背景:脑出血(ICH)导致毁灭性的发病率和死亡率,研究表明,血肿的毒性成分在脑出血后的脑损伤中起关键作用。近年来研究发现TLR9参与调节外周巨噬细胞的吞噬作用。目前的研究检查了TLR9在ICH后巨噬细胞/小胶质细胞(M/M)功能中的作用。
方法:RAW264.7(巨噬细胞),BV2(小胶质细胞),和HT22#(神经元)细胞系用慢病毒转染用于TLR9过表达。输注C57BL/6或EGFPTg/+小鼠的全血进行吞噬和损伤实验,和布鲁沙托尔用于实验。在ICH后第1、3、5、7和28天进行TLR9激动剂ODN1826或对照ODN2138的腹膜内注射,以研究TLR9在小鼠中的作用。此外,在M/M消除实验中共同注射氯膦酸盐。在ICH诱导后的不同时间点收集脑用于组织学和蛋白质实验。细胞和组织学方法用于测量血肿/铁残留,M/M变化,神经损伤,和脑组织损失。在模型前和ICH后第1、3、7和28天进行行为测试。
结果:TLR9的过表达在体外促进M/M吞噬作用并保护神经元免受血液来源的危害。此外,ODN1826增强M/M激活和吞噬功能,促进血肿/铁溶解,减少脑损伤,改善ICH小鼠的神经功能恢复,被注射氯膦酸盐废除了。实验结果表明,Nrf2/CD204通路参与了TLR9诱导的ICH后M/M吞噬。
结论:我们的研究表明,在ICH后,TLR9通过Nrf2/CD204途径增强M/M吞噬作用具有保护作用。我们的发现可以作为ICH治疗的潜在目标。
BACKGROUND: Intracerebral hemorrhage (ICH) causes devastating morbidity and mortality, and studies have shown that the toxic components of hematomas play key roles in brain damage after ICH. Recent studies have found that TLR9 participates in regulating the phagocytosis of peripheral macrophages. The current study examined the role of TLR9 in macrophage/microglial (M/M) function after ICH.
METHODS: RAW264.7 (macrophage), BV2 (microglia), and HT22# (neurons) cell lines were transfected with lentivirus for TLR9 overexpression. Whole blood from C57BL/6 or EGFPTg/+ mice was infused for phagocytosis and injury experiments, and brusatol was used for the experiments. Intraperitoneal injection of the TLR9 agonist ODN1826 or control ODN2138 was performed on days 1, 3, 5, 7, and 28 after ICH to study the effects of TLR9 in mice. In addition, clodronate was coinjected in M/M elimination experiments. The brains were collected for histological and protein experiments at different time points after ICH induction. Cellular and histological methods were used to measure hematoma/iron residual, M/Ms variation, neural injury, and brain tissue loss. Behavioral tests were performed premodeling and on days 1, 3, 7, and 28 post-ICH.
RESULTS: Overexpression of TLR9 facilitated M/M phagocytosis and protected neurons from blood-derived hazards in vitro. Furthermore, ODN1826 boosted M/M activation and phagocytic function, facilitated hematoma/iron resolution, reduced brain injury, and improved neurological function recovery in ICH mice, which were abolished by clodronate injection. The experimental results indicated that the Nrf2/CD204 pathway participated in TLR9-induced M/M phagocytosis after ICH.
CONCLUSIONS: Our study suggests a protective role for TLR9-enhanced M/M phagocytosis via the Nrf2/CD204 pathway after ICH. Our findings may serve as potential targets for ICH treatment.