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.
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。