Abstract：
OBJECTIVE: Ischemic stroke is a leading cause of death and disability in individuals worldwide. Cerebral ischemia-reperfusion injury (CIRI) typically results in severe secondary injury and complications following reperfusion therapy. Microglia play critical roles in the inflammatory reaction of CIRI. However, less attention has been given to microglial death in this process. Our study aims to explore microglial death in CIRI and the effects and mechanism of minocycline treatment on microglia.
METHODS: A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0 h, 24 h and 48 h post-operation, rats were intraperitoneally injected with 45 mg/kg minocycline. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, assessment of activated microglia and examination of mitochondrial structure were conducted and checked at 72 h after reperfusion. Additionally, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and function, and labile Fe2+ and ferroptosis-associated gene/protein levels were measured. Hemin was used for further validation after transcriptome analysis.
RESULTS: In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglia ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe2+ levels, lipid peroxidation, and expression of ferritin heavy chain (FTH) and it improved mitochondrial structure and function in vitro. Upregulation of HO-1 counteracted the protective effect of minocycline.
CONCLUSIONS: Ferroptosis is a major form of microglial death in CIRI. The protective mechanism of minocycline in CIRI partially hinges on its ability to effectively ameliorate microglia ferroptosis by downregulating HO-1 expression. Consequently, targeting microglia ferroptosis is a promising treatment for CIRI.
METHODS: A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0 h, 24 h and 48 h post-operation, rats were intraperitoneally injected with 45 mg/kg minocycline. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, assessment of activated microglia and examination of mitochondrial structure were conducted and checked at 72 h after reperfusion. Additionally, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and function, and labile Fe2+ and ferroptosis-associated gene/protein levels were measured. Hemin was used for further validation after transcriptome analysis.
RESULTS: In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglia ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe2+ levels, lipid peroxidation, and expression of ferritin heavy chain (FTH) and it improved mitochondrial structure and function in vitro. Upregulation of HO-1 counteracted the protective effect of minocycline.
CONCLUSIONS: Ferroptosis is a major form of microglial death in CIRI. The protective mechanism of minocycline in CIRI partially hinges on its ability to effectively ameliorate microglia ferroptosis by downregulating HO-1 expression. Consequently, targeting microglia ferroptosis is a promising treatment for CIRI.
摘要：
目的：缺血性卒中是全球人群死亡和残疾的主要原因。脑缺血再灌注损伤(CIRI)通常在再灌注治疗后导致严重的继发性损伤和并发症。小胶质细胞在CIRI的炎症反应中起关键作用。然而,在这一过程中，对小胶质细胞死亡的关注较少。我们的研究旨在探讨CIRI中的小胶质细胞死亡以及米诺环素治疗对小胶质细胞的影响和机制。
方法：采用大鼠大脑中动脉阻塞（MCAO）模型诱导CIRI。在0h时，术后24小时和48小时，大鼠腹腔注射45mg/kg米诺环素。神经功能缺损评分,氯化2,3,5-三苯基四唑（TTC）染色，再灌注后72h进行活化小胶质细胞评估和线粒体结构检查。此外,建立体外氧糖剥夺/再灌注(OGD/R)模型。用各种细胞死亡的药理学抑制剂或米诺环素处理BV-2细胞。细胞活力，脂质过氧化，线粒体结构和功能，并测量不稳定的Fe2和铁死亡相关基因/蛋白质水平。在转录组分析后,将血红素用于进一步验证。
结果：在MCAO和OGD/R模型中，铁死亡被确定为小胶质细胞死亡的主要形式。米诺环素通过降低HO-1表达抑制小胶质细胞铁性凋亡。此外,米诺环素改善线粒体膜电位，线粒体结构和体内小胶质细胞存活。米诺环素也降低了不稳定的Fe2+水平，脂质过氧化，和铁蛋白重链（FTH）的表达，并在体外改善线粒体结构和功能。HO-1的上调抵消了米诺环素的保护作用。
结论：铁凋亡是CIRI中小胶质细胞死亡的主要形式。二甲胺四环素在CIRI中的保护机制部分取决于其通过下调HO-1表达有效改善小胶质细胞铁性凋亡的能力。因此,靶向小胶质细胞铁性凋亡是CIRI的一种有前途的治疗方法。
方法：采用大鼠大脑中动脉阻塞（MCAO）模型诱导CIRI。在0h时，术后24小时和48小时，大鼠腹腔注射45mg/kg米诺环素。神经功能缺损评分,氯化2,3,5-三苯基四唑（TTC）染色，再灌注后72h进行活化小胶质细胞评估和线粒体结构检查。此外,建立体外氧糖剥夺/再灌注(OGD/R)模型。用各种细胞死亡的药理学抑制剂或米诺环素处理BV-2细胞。细胞活力，脂质过氧化，线粒体结构和功能，并测量不稳定的Fe2和铁死亡相关基因/蛋白质水平。在转录组分析后,将血红素用于进一步验证。
结果：在MCAO和OGD/R模型中，铁死亡被确定为小胶质细胞死亡的主要形式。米诺环素通过降低HO-1表达抑制小胶质细胞铁性凋亡。此外,米诺环素改善线粒体膜电位，线粒体结构和体内小胶质细胞存活。米诺环素也降低了不稳定的Fe2+水平，脂质过氧化，和铁蛋白重链（FTH）的表达，并在体外改善线粒体结构和功能。HO-1的上调抵消了米诺环素的保护作用。
结论：铁凋亡是CIRI中小胶质细胞死亡的主要形式。二甲胺四环素在CIRI中的保护机制部分取决于其通过下调HO-1表达有效改善小胶质细胞铁性凋亡的能力。因此,靶向小胶质细胞铁性凋亡是CIRI的一种有前途的治疗方法。
