Zheng Fenglei, Tao Yijing, Liu Jingjing, Geng Zhimin, Wang Ying, Wang Yujia, Fu Songling, Wang Wei, Xie Chunhong, Zhang Yiying, Gong Fangqi
Department of Cardiology, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, 310052, People's Republic of China.
J Inflamm Res. 2021 Mar 5;14:719-735. doi: 10.2147/JIR.S297131. eCollection 2021.
Macrophages-mediated inflammation is linked with endothelial damage of Kawasaki disease (KD). KCa3.1, a calcium-activated potassium channel, modulates inflammation of macrophages. However, little is known about the role of KCa3.1 in inflammation by macrophages involved in KD. Hence, this study is aimed to explore the potential role of KCa3.1 in regulating inflammatory response by macrophages and subsequent vascular injury in an in vitro model of KD.
RAW264.7 cells were stimulated with cell wall extract (LCWE) with or without TRAM-34 or PDTC or AG490. Subsequently, mouse coronary artery endothelial cells (MCAECs) were incubated with RAW264.7 cells-conditioned medium to mimic local inflammatory lesions in KD. CCKi8 assay was used to evaluate cell viability. The mRNA levels of inflammatory mediators were detected by qRT-PCR. Expressions of KCa3.1, MCAECs injury-associated molecules, proteins involved in signal pathways of nuclear factor-κB (NF-κB), signal transducers and activators of transcription (STAT) 3 and p38 were evaluated by Western blot.
Our study showed that LCWE increased KCa3.1 protein level in RAW264.7 macrophages and KCa3.1 inhibition by TRAM-34 notably suppressed the expression of pro-inflammatory molecules in LCWE-treated macrophages via blocking the activation of NF-κB and STAT3 pathways. Besides, the inflammation and damage of MCAECs were attenuated in the TRAM-34-treated group compared with the KD model group. This vascular protective role was dependent on the down-regulation of NF-κB and STAT3 signal pathways, which was confirmed by using inhibitors of NF-κB and STAT3.
This study demonstrates that KCa3.1 blockade of macrophages suppresses inflammatory reaction leading to mouse coronary artery endothelial cell injury in a cell model of KD by hampering the activation of NF-κB and STAT3 signaling pathway. These findings imply that KCa3.1 may be a potential therapeutic target for KD.
巨噬细胞介导的炎症与川崎病（KD）的内皮损伤有关。KCa3.1是一种钙激活钾通道，可调节巨噬细胞的炎症反应。然而，关于KCa3.1在参与KD的巨噬细胞炎症中的作用知之甚少。因此，本研究旨在探讨KCa3.1在KD体外模型中调节巨噬细胞炎症反应及随后血管损伤中的潜在作用。
用或不用TRAM-34、PDTC或AG490处理RAW264.7细胞，并用细胞壁提取物（LCWE）刺激。随后，将小鼠冠状动脉内皮细胞（MCAECs）与RAW264.7细胞条件培养基一起孵育，以模拟KD中的局部炎症病变。采用CCKi8法评估细胞活力。通过qRT-PCR检测炎症介质的mRNA水平。通过蛋白质印迹法评估KCa3.1、MCAECs损伤相关分子、参与核因子κB（NF-κB）信号通路的蛋白质、信号转导和转录激活因子（STAT）3和p38的表达。
我们的研究表明，LCWE增加了RAW264.7巨噬细胞中KCa3.1蛋白水平，TRAM-34对KCa3.1的抑制通过阻断NF-κB和STAT3通路的激活，显著抑制了LCWE处理的巨噬细胞中促炎分子的表达。此外，与KD模型组相比，TRAM-34处理组中MCAECs的炎症和损伤减轻。这种血管保护作用依赖于NF-κB和STAT3信号通路的下调，这通过使用NF-κB和STAT3抑制剂得到证实。
本研究表明，在KD细胞模型中，巨噬细胞的KCa3.1阻断通过阻碍NF-κB和STAT3信号通路的激活，抑制了导致小鼠冠状动脉内皮细胞损伤的炎症反应。这些发现意味着KCa3.1可能是KD的一个潜在治疗靶点。
