Short-chain fatty acids abrogate Japanese encephalitis virus-induced inflammation in microglial cells via miR200a-3p/ZBTB20/Ikßa axis

Japanese encephalitis virus (JEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in humans. Survivors of this infection often develop life-long neurological sequelae. Short-chain fatty acids (SCFAs) produced in the gut are vital mediators of the gut-brain axis. We aimed to study microRNA-based mechanisms of SCFAs in an in vitro model of JEV infection. N9 microglial cells were pre-treated with SCFA cocktail before JEV infection. Cytokine bead analysis (CBA), immunoblotting and PCR were performed to analyse relevant inflammatory markers. microRNA sequencing was performed using Illumina Hiseq and Bioinformatical tools were used for differentially expressed (DE) miRNAs and Weighted gene co-expression network analysis (WGCNA). microRNA mimic/inhibitor experiments and luciferase assay were performed to study miRNA-target interaction. A significant reduction in MCP1 and TNFa along with reduced expression of phospho-NF-?B was observed in SCFA conditions. Significant attenuation of HDAC activity and protein expression was recorded. miRNA sequencing revealed 160 DE miRNAs in SCFA+JEV treated cells at 6 hours post infection (HPI). WGCNA revealed miR-200a-3p, a hub miRNA significantly upregulated in SCFA conditions. Transcription factor ZBTB20 was bioinformatically predicted and validated as a gene target for miR200a-3p. Further miRNA mimic/inhibitor assay demonstrated that miR-200-3p regulated ZBTB20 along with I?ßa that possibly dampened NF-?B signal activation downstream. Overall design: N9 microglial cells were pre-treated with SCFA for 12 hours followed by JEV infection for 6hours and 24 hours. There were 4 groups in both the infection timeline namely, Control, SCFAControl, JEVcontrol, SCFAJEV. Total RNA extraction was performed using phenol chloroform method. Differential expression analysis in all 24 conditions was performed by small RNA sequencing (Illumina) according to the manufacturer's instructions.

日本脑炎病毒（Japanese encephalitis virus, JEV）属于黄病毒科（Flaviviridae），是引发人类病毒性脑炎的主要病原体之一。感染存活者常遗留终身神经系统后遗症。肠道产生的短链脂肪酸（Short-chain fatty acids, SCFAs）是肠-脑轴的关键介导因子。本研究旨在探究短链脂肪酸在日本脑炎病毒感染体外模型中的microRNA调控机制。

研究人员使用短链脂肪酸混合物预处理N9小胶质细胞，随后进行日本脑炎病毒感染。通过细胞因子磁珠分析（Cytokine bead analysis, CBA）、免疫印迹与聚合酶链式反应（PCR）检测相关炎症标志物。采用Illumina Hiseq平台开展microRNA测序，并借助生物信息学工具分析差异表达（differentially expressed, DE）microRNA，同时进行加权基因共表达网络分析（Weighted gene co-expression network analysis, WGCNA）。通过microRNA模拟物/抑制剂实验与荧光素酶报告实验，研究microRNA与靶标的相互作用。

实验结果显示，短链脂肪酸处理组中，单核细胞趋化蛋白1（MCP1）与肿瘤坏死因子α（TNF-α）的表达水平显著降低，磷酸化核因子κB（phospho-NF-κB）的表达也随之减弱；同时组蛋白去乙酰化酶（Histone deacetylase, HDAC）活性与蛋白表达水平均显著下降。microRNA测序结果表明，感染后6小时（hours post infection, HPI）的短链脂肪酸+日本脑炎病毒处理组细胞中共检测到160个差异表达microRNA。加权基因共表达网络分析显示miR-200a-3p为核心microRNA，在短链脂肪酸处理组中其表达显著上调。经生物信息学预测并验证了转录因子ZBTB20为miR-200a-3p的靶基因。后续microRNA模拟物/抑制剂实验证实，miR-200a-3p可调控ZBTB20与核因子κB抑制蛋白α（IκBα），进而可能抑制NF-κB信号通路的下游激活。

整体实验设计：N9小胶质细胞经短链脂肪酸预处理12小时，随后进行日本脑炎病毒感染，分别在感染后6小时与24小时两个时间点取样。两个时间点均设置4组实验：对照组（Control）、SCFA对照组（SCFAControl）、JEV感染对照组（JEVcontrol）、SCFA+JEV处理组（SCFAJEV）。总RNA采用酚-氯仿法提取。所有24组样本的差异表达分析均通过小RNA测序（Illumina平台）完成，实验操作严格遵循厂商说明书。