丙种球蛋白对川崎病小鼠心脏核因子κB与MMP-9表达及活性的影响

Effects of intravenous immunoglobulin upon the overexpression and over-activation of nuclear factor-κB and matrix metalloproteinase-9 in murine model of Kawasaki disease

目的 探讨静脉注射丙种球蛋白（IVIG）对川崎病小鼠心脏核因子（NF）-κB及其下游基质金属蛋白酶9 （MMP-9）的表达及活性的影响.方法 将72只3～4周龄C57BL/6小鼠按随机数字表法分为IVIG组、模型组和对照组,每组24只.前两组予干酪乳杆菌细胞壁提取物（LCWE）0.5 mg单次腹腔注射进行造模,对照组同法予生理盐水0.5 ml.IVIG组在试验第5天予IVIG（2 mg/g）腹腔注射进行干预,其他两组同样方法注射同浓度生理盐水.在实验第14、28、56天时,每组取8只小鼠分别行超声心动图检测,留取心脏标本进行病理分析.通过蛋白质印迹和电泳迁移率实验（EMSA）法检测心脏及脾脏组织NF-κB的表达.通过蛋白质印迹法和明胶酶谱法检测心脏及冠状动脉组织MMP-9表达量和生物活性.采用f检验及方差分析进行组间比较.结果 LCWE注射后HE染色可见模型组小鼠冠状动脉局部出现大量炎性细胞浸润,弹力纤维染色可见模型组冠状动脉弹力层断裂浅染.超声心动图可见实验第14天及28天模型组小鼠左侧冠状动脉的内径均值均高于IVIG组及对照组[第28天：（0.48±0.07）比（0.41 ±0.03）和（0.35 ±0.02）mm,均P＜0.01],后两组差异均无统计学意义（均P ＞0.05）.蛋白质印迹测得各时间点模型组心脏组织NF-κB以及MMP-9的蛋白表达水平均明显高于对照组和IVIG组[第28天：（58±14）比（19±11）和（25±14） μg/L,（100±41）比（35±19）和（39±19）μg/L,均P＜0.01].EMSA和明胶酶谱检测心脏组织NF-κB和MMP-9的蛋白活性水平,可见各时间点模型组均明显高于对照组和IVIG组[第28天：（84 788±2 081）比（27 220±4 990）和（50 192±1 586）μg/L,（18 560±7963）比（9 112±3 398）和（11 834±4 996） μg/L,均P＜0.05].结论 川崎病小鼠模型心脏组织的NF-κB和（或）MMP-9呈过度表达及激活状态.IVIG在川崎病小鼠模型上可有效抑制冠状动脉周围炎症细胞浸润,缓解冠状动脉损伤,其机制可能是通过抑制NF-κB和（或）MMP-9的过度表达及激活来实现的.

Objective To explore the changes of expression and biological activity of nuclear factorkappa B （NF-κB） and matrix metalloproteinase-9 （MMP-9） after using intravenous immunoglobulin （IVIG） in a murine model of Kawasaki disease （KD） and elucidate the therapeutic mechanism of IVIG for the treatment of KD.Methods A total of 72 mice were categorized randomly into IVIG,KD and control groups.Lactobacillus casei cell wall extract （LCWE） was prepared and injected intraperitoneally into C57BL/6 mice to induce KD （0.5 mg single injection）.IVIG group received an intraperitoneal injection of IVIG （2 mg/g） while KD model group had an intraperitoneal injection of normal saline.At Days 14,28 and 56,the diameter of coronary artery was by echocardiography in 8 mice of each group.At the same time,the stains of hematoxylin ＆amp; eosin and elastic fiber were used to observe the pathological damage of coronary artery.Western blot was used to evaluate the expressions of NF-κB and MMP-9,electrophoretic mobility shift assay （EMSA） was used to measure the activity of NF-κB and Gelatin zymography was used to evaluate the activity of MMP-9 in heart samples of murine model of KD.Results The local inflammatory infiltrate,composed predominantly of mononuclear lymphocytes,of coronary artery trunk and branches was observed at Days 14 and 28 while broken elastin was observed at Day 56.And the inflammatory cell infiltrate was less severe and no apparent broken elastin was observed in IVIG and control groups.On echocardiography,the average value of diameter of left coronary artery in KD model group was higher than that in IVIG and control groups （28 d：（0.48 ± 0.07） vs （0.41 ± 0.03） and （0.35 ± 0.02） mm,all P 〈 0.01）.Compared with the other two groups,the result of Western blot showed that the expressions of NF-κB and MMP-9 in KD model group were markedly higher than those in IVIG treatment group and that in control group at each time point （28 d：（58±14） vs （25±14） ＆amp; （19±11） μg/L,（100±41） vs （39±19） ＆amp; （35±19） μg/L,all P 〈0.01）.The activity of NF-κB by EMSA and the result from KD model group were much higher than those from the control and IVIG groups （28 d：（84 788 ±2 081） vs （27 220 ±4 990） ＆amp; （50 192-± 1 586）μg/L,all P 〈 0.01].And it was in accord with the expression of NF-κB.The outcome of gelatin zymography demonstrated that the activity of MMP-9 had similar change with the expression of MMP-9 （18560±7963） vs （9 112±3 398） ＆amp; （11 834±4996） μg/L,allP〈0.05）.Conclusions NF-κB/MMP-9 is overexpressed and over-activated in the heart of KD mouse models.IVIG may inhibit the inflammatory cell infiltration and alleviate coronary artery.And such a therapeutic effect is possibly achieved by a suppression of the overexpression and over-activation of NF-κB/MMP-9 pathway.