自身免疫性心肌炎心肌基质金属蛋白酶的其抑制因子表达改变的意义

Effects and significances of the balance between MMPs and TIMPs in experimental autoimmune myocarditis mice model

目的:以人工合成多肽诱导的实验性自身免疫性心肌炎(experimental autoimmune myocar-ditis,EAM)小鼠为实验模型,分析心肌内基质金属蛋白酶(matrix metalloproteinases,MMPs)及组织基质金属蛋白酶抑制因子(tissue inhibitors of matrix mentalloproteinases,TIMPs)表达变化,探讨病理性自身免疫可能的致病机制。方法:人工合成多肽诱导易感品系(BALB/C)小鼠构建EAM模型。超声评价2组小鼠血流动力学变化情况。分别以免疫组化法、逆转录多聚酶链反应(RT-PCR)法,比较2组小鼠心肌组织中MMP-3、MMP-9及TIMP-1在蛋白质水平与基因水平的表达变化。Masson三色染色法分析2组心肌胶原含量情况。结果:病理切片观察模型组小鼠心肌炎症反应明显。超声检查结果显示,模型组较对照组心功能明显恶化。免疫组化与RT-PCR结果分析显示,模型组MMP-3、MMP-9蛋白质及基因表达水平明显高于对照组,而TIMP-1的蛋白质及基因表达水平均相对下降。Masson染色切片分析显示,2组心肌胶原含量差异无统计学意义。结论:EAM模型小鼠心肌组织MMPs表达水平增高,而TIMPs表达水平下降,2者表达比例失衡。该失衡状态可能影响心肌胞外基质的正常功能,并进一步影响心功能。

Objective：Using the experimental autoimmune myocarditis BALB/C mouse model（EAM）,which was induced with peptide antigen derivatized from cadiac myosin,as the tool for investigating the change of cardiac extracellular matrix in experimental autoimmue myocarditis.To analysis the alteration of MMPs and TIMPs expression in cardiac extracellular matrix of the EAM model and the impact of this phenomenon.Meth-ods：Thirty-two eight-week-old BALB/C mice were divided into two groups.Peptide antigen was synthesized by methods of standard FMOC chemistry and used for immunization of genetically predisposed BALB/C mice to provoke autoimmune myocarditis.Cardiac systolic function indices,such as peak velocity of the aorta（Vp） and flow velocity integral of the aorta（Vi）,were determined by echocardiography.Expression of MMP-3、MMP-9 and TIMP-1 in myocardium were analyzed by semi-quantitative RT-PCR.The protein abundance of MMP-3、 MMP-9 and TIMP-1 were analyzed by immunohistochemistry.Histological cross sections of the hearts were stained with hematoxylin-eosin and masson method to evaluate myocardial histopathological scores and amount of myocardial collagen.Autoantibodies of cadiac myosin were analysised by immunohistochemistry.Results： In EAM model mice,cardiac function indices（Vp,Vi） were all significantly lower compared to the control group 〔（78.71 ±13.25） vs.（87.63 ±8.47）,（2.23 ±0.66） vs.（3.55 ±0.43）,P 0.05〕.In addition,mRNAabundance for MMP-3 and MMP-9 was upregulated（P 0.05）,whereas mRNA abundance for TIMP-1 was downregulated（P 0.01）in model mice.The protein abundance for MMP-3 was upregulated 〔（1.99 ± 0.38） vs.（0.89 ± 0.27）,P 0.01〕in model mice.The protein abundance for MMP-9 was also upregulated 〔（1.19 ± 0.42） vs.（3.55 ± 0.43）,P 0.05〕in model mice.But the protein abundance for collagen（analyzed with histological cross sections of the hearts stained with masson method）remained unaltered in model mice 〔（4.75 ± 0.72） vs.（4.33 ± 0.65）,P 0.05〕.Conclusion：Respecting the important role of MMPs/TIMPs system in metabolism of cardia extracellular matrix,the increment of MMPs in the absence of counterbalance by TIMPs in EAM models may impact the normal fuction of cardia extracellular matrix and contribute to the development of left ventricular dysfunction in EAM models.