miR-141-3p 通过下调 HMGB1 抑制细菌性脑膜炎中天麻细胞的激活和炎症细胞因子的释放。
miR-141-3p inhibits the activation of astrocytes and the release of inflammatory cytokines in bacterial meningitis through down-regulating HMGB1.
机构信息
Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Department of Pediatrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
出版信息
Brain Res. 2021 Nov 1;1770:147611. doi: 10.1016/j.brainres.2021.147611. Epub 2021 Aug 14.
BACKGROUND
Bacterial meningitis (BM) is a serious infectious disease of the central nervous system that often occurs in children and adolescents. Many studies have suggested that microRNAs (miRNAs) are involved in BM. This study aimed to address the effects of miR-141-3p on astrocyte activation and inflammatory response in BM through HMGB1.
METHODS
The 3-week-old rats were injected with Streptococcus pneumoniae (SP) into the lateral ventricle to establish a BM model. Loeffler scoring method was used to evaluate the recovery of neurological function. Brain pathological damage was observed by hematoxylin and eosin (H&E) staining. Primary astrocytes were isolated from brain tissues of BM or non-infected SD rats. The levels of TNF-α, IL-1β, and IL-6 in brain tissues and astrocyte culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between miR-141-3p and HMGB1 was tested using dual-luciferase reporter assay. The expression of miR-141-3p, HMGB1, and the astrocytic marker glial fibrillary acidic protein (GFAP) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blotting. Methylation-specific PCR (MSP) analysis was performed to measure the methylation status of miR-141 promoter.
RESULTS
The results showed that lower Loeffler scores were exhibited in rats with BM. The subarachnoid space of brain tissues of BM rats was widened, and obvious inflammatory cells were observed. miR-141-3p expression was reduced in BM rats and SP-treated astrocytes. Additionally, we found that overexpression of miR-141-3p led to the downregulation of HMGB1, GFAP, and inflammatory cytokines (TNF-α, IL-1β, and IL-6) in astrocytes. Furthermore, the results of dual-luciferase reporter assay confirmed that miR-141-3p directly targeted HMGB1. Overexpression of miR-141-3p inhibited the levels of GFAP, TNF-α, IL-1β, and IL-6 in astrocytes, which was eliminated by the up-regulation of HMGB1. The results of MSP analysis indicated that miR-141 promoter was highly methylated in brain tissues and astrocytes. DNMT1 was involved in the methylation of miR-141 promoter in BM.
CONCLUSION
The present study verified that miR-141-3p affected inflammatory response by suppressing HMGB1 in SP-induced astrocytes and BM rat model.
背景
细菌性脑膜炎（BM）是一种常见于儿童和青少年的中枢神经系统严重感染性疾病。许多研究表明 microRNAs（miRNAs）参与了 BM 的发病过程。本研究旨在通过高迁移率族蛋白 B1（HMGB1）探讨 miR-141-3p 对 BM 中天 鼠星型胶质细胞激活和炎症反应的影响。
方法
将 3 周龄大鼠经侧脑室注射肺炎链球菌（SP）建立 BM 模型。采用 Loeffler 评分法评估神经功能恢复情况。苏木精-伊红（H&E）染色观察脑组织病理损伤。从 BM 或非感染 SD 大鼠脑组织中分离原代星形胶质细胞。酶联免疫吸附试验（ELISA）检测脑组织和星形胶质细胞培养上清中 TNF-α、IL-1β 和 IL-6 的水平。双荧光素酶报告基因检测验证 miR-141-3p 与 HMGB1 的靶向关系。实时定量聚合酶链反应（qRT-PCR）或 Western blot 检测 miR-141-3p、HMGB1 和星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）的表达。甲基化特异性 PCR（MSP）分析检测 miR-141 启动子的甲基化状态。
结果
结果显示，BM 大鼠的 Loeffler 评分较低。BM 大鼠蛛网膜下腔增宽，可见明显的炎性细胞。BM 大鼠和 SP 处理的星形胶质细胞中 miR-141-3p 表达下调。此外，过表达 miR-141-3p 可下调星形胶质细胞中 HMGB1、GFAP 和炎症细胞因子（TNF-α、IL-1β 和 IL-6）的表达。双荧光素酶报告基因检测结果证实 miR-141-3p 可直接靶向 HMGB1。过表达 miR-141-3p 抑制星形胶质细胞中 GFAP、TNF-α、IL-1β 和 IL-6 的水平，而 HMGB1 的上调则消除了这种抑制作用。MSP 分析结果表明，miR-141 启动子在脑组织和星形胶质细胞中高度甲基化。DNMT1 参与了 BM 中 miR-141 启动子的甲基化。
结论
本研究证实，miR-141-3p 通过抑制 SP 诱导的星形胶质细胞和 BM 大鼠模型中的 HMGB1 来影响炎症反应。
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