miR-141-3p inhibits the activation of astrocytes and the release of inflammatory cytokines in bacterial meningitis through down-regulating HMGB1,Brain Research

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miR-141-3p inhibits the activation of astrocytes and the release of inflammatory cytokines in bacterial meningitis through down-regulating HMGB1
Brain Research ( IF 2.7 ) Pub Date : 2021-08-14 , DOI: 10.1016/j.brainres.2021.147611
Xiao Fang ₁ , Huaili Wang ₁ , Zhihong Zhuo ₁ , Peichao Tian ₁ , Zheng Chen ₁ , Yue Wang ₁ , Xiuyong Cheng ₁

Affiliation

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.

中文翻译：

miR-141-3p通过下调HMGB1抑制细菌性脑膜炎星形胶质细胞的活化和炎性细胞因子的释放

背景

细菌性脑膜炎（BM）是一种严重的中枢神经系统传染病，多发于儿童和青少年。许多研究表明，微RNA（miRNA）与BM有关。本研究旨在通过 HMGB1 探讨 miR-141-3p 对 BM 中星形胶质细胞活化和炎症反应的影响。

方法

3周龄大鼠注射肺炎链球菌（SP ）进入侧脑室建立BM模型。Loeffler评分法用于评价神经功能恢复情况。通过苏木精和伊红（H＆E）染色观察脑病理损伤。从 BM 或未感染的 SD 大鼠的脑组织中分离出原代星形胶质细胞。采用酶联免疫吸附试验（ELISA）测定脑组织和星形胶质细胞培养上清液中TNF-α、IL-1β和IL-6的含量。miR-141-3p 和 HMGB1 之间的靶向关系使用双荧光素酶报告基因测定法进行测试。通过定量实时聚合酶链反应 (qRT-PCR) 或蛋白质印迹检测 miR-141-3p、HMGB1 和星形胶质细胞标记胶质纤维酸性蛋白 (GFAP) 的表达。

结果

结果表明，BM 大鼠的 Loeffler 评分较低。BM大鼠脑组织蛛网膜下腔增宽，可见明显炎症细胞。BM 大鼠和 SP 处理的星形胶质细胞中 miR-141-3p 表达降低。此外，我们发现 miR-141-3p 的过表达导致星形胶质细胞中 HMGB1、GFAP 和炎性细胞因子（TNF-α、IL-1β 和 IL-6）的下调。此外，双荧光素酶报告基因检测结果证实 miR-141-3p 直接靶向 HMGB1。miR-141-3p 的过表达抑制了星形胶质细胞中 GFAP、TNF-α、IL-1β 和 IL-6 的水平，而这被 HMGB1 的上调所消除。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。