Tian Jing, An Xinjiang, Niu Ling
Department of Cardiology, Xuzhou Children's Hospital, Xuzhou, Jiangsu 221002, P.R. China.
Exp Ther Med. 2017 Jun;13(6):3333-3336. doi: 10.3892/etm.2017.4409. Epub 2017 Apr 28.
The aim of the study was to investigate the role and mechanisms of action of nuclear factor-κB (NF-κB)-mediated caspase-4 activation in the induction of inflammatory cytokines during Kawasaki disease (KD) and coronary artery endothelial cell injury. Peripheral blood mononuclear cells (PBMCs) were isolated from KD patients and healthy controls and cultured. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was applied to detect tumor necrosis factor (TNF)-α levels in activated PBMC-conditioned culture media. To establish a culture model for human coronary artery endothelial cells (HCAECs), we employed KD patient-origin PBMC culture-conditioned media to induce HCAEC transformation and detected the nuclear activation of NF-κB p65 and intracellular caspase-4 protein concentrations using western blot analysis. We also investigated the nuclear transfer of NF-κB p65 using immunofluorescence, as well as HCAEC interleukin (IL)-6 and IL-1β secretion using ELISA. Finally, we investigated HCAEC apoptosis using using Annexin V/PI double staining. After PBMCs were stimulated , TNF-α secretion was significantly higher in the KD group versus controls (P<0.01). HCAEC cells treated with supernatant conditioned by cells from KD patients showed a significant elevation of NF-κB p65 and caspase-4 protein expression versus HCAEC cells treated with supernatant conditioned by control cells (P<0.01). Similarly, IL-6 and IL-1β secretion, as well as apoptotic rate, were significantly elevated (P<0.01). SN50, an NF-κB inhibitor, significantly attenuated caspase-4 expression, secretion of IL-6, IL-1β, and TNF-α, as well as HCAEC apoptosis in cells treated with KD patient PBMC-conditioned media. NF-κB can induce the generation of various inflammatory factors including IL-6 and IL-1β, mediate the expression of caspase-4 in HCAEC cells, and affect apoptosis and injury of HCAEC cells. Therefore, the expression of caspase-4, mediated by NF-κB signal pathway, plays a critical role in KD.
本研究旨在探讨核因子-κB（NF-κB）介导的半胱天冬酶-4激活在川崎病（KD）炎症细胞因子诱导及冠状动脉内皮细胞损伤过程中的作用及作用机制。从KD患者和健康对照中分离外周血单个核细胞（PBMC）并进行培养。应用双抗体夹心酶联免疫吸附测定（ELISA）检测活化的PBMC条件培养基中肿瘤坏死因子（TNF）-α水平。为建立人冠状动脉内皮细胞（HCAEC）培养模型，我们采用KD患者来源的PBMC培养条件培养基诱导HCAEC转化，并使用蛋白质印迹分析检测NF-κB p65的核激活及细胞内半胱天冬酶-4蛋白浓度。我们还使用免疫荧光研究NF-κB p65的核转位，并使用ELISA研究HCAEC白细胞介素（IL）-6和IL-1β分泌。最后，我们使用膜联蛋白V/碘化丙啶双染法研究HCAEC凋亡。PBMC受到刺激后，KD组TNF-α分泌显著高于对照组（P<0.01）。与用对照细胞条件上清处理的HCAEC细胞相比，用KD患者细胞条件上清处理的HCAEC细胞中NF-κB p65和半胱天冬酶-4蛋白表达显著升高（P<0.01）。同样，IL-6和IL-1β分泌以及凋亡率也显著升高（P<0.01）。NF-κB抑制剂SN50显著减弱了用KD患者PBMC条件培养基处理的细胞中半胱天冬酶-4表达、IL-6、IL-1β和TNF-α分泌以及HCAEC凋亡。NF-κB可诱导包括IL-6和IL-1β在内的多种炎症因子生成，介导HCAEC细胞中半胱天冬酶-4的表达，并影响HCAEC细胞的凋亡和损伤。因此，由NF-κB信号通路介导的半胱天冬酶-4表达在KD中起关键作用。
