【作者】 李香；

【导师】 易凡；

【作者基本信息】 山东大学 ， 医学基础药理学， 2014， 博士

【摘要】 研究目的心室重构是指心室受到损伤时发生的结构和功能的改变,出现心肌细胞肥大、死亡、间质纤维化、心肌肥厚、心肌细胞膜离子通道、交感神经支配的变化等。许多心血管疾病如高血压、心肌梗死、心力衰竭、扩张型心肌病、心脏瓣膜病等都存在心室重构现象,另外在高同型半胱氨酸血症、动脉粥样硬化、糖尿病等其他疾病并发症中也存在心室重构现象。心室重构是多种因素共同作用所产生的形态学、分子生物学、病理生理学、电生理环境的变化,其机制主要包括炎症反应、血流动力学负荷、细胞因子生成和蛋白酶诱导的细胞外基质(extracellular matrix, ECM)降解。NOD样受体(NOD-like receptor, NLRs)是一类新型胞浆内的固有免疫模式识别受体,识别细胞内病原体相关分子模式(pathogen-associated molecular patterns, PAMPs)或内源性危险信号相关分子(dangers-associated molecular patterns, DAMPs),从而启动免疫应答,在自身免疫调节中起重要作用。到目前为止,共发现了22个NLR家族成员,其中最具代表性的是NOD1(nucleotide-binding oligomerization domain containing1)和NOD2(nucleotide-binding oligomerization domain containing2).有文献报道,NOD1激动剂iEDAP注射小鼠心脏,激活NF-κB (nuclear transcription factor κB)和TGF-β (transforming growth factor β)信号通路,诱导心肌细胞坏死和凋亡进而导致心脏功能异常。但有关NOD2在心脏中表达及在心室重构中的作用,迄今尚未见报道。为此,本课题选用高同型半胱氨酸血症(hyperhomocysteinemia, hHcys)及心肌梗死(myocardial Infarction, MI)两种不同程度的心室重构模型,探讨NOD2是否参与心室重构过程,并就NOD2调节心室重构过程的可能机制进行深入研究。研究方法第一部分：通过无叶酸饮食建立WT和CBS+/-小鼠高同型半胱氨酸血症模型,利用小动物超声心动图检测小鼠左心室内径及心功能,Western Blot、RT-PCR和免疫组化等方法检测NOD2在心肌组织中的表达情况。体外实验用不同浓度的L-Hcy刺激大鼠心肌细胞H9C2和平滑肌细胞(smooth muscle cell,SMC),Western Blot方法检测NOD2的蛋白表达水平。为探讨NOD2在高同型半胱氨酸血症心室重构中的机制,我们采用NOD2敲基因鼠建立高同型半胱氨酸血症模型,进一步利用小动物超声心动图检测小鼠左心室内径及心功能指标,观察NOD2对hHcys引起心室重构的影响；Masson染色观察小鼠纤维化程度；通过Western Blot方法检测心室重构标志物基质金属蛋白酶2(matrix metalloproteinase2,MMP-2)、基质金属蛋白酶9(matrix metalloproteinase9,MMP-9)及基质金属蛋白酶抑制剂1(tissue inhibitor of metalloproteinase1,TIMP-1)等指标的表达变化。第二部分：通过永久性结扎冠状动脉左前降支建立小鼠心肌梗死模型。在心梗后3、7、14和28天分别收集梗死区和非梗死区的心肌组织,用实时荧光定量PCR检测不同梗死时间后NOD2的mRNA表达水平,通过免疫组化染色和、Western Blot分析心梗NOD2的蛋白表达水平。为进一步明确NOD2在心梗左室重构中的作用,采用NOD2敲基因小鼠建立心肌梗死模型,小动物超声心动图检测心肌梗死的小鼠左心室内径及心功能。通过Masson和Tunel染色观察野生和NOD2-/-小鼠心梗后纤维化程度及细胞调亡情况,酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定组织的促炎细胞因子水平,心肌组织裂解液运用Western Blot和明胶酶谱法检测MMP9、MMP2蛋白及活性变化,并用免疫组化染色观察心梗后炎性细胞浸润和心脏成纤维细胞(cardiac fibroblast,CF)转分化成心肌成纤维细胞情况。体外实验采用小鼠原代成纤维细胞探讨NOD2参与心室重构的分子机制,运用Western Blot等方法测定胞壁酰二肽(Muramyl Dipeptide,MDP)刺激成纤维细胞所产生的p38、细胞外信号调节激酶(Extracellular regulated proteinkinases1/2,ERK1/2)磷酸化水平及成纤维细胞缺氧4h复氧不同时间后NOD2和MMP9蛋白表达水平；ELISA检测MDP刺激成纤维细胞及成纤维细胞转染后缺氧4h复氧24h产生的炎性因子变化水平。结果第一部分：①NOD2随着血浆Hcy浓度的升高蛋白表达增加,在CBS+/-模型组中表达最强,其次是WT模型组。免疫组化和PCR结果同样证实,NOD2在CBS+/-模型组中的表达最强。②利用不同浓度的L-Hcy刺激大鼠心肌细胞H9C2,发现NOD2的表达成浓度依赖性上调,其中,L-Hcy浓度在25μmol/L时,NOD2表达最强；不同浓度的L-Hcy刺激SMC, NOD2的表达同样显著上调。③心室重构标志物MMP9在WT模型中表达显著升高,NOD2敲除后MMP9表达降低；COL1在WT模型中表达降低,而NOD2敲除后COL1表达升高。虽然NOD2能影响MMP9、 C0L1的蛋白表达,但是NOD2缺失未能减轻hHcys引起的心室重构。相比于WT对照组,WT模型组的左室舒张末期内径(LVIDd),左室收缩末期内径(LVIDs)增大,说明左室肥大；左室舒张末期后壁厚度(LVPWd)和左室收缩末期后壁厚度(LVPWs)降低则表明左室壁变薄。而NOD2敲基因鼠高同型半胱氨酸血症后的左室肥大和室壁变薄现象并没有改变,说明NOD2基因敲出后未能减轻hHcys引起的心室重构。第二部分：①首次证实,NOD2在心梗组织中表达升高。与假手术组相比,MI组非梗死区和梗死区NOD2mRNA表达水平明显升高,其中梗死区NOD2的mRNA表达变化水平最高,而且呈时间依赖性变化；Western Blot和免疫组化结果同样证实了心梗后NOD2表达升高。②NOD2缺失能减轻心梗后心肌功能失常和心肌重构的发生。通过超声心动图发现,NOD2-/-,心梗组心功能损伤明显改善；WT心梗后变化的左室直径和室壁厚度在NOD2"/-敲基因鼠模型中也有所减轻,心肌重构减弱；TUNEL和Masson染色显示在NOD2-/-心梗组,细胞凋亡和心肌纤维化程度明显降低。③我们进一步发现,NOD2缺失能降低心梗区炎性因子水平,炎性细胞的浸润及MMP-9的活性。心梗发生后,TGF-β、白介素1β(interleukin,IL-1p)、肿瘤坏死因子α(Tumor Necrosis Factor,TNF-α)、单核细胞趋化蛋白1(monocyte chemoattractant protein, MCP-1)等炎性因子水平显著升高,而NOD2缺失能降低这些炎性因子的水平。小鼠心肌梗死后MMP9的蛋白和活性都升高, NOD2缺失后减弱了MI导致的MMP9蛋白和活性水平的提高。炎性细胞浸润在NOD2-/-心梗组中表达也显著降低。④MDP诱导心肌成纤维细胞有丝分裂原激活蛋白激酶(Mitogenaetivated proteinkinase, MAPK)信号通路的激活和促进促炎介质的产生。我们利用NOD2激活剂MDP刺激心肌成纤维细胞,发现P38、ERK-1/2等激酶的磷酸化水平增加；IL-1β、TNF-α、TGF-β、MCP-1、IL-8、细胞间黏附因子1(Intercellular adhesion molecule,ICAM-1)等促炎介质表达水平显著升高。⑤NOD2沉默减弱缺氧诱导的MMP9表达。在原代培养的心肌成纤维细胞中,缺氧／复氧显著增加NOD2和MMP9的表达,NOD2基因沉默后,缺氧诱导的MMP9表达和促炎介质的表达受到抑制。结论1.本文首次探讨了NOD2在高同型半胱氨酸血症(hHcys)导致心室重构中的作用,发现NOD2在CBS+/-造模的hHcys小鼠心肌组织中蛋白及mRNA水平显著升高；利用NOD2敲基因鼠进一步研究发现, NOD2缺失虽能降低心室重构标志物MMP9的表达,但对高同型半胱氨酸血症诱导的心室重构没有改善作用。分析原因一是高同型半胱氨酸血症对心脏的损伤较小,只影响左室舒张内径和左室后壁厚度而对心脏收缩及舒张功能没有影响,因此NOD2缺失后的改善作用未能体现出来。二是NOD2敲除后可能衍生出别的代偿机制从而影响高同型半胱氨酸血症引起的心脏损伤,具体机制需进一步明确。2.建立冠状动脉左前降支结扎心肌梗死模型,研究NOD2是否参与心肌梗死导致的心室重构过程。我们证实,在心肌梗死区和非梗死区NOD2mRNA表达水平明显升高,其中梗死区NOD2的mRNA表达变化水平最高。利用NOD2敲基因鼠进一步研究发现,NOD2缺失能减轻心梗后心功能障碍和心肌重构的发生,细胞凋亡和心肌纤维化程度也明显降低。研究表明,NOD2通过调控MMP9蛋白及活性、炎性介质水平和炎性细胞浸润来介导心肌梗死后左室重构过程。体外采用小鼠心脏原代成纤维细胞,研究表明MDP诱导激活了心肌成纤维细胞MAPK通路,p38,ERK1/2磷酸化水平升高,并且释放大量炎性介质；心肌成纤维细胞缺氧再灌注NOD2和MMP-9水平明显升高,而NOD2基因沉默后,缺氧诱导的MMP9蛋白表达及炎性介质水平明显降低。更多还原

【Abstract】 ObjectiveVentricular remodeling is the changes of structure and function in the incidence of heart damage, such as myocardial necrosis and apoptosis, cardiac hypertrophy, interstitial fibrosis, change in cardiac ion channels and sympathetic innervations. Many cardiovascular diseases such as hypertension, myocardial infarction, heart failure, dilated cardiomyopathy, valvular heart disease have the phenomenon of ventricular remodeling. In addition, hyperhomocysteinemia, atherosclerosis and diabetic complications also have the phenomenon of ventricular remodeling. Ventricular remodeling is the combined effects produced by a variety of morphological, molecular biology, pathophysiology changes, electrophysiological environment, its mechanisms including inflammation, hemodynamic load, neurohormonal activation, cytokine production and protease-induced extracellular matrix degradation.NOD-like receptors (NOD-like receptor, NLRs) are innate immune pattern recognition receptors which recognise intracellular pathogen-associated molecular patterns or endogenous danger signals related molecules, thereby initiating an immune response.NLRs plays an important role in the regulation of immune response. So far,22NLR family members are currently discovered, the most representative NLRs are NOD1and NOD2. It has been reported that activation of NOD1with the specific agonist iEDAP induces a time-and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in the heart. But so far, the expression of NOD2and the role of NOD2involved in ventricular remodeling in the heart keeps unknown. Therefore, in the present study we selected two different exent ventricular remodeling model--yperhomocysteinemia and myocardial infarction to investigate whether NOD2is involved in ventricular remodeling and to explore the possible mechanisms by which NOD2regulates the ventricular remodeling.MethodsSection one:Hyperhomocysteinemia model was established by WT and CBS+/" mouse non-folate diet, left ventricular internal diameter and cardiac function was acquired by echocardiography. Western Blot, RT-PCR and immunohistochemistry was done to evaluate the expression of NOD2in the myocardium. To investigate the mechanisms of cardiac dysfunction and ventricular remodeling caused by hHcys, we used NOD2gene knockout mouse to establish hyperhomocysteinemia model. Left ventricular internal diameter and cardiac function acquired by echocardiography was to observe whether there was a little amelioration in ventricuar remodeling in NOD2knockout mouse; Masson staining was to observe the degreement of fibrosis in mice; the protein level of matrix metalloproteinase MMP2, MMP9and matrix metalloproteinase inhibitor TIMP-1was detected by Western Blot.Section two:Mouse MI model was induced by permanent left anterior descending coronary artery ligation. Sham operated ones underwent the same procedure but ligation. Both infarct and remote myocardium from sham and post-MI3,7,14and28days were homogenized to evaluate mRNA expression of NOD2by quantitative real-time polymerase chain reaction(qPCR), and detect its protein level by Western Blot and immunohistochemistry post-MI28days. To further investigate the role of NOD2in post-MI LV remodeling process, NOD2gene knockout mouse were used to establish myocardial infarction model. Left ventricular internal diameter and cardiac function post-MI28days were acquired by echocardiography; Masson staining and Tunel staining were used to observe the degreement of fibrosis and apoptosis; the enzyme-linked immunosorbent assay (ELISA) measured tissue proinflammatory factor levels; the protein level and activity changes of matrix metalloproteinase MMP2, MMP9of cardiac tissue lysates were detected by Western Blot and gelatin zymography. The inflammatory cells infiltration after myocardial infarction and cardiac fibroblasts (cardiac fibroblast) trans-differentiation into cardiomyocytes fibroblasts were observed by immunohistochemical. In vitro, use mouse fibroblasts to investigate the molecular mechanisms of ventricular remodeling which NOD2was involved in.The phosphorylation levels of ERK1/2and p38in fibroblasts stimulated with MDP were detected by Western Blot which was also used to acquire NOD2and MMP9protein levels in cardiac fibroblasts under hypoxia/reoxygenation conditon; RT-PCR detected the transfection efficiency of NOD2; ELISA determined relative levels of proinflammtory mediators in cardiac fibroblasts with shRNA-NOD2transfection. ResultsSection one:First of all, NOD2expression was improved with increasing concentrations of Hcy. The level of NOD2in CBS+-/-model group was highest, and then WT model group. Immunohistochemistry and PCR results also confirmed that NOD2expression in CBS+-/-model group was the strongest.②NOD2expression was significantly increased in H9C2stimulated with different concentrations of L-Hcy. NOD2expression was the strongest when L-Hcy concentration was25μmol/L. The same result was found in SMC.③The expression of MMP9which was a ventricular remodeling marker was significantly increased in WT model group, while reduced in NOD2knockout model group; the expression of COL1was significantly decreased in WT model group, while increased in NOD2knockout model group. Although NOD2can affect the expression of MMP9, COL1, but failed to change hHcys-induced ventricular remodeling. Compared to WT controls group, LVIDd LVIDs increased in WT model group indicating left ventricular hypertrophy; LVPWd and LVPWs decreased indicating left ventricular wall thinning. NOD2gene knockout failed to alleviate the left ventricular hypertrophy and ventricular wall thinning.Section two:①NOD2expression was significantly increased in the cardiac infarcted area in post-MI mice. NOD2mRNA levels were significantly increased in the tissue from the cardiac infracted area in post-MI mice at different time points. The enhanced NOD2protein levels were further confirmed by Western blot and immunochemical.②NOD2deficiency exhibited improvements in cardiac dysfunction and remodeling after MI. Cardiac functions were examined by echocardiography at28days after MI.MI significantly decreased cardiac function in WT mice as evidenced by decreases in EF%, FS%, when compared with WT basal controls, which can be recovered in NOD2-/-mice. In NOD2-/-mice, the MI-induced LV enlargement and wall thinning were significantly improved. Furthermore, TUNEL staining and Masson trichrome staining indicated that NOD2deficiency protected against ischemia-induced cell death and cardiac fibrosis.③NOD2deficiency reduced the levels of proinflammatory mediators, inflammatory cell infiltration and MMP-9activity after MI. NOD2deficiency significantly reduced the levels of proinflammatory cytokines and chemokines including IL-1β, TGF-P, TNF-a and MCP-1in the cardiac infarcted area in mice after MI. Moreover, elevated MMP-9levels and activity in the cardiac tissues were blocked by NOD2deficiency after MI.The infiltration of inflammation cells and the expression of a-SMA was significantly reduced in NOD2-/-mice.④MDP induced activation of MAPK signaling pathways, production of proinflammatory mediators in primary cultured cardiac fibroblasts. MDP induced the activation of MAPKs as assessed by measuring the levels of phospho-specific ERK-1/2, p38MAPK. Also MDP enhanced the production of proinflammatory mediators in primary cultured cardiac fibroblasts.⑤Gene silencing of NOD2attenuated hypoxia-induced MMP-9expression.In primary cultured cardiac fibroblasts, hypoxia/reoxygenation significantly enhanced NOD2and MMP9enpression while hypoxia-induced MMP-9expression was markedly blocked by shRNA-NOD2. Additionly, hypoxia-induced the production of proinflammatory mediators was attenuated by gene silencing of NOD2.Conclusion1. We investigated for the first time the role of NOD2in ventricular remodeling caused by hHcys. In this study, we found that the mRNA and protein levels of NOD2significantly increasedin myocardial tissue of CBS+-/-hyperhomocysteinemia mouse. Further study observed that although NOD2deficiency reduced the levels of MMP expression which is a ventricular remodeling marker, but it had no improvement for hyperhomocysteinemia-induced ventricular remodeling.The possible reasons were that hyperhomocysteinemia induced less damage to heart, only had the effects on the left ventricular diastolic inner diameter and left ventricular posterior wall thickness and has no effect on systolic and diastolic function, thus the improving role of NOD2failed to be reflected. Secondly some other compensatory mechanisms may be involved in the development of hyperhomocysteinemia.2. We further explored whether NOD2participated in ventricular remodeling caused by mycardial infarction with the model of left anterior descending coronary artery ligation. We confirmed for the first time that mRNA levels of NOD2were significantly higher in the infarcted area and non-infarcted area, expecially in the infarcted area. Further study found that NOD2deficiency attenuated cardiac dysfunction and remodeling after myocardial infarction and protectd against ischemia-induced cell death, also cardiac fibrosis was significantly reduced in NOD2-/-mice. Studies had shown that NOD2mediated left ventricular remodeling after myocardial infarction by regulating the levels of MMP9protein and activity, inflammatory mediators production and inflammatory cell infiltration. In vitro studies had shown that NOD2agonist MDP induced the activation of MAPKs in cardiac fibroblasts as assessed by measuring the levels of phospho-specific ERK1/2p38MAPK. We also observed that MDP enhanced the production of proinflammatory mediators and hypoxia/reoxygenation significantly enhanced NOD2and MMP-9expression in primary cultured cardiac fibroblasts. After silencing NOD2gene, hypoxia-induced MMP9expression was markedly blocked and hypoxia-induced the production of proinflammatory mediators was attenuatd.更多还原