摘要
目的通过检测川崎病(KD)小鼠TNF-α、核因子-κB(NF-κB)、基质金属蛋白酶-9(MMP-9)表达及活性变化,探讨KD的发病机制。方法 3周龄C57BL/6雄性小鼠60只,分为模型组和对照组,每组各30只。模型组单次腹腔注射0.5 mL干酪乳杆菌细胞壁提取物(LCWE)制备KD模型,对照组注射等量生理盐水。检测两组小鼠注射后14、28和56 d时点外周血TNF-α水平,心脏、冠状动脉组织NF-κB、MMP-9及其组织抑制物-1(TIMP-1)表达水平,NF-κB、MMP-9表达活性。观察小鼠心脏超声改变,同时行病理学检查冠状动脉病变严重程度。结果①模型组建模后14和28 d时点超声心动图显示,冠状动脉血管内膜毛糙,血管壁与血管周围出现高回声,部分呈冠状动脉瘤样扩张改变;②模型组建模后14和28 d时点病理学检查可见冠状动脉主干周围管壁肿胀、以淋巴细胞为主的大量炎细胞浸润,弹力层显著破坏。模型组建模后14 d时点,血清TNF-α水平明显高于对照组[(389.3±0.3)vs(18.9±0.3)pg·mL-1,P<0.01];建模后14和28 d时点心脏及冠状动脉NF-κBp65蛋白表达水平模型组均显著高于对照组[14 d:(37.5±9.3)vs(14.6±5.6)μg·L-1,28 d;(57.6±13.7)vs(21.6±6.6)μg·L-1;P均<0.05];建模后14和28 d时点心脏及冠状动脉MMP-9/TIMP-1水平模型组显著高于对照组[14 d:(4.9±1.7)vs(0.5±0.4),28 d:(12.3±6.9)vs(0.09±0.1);P均<0.01]。结论 KD急性期TNF-α等炎性细胞因子分泌,NF-κB活化和MMP-9分泌上调可能是KD心脏及冠状动脉炎症发生的重要通路。
Objective To detect the changes of tumor necrosis factor α( TNF-α),nuclear factor kappa B( NF-κB) and matrix metalloproteinases-9( MMP-9) in acute phase of a murine model of mice with Kawasaki disease,and to investigate the pathogenesis of Kawasaki disease. Methods Lactobacillus casei cell wall extraction( LCWE) was prepared and injected intraperitoneally to 3 weeks old C57BL /6 mice to induce KD. On day 14,28 and 56,western blotting,electrophoretic mobility shift assay( EMSA), zymography and enzyme linked immunosorbent assay( ELISA) were used to detect serum TNF-α levels,the expression and activation of NF-κBp65 protein in cardiac tissue and coronary artery,the expression of MMP-9 and their inhibitors in cardiac tissue and coronary artery in murine model of KD. At the same time,coronary artery damage was assessed by echocardiography and pathological detection. Results Echocardiography identified that coarsed intima of coronary artery and high density echo images around the coronary artery wall were found after intraperitoneal injections of LCWE,and accompanied by local coronary artery aneurysm. Furthermore,swelling of vessal wall and focal inflammatory infiltrate in the murine model of KD group were identified in the coronary artery trunk and branches. Broken elastin was consistently observed in the murine model of KD group. The serum TNF-α levels in the murine model group of KD( 389. 3 ± 0. 3 pg·mL- 1on d14) were significantly higher as compared to control group( 18. 9 ± 0. 3 pg·mL- 1on D14)( P < 0. 01). The expression of NF-κBp65 in the model group( 37. 5 ± 9. 3μg·L- 1on d14, 57. 6 ± 13. 7 μg·L- 1on d28) was significantly higher than control group( 14. 6 ± 5. 6 μg·L- 1on d14,21. 6 ± 6. 6 μg·L- 1on d28) on day 14 and day 28 following LCWE injection( all P < 0. 05). The expressions of MMP-9 / TIMP-1 in control group( 0. 5 ±0. 4 on d14,0. 09 ± 0. 1 on d28) were significantly lower than the murine model group of KD( 4. 9 ± 1. 7 on d14,12. 3 ± 6. 9 on d28) on day 14 and day 28 after LCWE injection( P < 0. 01). Conclusion Up-regulation of TNF-α / NF-κB / MMP-9 pathway might be an important mechanism of inflammation of heart and coronary arteritis in the acute phase of KD. Further study is needed to clarify this pathogenesis.
出处
《中国循证儿科杂志》
CSCD
2014年第1期59-63,共5页
Chinese Journal of Evidence Based Pediatrics
基金
国家自然科学基金面上项目:81274109、30973238
北京自然科学基金B类/北京教育委员会重大科研项目:KZ201010025024
北京市教育委员会科技创新平台项目:PXM2011_014226_07_000085
北京市卫生系统高层次卫生技术人才培养计划项目:2009-3-38
关键词
川崎病
动物模型
肿瘤坏死因子Α
基质金属蛋白酶-9
Kawasaki disease
Model
Tumor necrosis factor-α
Nuclear factor kappa-B
Matrix metalloproteinases-9
