脂质运载蛋白2促进胶原沉积和炎症反应参与川崎病导致心肌损伤的实验研究

石曌玲; 殷玥; 李晨; 薛涵; 王一石; 杨峥; 马恒; 刘曼玲

doi:10.12125/j.chj.201806036

脂质运载蛋白2促进胶原沉积和炎症反应参与川崎病导致心肌损伤的实验研究

Lipocalin 2-induced myocardial collagen fibril deposition participates in Kawasaki disease-related myocardial injury

摘要

摘要:
目的探讨脂质运载蛋白（Lcn）2在川崎病(KD)时心肌损伤的作用和相关机制。
方法将40只（1月龄）SD大鼠随机分为对照组和干酪乳杆菌细胞壁成分（LCWE）模型组。建立KD大鼠模型4周后酶联免疫吸附剂测定（ELISA）法检测KD大鼠血浆和心肌组织匀浆中Lcn 2的含量；心脏超声检测大鼠心脏收缩舒张功能；Masson染色评估心肌胶原产生的程度；荧光实时定量PCR法检测心肌组织中白介素（IL）-6、CD3、CD45和胶原蛋白（Collagen）I的mRNA表达；Western blot方法检测心肌组织中核因子（NF）-кB和其抑制蛋白IкB的磷酸化水平和Collagen I的蛋白表达变化。采用IкB的抑制剂BAY 11-7082（2.5 μmol/L）处理可有效抑制Collagen I的表达。
结果 LCWE注射28 d后，成功诱导KD大鼠冠状动脉损伤（P<0.05）。在此基础上发现，KD大鼠心脏左室舒张末期内径（LVIDd）增加，左室射血分数（LVEF）降低（P<0.05）。Masson染色显示KD大鼠心肌出现一定程度心肌纤维化和胶原沉积。心肌中的IL-6、CD3和CD45等炎症相关分子的mRNA表达水平显著升高（P<0.05）。KD大鼠心脏组织和循环中Lcn 2水平升高（P<0.05）。给予心肌成纤维细胞Lcn 2（10 ng/ml）处理48 h，心肌成纤维细胞Collagen I 的mRNA和蛋白表达水平均升高（P<0.05）。并且Lcn 2处理可显著上调心肌成纤维细胞NF-кB和IкB的磷酸化水平。采用IкB的抑制剂BAY 11-7082（2.5 μmol/L）处理可有效抑制Lcn 2对Collagen I蛋白的上调作用。
结论 KD状态下Lcn 2显著升高，Lcn 2通过激活NF-кB信号途径诱发心肌成纤维细胞胶原合成增加和产生炎症反应，进而造成心肌损伤。

Abstract:
AIM To investigate whether lipocalin 2-induced myocardial collagen fibril deposition and inflammatory reactions participated in Kawasaki disease-related myocardial injury using the rat model of Kawasaki disease established by lactobacillus casei cell wall extract (LCWE).
METHODS Forty rats (1 month old) were randomly divided into control group and LCWE model group. In LCWE model group, LCWE was injected intraperitoneally to established Kawasaki disease rat model. ELISA was used to detect the plasma Lcn 2 levels in plasma and cardiac tissue homogenates of Kawasaki disease rats, cardiac ultrasound was used to detect systolic and diastolic functions in rats and Masson staining was used to evaluate the degree of myocardial fibrosis. The expressions of IL-6, CD3, CD45 and Collagen I mRNA in myocardial tissue were detected by real-time fluorescence quantitative PCR and the expressions of Collagen I P, NF-кB, and IкB were detected by Western blotting.
RESULTS Echocardiography confirmed that the left ventricular end-diastolic dimension (LVIDd) and left ventricular ejection fraction (LVEF) were decreased in Kawasaki disease rats compared with those in control group (P<0.05). Masson staining showed a certain degree of myocardial fibrosis and collagen deposition in Kawasaki disease rats. The mRNA expression levels of inflammation-related molecules, such as IL-6, CD3 and CD45, in the myocardium were significantly elevated (P<0.05). The cardiac tissue and circulation levels of Lcn 2 in Kawasaki disease rats were significantly higher than those in control group (P<0.05). More importantly, the cultured cardiac fibroblasts were treated with Lcn 2 (10 ng/ml) for 48 hours and then the mRNA and protein expression levels of Collagen I of the cultured cardiac fibroblasts were significantly increased (P<0.05) and the phosphorylation of NF-кB and IκB in cardiac fibroblasts was significantly up-regulated (P<0.05). Treatment with IκB inhibitor BAY 11-7082 (2.5 μmol/L) effectively inhibited Lcn 2 up-regulation of Collagen I protein.
CONCLUSION Lcn 2-induces myocardial collagen fibril deposition and inflammatory reactions by activating the NF-кB signal participates in Kawasaki disease-related myocardial injury.

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