Ritson Megan, Xia Dong, Wheeler-Jones Caroline, Stolp Helen B
Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK.
J Neurochem. 2025 Jun;169(6):e70130. doi: 10.1111/jnc.70130.
The mechanism by which chronic systemic inflammation contributes to cerebral endothelial dysfunction and neurological disorders is unclear, although endothelial inflammatory signalling is considered a cornerstone of this process. Here, we have performed transcriptomic analysis of published RNASeq datasets and identified consistent upregulation of the Tumour Necrosis Factor-C-X-C Motif Chemokine Ligand 10 (TNF-CXCL10) signalling pathway in mouse cerebral endothelial cells following a single inflammatory challenge. We subsequently investigated the effects of repeated low-level inflammation on the modulation of this pathway in a mouse cerebral endothelial cell line, analysing the effect on markers of endothelial cell activation and changes in cellular function, as a potential mechanism underlying the cerebrovascular response to low-level systemic inflammation. Mouse cerebral endothelial cells (bEnd.3) were exposed to hour-long treatments with phosphate buffered saline (PBS), a single low concentration of TNF (0.5 ng/mL), repeated low-concentration TNF (0.5 ng/mL, 1 h × 4 days) or a single cumulative concentration of TNF (2.0 ng/mL). RNA and protein were extracted 4 and 24 h after the final treatment for analysis of gene/protein expression using qRT-PCR and western blotting. Repeated inflammatory challenge significantly upregulated both Intercellular Adhesion Molecule 1 (ICAM1) and CXCL10 at the mRNA and protein levels. Signal transducer and activator of transcription 1 (STAT1) and phosphorylated-STAT1 (pSTAT1) protein levels were also increased at 4 and 24 h. Differentially, tumor necrosis factor receptor-associated factor 2 (TRAF2) and Interferon gamma (IFNγ) gene expression were decreased at 4 h, returning to control levels at 24 h. Functional analysis revealed significant increases in endothelial cell proliferation and apoptosis in the presence of repeated TNF exposure. CXCL10 knockdown with small interfering RNA (siRNA) reduced mean caspase 3/7 activity induced by the repeated inflammatory paradigm. These data suggest an upregulation of the TNF-CXCL10 pathway in response to low-level repetitive inflammation in mouse cerebral endothelial cells. Modulation of this pathway may represent a broad therapeutic target for neurovascular disease.
尽管内皮细胞炎症信号传导被认为是这一过程的基石，但慢性全身性炎症导致脑内皮功能障碍和神经疾病的机制尚不清楚。在此，我们对已发表的RNA测序数据集进行了转录组分析，并确定在单次炎症刺激后，小鼠脑内皮细胞中肿瘤坏死因子-C-X-C基序趋化因子配体10（TNF-CXCL10）信号通路持续上调。随后，我们研究了反复低水平炎症对小鼠脑内皮细胞系中该信号通路调节的影响，分析其对内皮细胞活化标志物和细胞功能变化的影响，作为脑血管对低水平全身性炎症反应的潜在机制。将小鼠脑内皮细胞（bEnd.3）用磷酸盐缓冲盐水（PBS）处理1小时、单次低浓度肿瘤坏死因子（TNF，0.5 ng/mL）、反复低浓度TNF（0.5 ng/mL，1小时×4天）或单次累积浓度TNF（2.0 ng/mL）处理。在最后一次处理后4小时和24小时提取RNA和蛋白质，使用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法分析基因/蛋白质表达。反复炎症刺激显著上调了细胞间黏附分子1（ICAM1）和CXCL10的mRNA和蛋白质水平。信号转导子和转录激活子１（STAT1）及磷酸化STAT1（pSTAT1）的蛋白质水平在4小时和24小时也有所增加。不同的是，肿瘤坏死因子受体相关因子2（TRAF2）和干扰素γ（IFNγ）基因表达在4小时时下降，并在24小时恢复到对照水平。功能分析显示，在反复暴露于TNF的情况下，内皮细胞增殖和凋亡显著增加。用小干扰RNA（siRNA）敲低CXCL10可降低反复炎症模式诱导的半胱天冬酶3/7平均活性。这些数据表明，在小鼠脑内皮细胞中，TNF-CXCL10信号通路在低水平重复性炎症反应中上调。对该信号通路的调节可能是神经血管疾病的一个广泛治疗靶点。
