小儿川崎病不同病理时期Casp4及NF-κB信号通路相关因子表达分析

Expression analysis of Casp4and NF-κB signaling pathway related factors in different pathological stages of Kawasaki disease in children

目的研究小儿川崎病(KD)不同病理时期Casp4及NF-κB信号通路相关因子表达,以及CASP4和NF-κB之间的关系。方法选择100例KD患儿和30例健康儿童进行研究,KD患儿均接受静脉注射免疫球蛋白(IVIG)(2g/kg)和阿司匹林口服治疗。按照病情发展时期将KD患儿分为急性期(发病后1-2周内)和缓解期(发病4周后)。通过ELISA试剂盒检测PBMC中NF-κB p65活性和血清中Casp4、TNF-α和IL-6活性。将HCAEC细胞分为Control组、TNF-α组、TNF-α+SN50组。TNF-α组细胞用10ng/mL TNF-α刺激细胞4h。TNF-α+SN50组细胞用10ng/mL TNF-α和5ng/ml NF-κB抑制剂SN50刺激细胞4h。Control组细胞正常培养。Western blot检测细胞中NF-κB p65、I-κBα、Casp4、TNF-α和IL-6的蛋白表达。此外,评估了HCAEC细胞与单核细胞的粘附。结果与健康儿童比较,KD患儿的PBMC中NF-κB p65活性及血清中TNF-α、IL-6和Casp4活性均显著升高(P<0.001)。与急性期比较,缓解期KD患儿的PBMC中NF-κB p65活性及血清中TNF-α、IL-6和Casp4活性均显著下降(P<0.001)。与TNF-α组相比,TNF-α+SN50组HCAEC细胞中的NF-κB p65、Casp4、TNF-α和IL-6的蛋白相对表达量均显著降低,而I-κBα显著升高(P<0.001)。与TNF-α组相比,TNF-α+SN50组粘附实验的相对荧光强度显著降低(P<0.001)。结论与急性期相比,缓解期KD患儿的Casp4和NF-κB信号通路相关因子的活性显著降低。此外,NF-κB抑制剂SN50可抑制TNF-α诱导的HCAEC细胞中Casp4和NF-κB信号通路相关因子的活性以及HCAEC细胞与单核细胞粘附。

Objective To study the expression of Casp4 and NF-κB signaling pathway related factors in different pathological stages of Kawasaki disease(KD)in children,and the relationship between CASP4 and NF-κB.Methods 100 children with KD and 30 healthy children were selected for the study.All children with KD received intravenous immunoglobulin(IVIG)(2 g/kg)and oral aspirin.According to the development period of the disease,children with KD were divided into the acute phase(within 1 to 2 weeks after the onset)and the remission phase(4 weeks after the onset).The activity of NF-κB p65 in PBMC and the activity of Casp4,TNF-αand IL-6 in serum were detected by ELISA kit.The HCAEC cells were divided into Control group,TNF-αgroup,and TNF-α+SN50 group.Cells in the TNF-αgroup were stimulated with 10 ng/mL TNF-αfor 4 h.Cells in the TNF-α+SN50 group were stimulated with 10 ng/mL TNF-αand 5 ng/ml NF-κB inhibitor SN50 for 4 h.Cells in the Control group were cultured normally.Western blot was used to detect the protein expression of NF-κB p65,I-κBα,Casp4,TNF-αand IL-6 in the cells.In addition,the adhesion of HCAEC cells to monocytes was evaluated.Results Compared with healthy children,the activity of NF-κB p65 in PBMC of children with KD and the activities of TNF-α,IL-6 and Casp4 in serum were significantly increased(P<0.001).Compared with the acute phase,the activities of NF-κB p65 in PBMC and the activities of TNF-α,IL-6 and Casp4 in serum of children with KD in remission stage were significantly decreased(P<0.001).Compared with the TNF-αgroup,the relative expressions of NF-κB p65,Casp4,TNF-αand IL-6 in HCAEC cells in the TNF-α+SN50 group were significantly reduced,while I-κBα was significantly increased(P<0.001).Compared with the TNF-αgroup,the relative fluorescence intensity of the adhesion experiment in the TNF-α+SN50 group was significantly reduced(P<0.001).Conclusion Compared with the acute phase,the activities of Casp4 and NF-κB signaling pathwayrelated factors in children with KD in remission are significantly reduced.In addition,the NF-κB inhibitor SN50 can inhibit the activity of Casp4 and NF-κB signaling pathway-related factors in HCAEC cells induced by TNF-αand the adhesion of HCAEC cells to monocytes.