objective By using electrocoagulation to block the middle cerebral artery (MCA) in mice, a middle cerebral artery occlusion model (MCAO) was established in mice. The degree of neuronal cell damage and the expression levels of ferroptosis-related molecules in the model mice were compared with those in the model mice after exosome intervention to explore the mechanism by which exosomes regulate ferroptosis to improve ischemic stroke. Methods A total of 32 male SPF-grade C57BL/6 mice at 6-8-week-old were randomly divided into 4 groups, with 8 mice in each group. The sham operation group (Sham group), the model group (MCAO group), the model + normal saline group (MCAO + NACL group), and the model + exosome group (MCAO + EXO group). Exosomes were extracted from the supernatant of human amniotic mesenchymal stem cell culture medium by centrifugation and filtration,and the extracted exosomes were observed under an electron microscope by analyzing particle size (NTA). The MCAO + EXO group mice were intravenously injected with 1 ml of exosomes, and the MCAO + NACL group mice were injected with the same volume of normal saline. The MCAO model was established by electrocoagulation of the middle cerebral artery,while the artery was exposed without electrocoagulation in sham group. The Longa neurological deficit score was used to evaluate the degree of neurological impairment, followed by cardiac perfusion and brain collection. The percentage of cerebral infarction volume was evaluated by TTC staining; the morphological characteristics of nerve cells in the brain were evaluated by H&E staining; the expression levels of ferroptosis-related factors (NRF2, SLC7A11, GPX4) in the cerebral infarction area and its surrounding tissues were examined by RT-qPCR. Results Compared with the MCAO group, the Longa score of the MCAO + EXO group was significantly lower (P < 0.01). TTC staining revealed that there were obvious infarction foci in the brain tissue of the MCAO group, and the percentage of cerebral infarction volume in the MCAO + EXO group was significantly reduced compared with the MCAO group (P < 0.001). HE staining showed that the MCAO group showed vacuolar degeneration of neuronal cell bodies, nuclear shrinkage, dark staining, unclear nuclear structure, and disordered neuronal arrangement,but these alterations were largely restored in the MCAO + EXO group. Critically, the contents of Fe²⁺ and MDA in the cerebral infarction area and its surrounding tissues of the MCAO group was significantly higher than those of the sham group (P < 0.001),but significantly reduced in the MCAO + EXO group (P < 0.01). In addition, compared with the Sham group, the total of GSH, GSSG and GSH contents were significantly decreased in the MCAO model group (P < 0.01), but the GSH and GSH contents were largely recovered in the MCAO + EXO group (P < 0.001) with unchanged GSSG content. Real-time RT-qPCR revealed that the mRNA expression levels of NRF2, SLC7A11, and GPX4 were significantly decreased in the MCAO group compared with the Sham group, (P < 0.01), but they were significantly increased in the MCAO + EXO group (P < 0.05). Western blot showed that the protein expression levels of NRF2, SLC7A11 and GPX4 were significantly decreased in the MCAO group compared with the Sham group (P < 0.001), and they were also significantly increased in the MCAO + EXO group (P < 0.05). Conclusion In the mouse MCAO model, tail vein injection of exosomes can improve motor function, reduce infarct area, protect neuronal cell morphology, and reduce the degree of nerve cell injury.The infarct area and surrounding tissues of the mouse MCAO model undergoes ferroptosis, and the exosomes can reduce ferroptosis of nerve cells possibly through the NRF2/SLC7A11/GPX4 pathway, protecting the survival of nerve cells in ischemic condition.

Key words: Animal model, Ischemic stroke, Ferroptosis, Exosome, Mouse

中图分类号:

Q95-33

徐英韬,王蒙蒙,林平,等.外泌体通过NRF2/SLC7A11/GPX4通路调控铁死亡治疗小鼠缺血性脑卒中[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2025.034.

XU Yingtao,WANG Mengmeng,LIN Ping,et al. Exosomes Improve Ischemic Stroke by Regulation of Ferroptosis Through the NRF2/SLC7A11/GPX4 Pathway in Mice[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.034.

图/表 7

表1 引物序列

Table1 Sequence of primers

引物名称 Primer name	引物序列 Sequence（5’→3’）
GAPDH-F	AGGTCGGTGTGAACGGATTTG
GAPDH-R	GGGGTCGTTGATGGCAACA
NRF2-F	CTTTAGTCAGCGACAGAAGGAC
NRF2-R	AGGCATCTTGTTTGGGAATGTG
SLC7A11-F	CACCGGGGTCGGTTTTCTTA
SLC7A11-R	GGCAGATGGCCAAGCTTTTG
GPX4-F	CTATGGTCCCATGGAGGAGC
GPX4-R	AGGCAGACCTTCATGAGTGC

图1 电镜观察和粒径分析提取的外泌体注：A，电镜观察下的外泌体，直径100 nm左右，成茶托状；B，粒径分析（NTA）提取到的外泌体悬液，横坐标为直径/纳米（diameter/nm），纵坐标为颗粒数/毫升（particle/mL）。

Figure 1 Electron microscopy observation of extracted exosome and analysis of particle sizeNote：A， the exosomes observed under the electron microscope have a diameter of approximately 100 nm and are in the shape of a tea tray； B， the exosome suspension extracted by particle size analysis （NTA）， with the abscissa representing the diameter in nanometers （diameter/nm） and the ordinate representing the number of particles per milliliter （particle/mL）.

图2 MCAO模型建立过程MCA血流变化和梗死区域TTC染色表现注：A，黑色箭头所指为MCA，呈Y字型；B～D，电凝使MCA被逐渐阻断，血供逐渐消失；E，MCAO模型取脑进行TTC染色处理，黑色箭头所指白色区域即为梗死区域；A～D图比例尺为3 mm，E图为5 mm。

Figure 2 The process of establishing the MCAO model， changes in MCA blood flow and TTC staining manifestations of the infarction areaNote：A， The black arrow indicates MCA， which is in a Y-shaped configuration； B-D， Electrocoagulation gradually blocks MCA， and blood supply gradually disappears； E， In the MCAO model， the brain is subjected to TTC staining treatment. The white area indicated by the black arrow is the infarction area； The scale of A-D is 3 mm， and that of E is 5 mm.

图3 小鼠神经功能缺损评分及取脑后切片TTC染色检测脑梗死体积百分比注：A，各组小鼠造模和外泌体干预后的Longa评分；B，小鼠取脑后切片，进行TTC染色的结果，每1列6张切片为1个完整大脑，正常脑组织为红色，梗死区域脑组织为白色；C，计算脑梗死体积百分比；每组8只小鼠。**P＜0.01， ***P＜0.001。

Figure 3 The neurological deficit score of mice and the percentage of cerebral infarction volume were detected by TTC staining of brain slices after brain removalNote：A， Longa scores of each group of mice after modeling and exosome intervention； B， after extracting the brains of the mice， the slices were stained with TTC. The results showed that each column contained 6 slices representing a complete brain. Normal brain tissue was red， while brain tissue in the infarcted area was white； C， the percentage of brain infarction volume was calculated； there were 8 mice in each group. **P＜0.01， ***P＜0.001.

图4 小鼠取脑后切片HE染色观察神经细胞形态（×40、×100）注：图中第1行×40视野比例尺为100 μm，第2行×100视野比例尺为40 μm；每组8只小鼠。

Figure 4 After removing the brain of the mice， the sections were stained with HE to observe the morphology of nerve cells（×40、×100）Note：The scale of the ×40 field of view in the first row of the figure is 100 μm， and the scale of the ×100 field of view in the second row is 40 μm； there were 8 mice in each group.The scale of the ×40 field of view in the first row is 100 μm， and the scale of the ×100 field of view in the second row is 40 μm； the area within the red frame in the first row ×40 field of view corresponds to the overall field of view in the second row ×100； there are 8 mice in each group.

图5 小鼠取脑后微量法检测脑梗死区域及周围脑组织Fe2+、MDA、Total GSH、GSSG、GSH含量注：A，检测Fe2+含量；B，检测MDA含量；C，检测Total GSH含量；D，检测GSSG含量；E，计算GSH含量，GSH的含量为Total GSH和GSSG的差值；每组8只小鼠。**P＜0.01，***P＜0.001，****P＜0.0001。

Figure 5 After extracting the brain of the mice， the content of Fe2+， MDA， Total GSH， GSSG and GSH in the brain infarction area and the surrounding brain tissues was detected using the micro-methodNote：A， detect Fe2+ content； B， detect MDA content； C， measure the total GSH content； D， measure the GSSG content； E， calculate the GSH content， which is the difference between the total GSH and GSSG； There were 8 mice in each group. **P＜0.01，***P＜0.001，****P＜0.0001.

图6 小鼠取脑后检测梗死区域及周围脑组织NRF2、SLC7A11、GPX4的mRNA及蛋白表达注：A，NRF2的mRNA表达及其相对量分析；B，SLC7A11的mRNA表达及其相对量分析；C，GPX4的mRNA表达及其相对量分析；D，Western Blot各组小鼠NRF2、SLC7A11、GPX4蛋白和GAPDH（内参蛋白）条带，右侧为蛋白对应分子量；E， NRF2的蛋白表达及其相对量分析；F，SLC7A11的蛋白表达及其相对量分析；G，GPX4的蛋白表达及其相对量分析；每组8只小鼠。*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.000 1。

Figure 6 After the brain of mice was removed， the mRNA and protein expressions of NRF2， SLC7A11 and GPX4 in the infarct area and the surrounding brain tissue were detected.Note：A， analysis of NRF2 mRNA expression and its relative quantity； B， analysis of SLC7A11 mRNA expression and its relative quantity； C， analysis of GPX4 mRNA expression and its relative quantity； D， protein bands of NRF2， SLC7A11， GPX4 and GAPDH （reference protein） in each group of Western Blot analysis of mice； on the right side， the corresponding molecular weights of the proteins are indicated； E， protein expression and relative quantification analysis of NRF2； F， protein expression and relative quantification analysis of SLC7A11； G， protein expression and relative quantification analysis of GPX4； 8 mice in each group. *P＜0.05，**P＜0.01，***P＜0.001，****P＜0.000 1.

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