Chen Weiping, Wu Zhiping, Cheng Zhijuan, Zhang Yangbo, Luo Qinghua, Yin Min
Department of Neurology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330006, Jiangxi Province, PR China; Institute of Neuroscience, Nanchang University, Nanchang 330031, Jiangxi Province, PR China; Jiangxi Provincial Clinical Medical Research Center for Neurological Disorders, Nanchang 330031, Jiangxi Province, PR China.
Department of Neurology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330006, Jiangxi Province, PR China; Institute of Neuroscience, Nanchang University, Nanchang 330031, Jiangxi Province, PR China; Jiangxi Provincial Clinical Medical Research Center for Neurological Disorders, Nanchang 330031, Jiangxi Province, PR China.
Neuroscience. 2025 Feb 6;566:17-27. doi: 10.1016/j.neuroscience.2024.12.020. Epub 2024 Dec 11.
Microglia polarization plays a crucial role in inflammatory injury of brain following intracerebral hemorrhage (ICH). Heme oxygenase-1 (HO-1) has demonstrated protective properties against inflammation and promote hematoma clearance after ICH. The objective of this study was to explore impacts of HO-1 on microglia polarization and phagocytosis after ICH, along with the underlying mechanism.
ICH model was constructed in C57BL/6 mice. Neurological deficit of ICH mice was evaluated. HE detected pathological changes of mouse brain tissue. Immunofluorescence staining tested co-localization between HO-1 or NF-κB p65 and IBA1. The expressions of gene and proteins were detected by RT-qPCR and Western blot, respectively. Flow cytometry determined microglial polarization phenotype and neuron apoptosis. Cell viability of neuron was assessed by CCK-8. Red blood cells labeled by PKH-26 and co-cultured with microglia for examining microglial erythrophagocytosis.
Both HO-1 and NF-κB p65 phosphorylation were elevated in brain tissues of ICH mice. ZnPP, a HO-1 inhibitor, could exacerbate microglial M1 polarization and nerve injury, as well as repress microglial erythrophagocytosis in vitro and hematoma clearance in vivo. On the contrary, Tat-NBD, a NF-κB inhibitor, greatly suppressed microglial M1 polarization, and induced M2 polarization and microglial erythrophagocytosis, thus improving nerve injury and hematoma clearance after ICH. Notably, it was observed that NF-κB p65 could be activated by ZnPP treatment, and the regulatory roles of ZnPP on microglial polarization and erythrophagocytosis after ICH in vivo and in vitro were all diminished by Tat-NBD.
Therefore, our data demonstrated that HO-1 alleviated nerve injury and induced M2 polarization and phagocytosis of microglia after ICH via inhibiting NF-κB signaling pathway, which could provide deepen the pathological understanding of ICH and provide potential intervention targets and drug candidate for ICH.
小胶质细胞极化在脑出血（ICH）后脑的炎症损伤中起关键作用。血红素加氧酶-1（HO-1）已显示出对炎症的保护作用，并促进ICH后的血肿清除。本研究的目的是探讨HO-1对ICH后小胶质细胞极化和吞噬作用的影响及其潜在机制。
在C57BL/6小鼠中构建ICH模型。评估ICH小鼠的神经功能缺损。苏木精-伊红（HE）染色检测小鼠脑组织的病理变化。免疫荧光染色检测HO-1或核因子-κB p65（NF-κB p65）与离子钙结合衔接分子1（IBA1）的共定位。分别通过逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测基因和蛋白表达。流式细胞术测定小胶质细胞极化表型和神经元凋亡。通过细胞计数试剂盒-8（CCK-8）评估神经元的细胞活力。用PKH-26标记红细胞并与小胶质细胞共培养以检测小胶质细胞的红细胞吞噬作用。
ICH小鼠脑组织中HO-1和NF-κB p65磷酸化水平均升高。HO-1抑制剂锌原卟啉（ZnPP）可加剧小胶质细胞M1极化和神经损伤，并在体外抑制小胶质细胞的红细胞吞噬作用以及在体内抑制血肿清除。相反，NF-κB抑制剂Tat-NBD可显著抑制小胶质细胞M1极化，并诱导M2极化和小胶质细胞的红细胞吞噬作用，从而改善ICH后的神经损伤和血肿清除。值得注意的是，观察到ZnPP处理可激活NF-κB p65，并且Tat-NBD可消除ZnPP对体内外ICH后小胶质细胞极化和红细胞吞噬作用的调节作用。
因此，我们的数据表明，HO-1通过抑制NF-κB信号通路减轻ICH后的神经损伤并诱导小胶质细胞的M2极化和吞噬作用，这可能会加深对ICH病理的理解，并为ICH提供潜在的干预靶点和候选药物。
