摘要
背景
心肌炎广义是指心脏的炎症状态,是心脏病学最具代表性和挑战性的临床课题之一。其发病机制复杂,临床上可有轻度呼吸困难、急性心衰,甚至猝死等多种临床表现形式。年青人发病率较高,其中包括无症状患者,占40岁以下猝死病例的12%。虽然大多数心肌炎患者能够恢复健康,但是部分遗传易感性患者可逐步进展为慢性心肌炎和扩张性心肌病(DCM),并伴有充血性心力衰竭。虽然心肌炎定义看似简单,但是其诊断和治疗仍然是临床面临的重大课题。
构建一种好的心肌炎实验动物模型是进一步研究其分子和免疫学发病机制的关键。用心肌肌球蛋白或心肌α-肌球蛋白重链的肽段或用心肌肌钙蛋白Ⅰ与完全弗氏佐剂乳化后免疫遗传易感性小鼠,构建实验性自身免疫性心肌炎(EAM)动物模型。该模型能够很好的模拟人类急性发病期爆发性心肌炎和慢性发病期扩张性心肌病心脏病理损害,并为研究心脏特异性免疫反应和心肌炎症损害提供了极好的实验动物模型。这些实验模型能够为研究新的诊断方法和治疗策略提供帮助。
目的
本研究中,选用BALB/c小鼠构建实验性自身免疫性心肌炎动物模型,
方法
六周龄雌性BALB/c小鼠(野生型；体重13-18g,购自山东大学实验动物中心)饲养在专门的无病原微生物的实验动物中心。所有实验动物操作流程均获得实验动物保护权威机构的批准,并遵循山东大学医学院动物实验指导准则。
所有BALB/c小鼠被随机分成4组,其中包括C组：正常对照组；L组：低剂量短肽组；M组：中剂量短肽组；H组：高剂量短肽组。后3组小鼠共45只用特异的短肽免疫2次构建实验性自身免疫性心肌炎动物模型,特异的短肽来自小鼠心肌α-肌球蛋白重链(MyHc-a614-629[Ac-S LKLM ATLFSTYAS AD-OH])。该短肽具有抗原性(纯度≥98.75%,上海吉尔生物化学有限公司),先将其溶解在生理盐水中,再与等体积完全弗氏佐剂(美国Sigma公司)充分乳化。每只小鼠分别在试验第0天和第7天背部皮下注射上述含有短肽的乳化剂共2次。首次免疫后的第16天、21天和26天做心脏超声检察,心肌组织切片行苏木素-伊红染色及组织病理学评分；63天行Masson三原色染色,对间质纤维化进行评估。
结果
在实验性自身免疫性心肌炎动物模型中,用小鼠心肌α-肌球蛋白重链来源的短肽和完全弗氏佐剂的乳化液免疫BALB/c小鼠诱导的心肌炎,其炎症高峰期是在首次免疫后的第21天。第63天心肌组织中的炎症基本消退,但是病理性重塑仍持续存在。
结论
尽管实验行自身免疫性心肌炎动物模型是人为构建,但是该模型为在活体内且无外源病原微生物感染的情况下,研究心肌炎发病机制和自身免疫损伤机制提供了很大的便利条件。通过研究实验性自身免疫性心肌炎动物模型，我们不仅可以了解自身免疫机制在心肌炎发病发展中所起到的作用,同时也为研究炎症性心脏病的病理生理改变和开展新的免疫调节治疗提供了可能性。另外,实验行自身免疫性心肌炎也为提高炎症性心肌病当前及未来影像学检查诊断的准确性提供了潜在的研究工具。
背景
心肌炎是心肌炎症病变并伴随后续的心肌组织损害性疾病,它可导致心源性猝死。其中大约10-20%有心肌炎组织病理学证据,临床表现无特异性,部分患者甚至没有临床症状,由于病情迁延不愈最终转化为扩张型心肌病(dilated cardiomyopathy, DCM)。心肌炎病因复杂,但在多数情况下是与肠道病毒感染有关,常见有柯萨奇病毒B3、腺病毒与细小病毒B19。用心肌肌球蛋白皮下注射免疫易感啮齿类动物,构建实验性自身免疫性心肌炎动物模型(experimental autoimmune myocarditis, EAM),该模型是人类心肌炎和扩张型心肌病实验动物模型。在病毒诱导的小鼠心肌炎模型中,心肌肌球蛋白是小鼠体内主要的自身抗原。实验表明,用纯化的小鼠心肌肌球蛋白免疫诱导易感小鼠心肌炎发病(在没有合并肠道病毒感染的情况F),与用肠道病毒感染诱导的心肌炎心脏组织病理损害相似。由BALB/c小鼠构建的实验性自身免疫性心肌炎模型为探讨心肌炎发病机制和研发有效的治疗药物提供了良好的实验平台。他汀是一类羟甲基戊二酸单酰辅酶A (HMG-CoA)还原酶抑制剂药物,不仅具有降脂作用,而且还有多功能免疫调节作用,通过抑制可诱导的MHC二类分子和抗原递呈细胞共刺激分子的表达,以及促进辅助性T淋巴细胞由Th1向Th2分化,可有效治疗T淋巴细胞介导的炎症性疾病。他汀类药物也能保护内皮细胞功能和微血管系统的完整,增加一氧化氮生物利用度和抗氧化作用。基于他汀类药物诸多的有益作用,该实验选用BABL/c小鼠构建实验性自身免疫性心肌炎模型,来研究瑞舒伐他汀对心肌炎症反应程度、心肌细胞凋亡及心功能等方面的影响作用。
目的
1.研究瑞舒伐他汀对急性期心肌炎的影响作用。
2.研究瑞舒伐他汀对心肌炎小鼠心肌细胞凋亡的影响作用。
3.研究瑞舒伐他汀对心肌炎小鼠心功能的影响。
方法
1.实验性自身免疫性心肌炎动物模型和药物治疗
雌性六周龄BABL/c小鼠饲养在特定无病原微生物的实验动物中心内,所有小鼠被随机分成4组,分别是正常对照组(group C),低剂量瑞舒伐他汀组(groupL),高剂量瑞舒伐他汀组(group H)和非药物治疗的EAM组(group N)。其中group L, group H, group N三组小鼠分别在试验开始的首日和第七天,给予背部皮下注射200μg/200μl心肌肌球蛋白短肽,构建实验性自身免疫性心肌炎模型。在小鼠首次免疫当天,两个药物治疗组group L和group H开始接受瑞舒伐他汀药物治疗。Group N组小鼠仅接受生理盐水灌胃，替代瑞舒伐他汀药物灌胃治疗。首次免疫后的第21天,进行心肌炎急性期实验分析研究。
2.超声心动图
测量完每只小鼠体重后,再将这些小鼠先麻醉,后经胸做超声心动图检测,分别测量收缩末期和舒张末期左室内径,通过仪器计算自动获取左室内射血分数和左室短袖缩短率。
3.组织学检查和心肌炎严重程度评估
做完上述检查后,安乐死所有实验小鼠,测量小鼠心脏重量并计算心脏重量/体重(HW/BW)比值,将心脏标本浸泡在福尔马林中24小时,后经石蜡包理、切片,行苏木素和伊红染色,镜检发现有单核细胞浸润和心肌细胞坏死区域为心肌炎病变区，用半定量分析法评价心肌炎症区域占整个心脏的百分比。
4.酶联免疫吸附试验检测炎症细胞因子
酶联免疫吸附试验试剂盒检测各组小鼠血清中的白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平。
5.血清脂质水平
检测21天血清中总胆固醇(TC)、甘油三酯(TGs)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)浓度。
6. TUNEL检测
TUNEL检测心肌组织中的凋亡细胞。
7.免疫组化染色
免疫组化检测活化的caspase3的表达。
8. Western blotting实验分析
Western blotting检测活性的caspase8and caspase9的表达水平。
结果
1.瑞舒伐他汀对心脏重量/体重比值和血清脂质水平的影响
与正常对照组相比,在group N组心脏重量/体重比值明显增加；而在group L、group H组该比值明显下降,说明瑞舒伐他汀治疗能够明显抑制心脏重量的相对增加,并且治疗效果呈剂量依赖性增强。21天各实验组血清脂质水平无统计学差异。
2.瑞舒伐他汀改善心功能情况
首次免疫后第21天,心脏超声图像分析显示,心肌炎小鼠心功能不全,经瑞舒伐他汀治疗可延缓心衰进程。
3.瑞舒伐他汀对炎症范围的影响和组织病理学评分
首次免疫后第21天,实验结果显示,瑞舒伐他汀能抑制炎症细胞浸润,减轻炎症反应程度和范围,瑞舒伐他汀治疗效果呈剂量依赖性加强。
4.抑制炎症因子的表达
用ELISA试剂盒检测4组血清细胞因子,结果显示瑞舒伐他汀能抑制心肌炎小鼠血清中炎性细胞因子的表达,且该作用呈剂量依赖性增强。
5.心肌细胞凋亡和心肌活性caspase3表达
用TUNEL试剂盒检测凋亡细胞的数量；免疫组化染色显示,含有活性的caspase3蛋白表达的阳性细胞分布在炎症区域,另外,许多淋巴细胞核内也有活性的caspase3蛋白的表达。两种染色方法均显示,相对group C, group N、groupL和group H三组检测到的凋亡细胞数量依次递减,group C组未见到两种染色方法的阳性细胞。该结果显示瑞舒伐他汀能抑制细胞凋亡。
6.瑞舒伐他汀对细胞内caspase表达的影响作用
在心肌炎症反应高峰期也即首次免疫后第21天,检测具有活性的caspase8和caspase9蛋白表达情况。结果发现相对治疗组和正常对照组,上述两种活性蛋白分子在非治疗组小鼠的心肌组织中高表达,瑞舒伐他汀能呈现剂量依赖性下调这两种活性蛋白分子的表达水平。
结论
本研究首次表明,瑞舒伐他汀能够通过抑制心肌炎性细胞的浸润及促炎细胞因子的释放、减少心肌细胞的凋亡来显著干预实验性自身免疫性心肌炎的进程,改善左心室功能,减缓左心室重塑。
背景
心肌炎是指心肌炎症性病变及伴随而来的心肌组织损伤,其病理损害表现为炎症细胞浸润、心肌细胞凋亡、坏死和心肌组织间质纤维化。心肌炎后期常常进展为扩张型心肌病(DCM),临床症状以心衰为主。多临床观察和动物实验结果均显示自身免疫反应在心肌炎的发病中起着重要的作用。
实验性自身免疫性心肌炎是感染后心肌炎实验动物模型,该模型是通过给遗传易感性小鼠免疫心肌肌球蛋白或者心肌α肌球蛋白重链来源的短肽或者心肌肌钙蛋白诱发免疫性心肌炎发病而构建的一类实验动物模型。该动物模型已被证实其慢性期心肌病理损害和扩张性心肌病临床病理损害相似,也表现为心脏扩大、心室腔扩张、弥漫性心肌纤维化、心肌细胞肥厚和萎缩性表现,类似于人类心肌病临床表现和病理损害。我们选用易感的BALB/c小鼠构建实验性自身免疫性心肌炎动物模型，来研究由免疫诱导的心肌炎其慢性期的发病机制。
3-羟基-3甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,通常称为“他汀类药物”,是一类众所周知的强效调脂药物。除了降脂作用外,他汀类药物目前还被证实具有多种心血管保护作用,其中包括疫调节,抗炎、抗氧化、保护内皮细胞、延缓细胞衰老、改善心肌重塑。基于上述他汀类药物对心血管的有益作用，本研究旨在探讨瑞舒伐他汀是否改善由BALB/c小鼠构建的实验性自身免疫性心肌炎慢性期病理损害和心肌重塑,进而阐明该药物的作用机理。
目的
1.评价瑞舒伐他汀对慢性期实验性自身免疫性心肌炎的影响作用。
2.探讨瑞舒伐他汀对心肌炎心功能的影响作用。
3.探讨瑞舒伐他汀对心肌重塑,尤其是心肌细胞肥大、间质纤维化发挥治疗作用的分子机制。
方法
1.实验性自身免疫性心肌炎动物模型
用心肌肌球蛋白α重链上具有抗原活性的短肽(MyHc-α614-629[Ac-SLKLMATLFSTYASAD-OH])免疫六周龄BALB/c小鼠构建实验性自身免疫性心肌炎动物模型(具体步骤同第一部分),将构建好的动物模型分组：非药物治疗组(group N);低剂量瑞舒伐他汀药物治疗组(group L);高剂量瑞舒伐他汀药物治疗组(group H);剩余的BALB/c小鼠(既没有接受免疫诱导构建心肌炎模型,也没用药物治疗)被划分到正常对照组中(group C)。
2.瑞舒伐他汀药物治疗
瑞舒伐他汀药物治疗与首次免疫诱导心肌炎同步进行,也即从实验开始的当天到实验结束的第63天,前后共9周时间,通过鼻饲管给小鼠灌胃注入瑞舒伐他汀,非药物治疗组和正常对照组小鼠予生理盐水代替药物灌胃。
3.心脏超声检查
各组小鼠是在首次免疫后的第21天和第63天分别进行心脏超生检查,用二维M超获取各组小鼠心脏超声图像和测量数据,胸骨旁长轴和短轴图像分别在二尖瓣和左室乳突肌水平获取。测量左心室舒张末期内径、左心室收缩末期内径、舒张末期室间隔厚度和舒张末期左室后壁厚度,每个测量数值是通过测量3个心动周期取平均值而获得。射血分数和短轴缩短率通过计算获得。
4.心肌炎组织学评价
心肌炎病理损害的严重性是在首次免疫后的第21天进行评估,用苏木素和伊红复染心脏组织切片,用半定量图像分析法评估心肌炎症损害程度。
5.酶联免疫吸附试验测量炎症细胞因子的表达水平
首次免疫后的第21天和第63天,用IL-6和TNF-α酶联免疫吸附试验试剂盒测量实验小鼠血清中的IL-6和TNF-α浓度。
6.心肌细胞肥厚和心肌组织纤维化的组织学评估
在试验第63天,也即实验性自身免疫性心肌炎慢性期,将小鼠安乐死,制备心脏标本,取5μm厚的心肌组织切片脱蜡进行组织学分析,HE染色评估心肌细胞肥厚，Masson三原色染色评估纤维化程度，天狼星红染色评估胶原沉积。
7. Western blotting分析
在心肌炎症高峰期的第21天，采用Western blotting分析法检测pERK1/2. tERK1/2、pJNK、tJNK、p-p38及t-p38;在心肌炎症慢性期的第63天用同样方法检测TGF-β1及pSmad2/3各蛋自表达水平的变化情况。
结果
1．瑞舒伐他汀对心肌结构和功能的影响
心脏超声显示，心肌炎后左心室功能紊乱并伴有心肌重构，相对于低剂量药物治疗组，高剂量瑞舒伐他汀药物治疗可明显改善实验性自身免疫性心肌炎小鼠左心室重构并减缓心衰的进程，而且连续治疗63天能进一步改善心肌炎小鼠的心功能。
2.瑞舒伐他汀改善小鼠实验性自身免疫性心肌炎的进程
在group N组中，能够观察到严重的心肌炎症损害。而在group L和group H组中，心肌炎症细胞浸润明显减少，组织病理学评分进一步证实基于瑞舒伐他汀药物治疗能抑制心肌炎症反应，而且这种抑制作用呈剂量依赖性增强。
3．瑞舒伐他汀对血清中炎症细胞因子的影响用作
ELISA方法检测四组小鼠血清中细胞因子的表达水平，两组瑞舒伐他汀药物治疗组(group L、group H)小鼠血清中IL-6和INF-α较group N[明显下降，而且这种抑制细胞因子产生的作用随瑞舒伐他汀药物剂量的增加而增加。
4.瑞舒伐他汀对心肌重塑的影响
显著的间质纤维化及胶原沉积在实验性自身免疫性心肌炎各实验组均能观察到，然而瑞舒伐他汀药物治疗组较group N组能明显减少心肌间质纤维化及胶原沉积的程度，且药物剂量越大上述作用越强。
5.瑞舒伐他汀对细胞内信号激酶和分子的影响作用
瑞舒伐他汀能呈剂量依赖性减少心脏p-ERK1/2、p-JNK、p-p38、TGF-β1和p-Smad2/3的表达，该结果显示瑞舒伐他汀能抑制实验性自身免疫性心肌炎小鼠心肌组织丝裂原激活蛋白激酶和TGF-β1-Smad2/3信号通路的激活。
结论
该研究首次证实瑞舒伐他汀能改善实验性自身免疫性心肌炎心肌重塑，主要表现在抑制心肌细胞肥厚和间质纤维化，进而改善左心室功能，这些有益的作用部分与下调丝裂原激活蛋白激酶和TGF-β1-Smad2/3信号通路的表达相关。当然,在临床实践中是否也可获得相似的治疗效果还需进一步的临床研究去证实。
Background
Myocarditis (see Glossary) is broadly defined as an inflammatory condition of the heart. It represents one of the most challenging clinical problems in cardiology, associated with a broad spectrum of pathological triggers and a wide range of clinical presentations that vary from mild dyspnea to acute heart failure and sudden death. It frequently affects young, previously healthy individuals and has been estimated to account for up to12%of sudden deaths in patients under40years of age. Although most individuals recover from acute myocarditis, genetically susceptible individuals may go on to develop chronic myocarditis and dilated cardiomyopathy (DCM) resulting in congestive heart failure. Despite this simplistic definition, the diagnosis and treatment of myocarditis continue to present clinical problems.
Developing good animal models for myocarditis is crucial to advance understanding of molecular and immunological disease mechanisms. Experimental autoimmune myocarditis (EAM) in genetically susceptible mice may be elicited by immunization of cardiac myosin or a myocardiotogenic peptide derived from the cardiac a-myosin heavy chain or cardiac troponin I emulsified in complete Freund's adjuvant, and EAM in mice mimics human fulminant myocarditis in the acute phase and human DCM in the chronic phase. The animal models of experimental autoimmune myocarditis offer an excellent tool to study heart-specific autoimmune responses and cardiac inflammation. These models hopefully will aid in the development of new diagnostic and therapeutic strategies.
Aims
In our study, we will construct an EAM derived from BALB/c mice.
Methods
Female BALB/c mice (wild-type;13~18g body weight, purchased from the animal experiment center of Shandong University, China) were housed under specific pathogen-free conditions at the Laboratory Animal Center. Six-week-old mice were used in all experiments. All of the animal procedures were approved by the Institutional Authority for Laboratory Animal Care and were performed in accordance with the Guidelines for Animal Experiments of Medical College of Shandong University.
All BALB/c mice were divided into four groups randomly, including normal control group, low-dose peptide group, middle-dose peptide group and high-dose peptide group. Forty-five mice were immunized twice to establish LAM. A specific peptide derived from murine cardiac u-myosin heavy chain (MyIIc-u614-629[Ac-SLKLMATLFSTYASAD-OH|) was used as an antigen. The peptide (purity≥98.75%; GL Biochem (Shanghai) Ltd, China) was dissolved in physiological saline and emulsified in an equal volume of Freund's complete adjuvant (Sigma-Aldrich. St Louis. MO, USA). Lach mouse was injected subcutaneously in the back with peptide on days0and7respectively. On days16,21.26and63after immunization, echocardiography was carried out. the severities of myocarditis and interstitial fibrosis were detected by histopathological evaluation.
Results
In the LAM model, immunization of susceptible mice with a myocardiotogenic peptide derived from the α-cardiac heavy chain emulsified in complete Fround's adjuvant (FCA) induces myocarditis in mice with a peak of inflammation in the heart around day21. On day63, inflammation largely resolve, but the process of pathological remodelling continues and many animals develop ventricular dilation and heart failure on follow-up.
Conclusion
Despite the fact that the EAM models are rather artificial, they offer the great advantage to study disease pathogenesis and autoimmune mechanisms in vivo in the absence of an infective agent. From the EAM model we can not only learn about autoimmune mechanisms contributing to disease development, but the model also allows us to study the pathophysiology of inflammatory heart disease and helps us in the design of novel, immunomodulating treatment strategies. In addition, the EAM model might offer a potential tool to refine the diagnostic accuracy of currently available and future imaging modalities in inflammatory cardiomyopathy.
Background
Myocarditis is defined as inflammation of the myocardium with consequent myocardial injury. It can lead to sudden death, and about10-20%of patients with histological evidence of myocardial inflammation, even when asymptomatic, will develop a chronic disease eventually leading to dilated cardiomyopathy (DCM). Many cases are associated with enteroviruses infections such as cardiotropic coxsackievirus B3, adenoviruses, or parvovirus B19. EAM induced in susceptible rodent animals by injection of cardiac myosin is an animal model of human myocarditis and post-myocarditis DCM. Cardiac myosin is one of the dominant autoantigens in virus-induced myocarditis in mice. The later phase of enteroviruses-induced heart disease can be mimicked by immunization of mice with purified murine cardiac myosin in the absence of viral infection. An EAM derived from BALB/c mice was constructed to understand the mechanisms of myocardial injury and to develop an effective therapeutic strategy for myocarditis. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatroy effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Published studies suggest they may be beneficial for T cell-mediated diseases by suppressing inducible class II MHC expression and costimulator on APCs, favoring Th2versus Thl differentiation of helper T cell. Statins can also protect endothelial function and the integrity of the microvasculature, increase nitric oxide (NO) bioavailability and exert antioxidant effects. Based on these beneficial effects of statins, this study tested whether rosuvastatin in an RAM BALB/c mouse model can affect the progression of myocarditis and apoptosis of the myocardium cell in vivo, and further enhance cardiac function.
Aims
1.To investigate the effect of rosuvastatin on the acute phase of myocarditis.
2.To test the effect of rosuvastatin on the progression of apoptosis of the myocardium cell in vivo.
3.To investigate the effect of rosuvastatin on cardiac function.
Methods
1.ModeI of EAM and medication
Female BALB/c mice were housed under specific pathogen-free conditions at the Laboratory Animal Center, six-week-old mice were used in all experiments. All BALB/c mice were divided into four groups randomly, including normal control group (group C). low-dose rosuvastatin group (group L), high-dose rosuvastatin group (group H) and non-treated LAM group (group N). Group L. group H and group N mice were immunized twice to establish LAM. Lach mouse was injected subcutaneously in the back with200ug/200μ1of peptide on days0and7respectively. Rosuvastatin therapy started at the same time of immunization. Group N received physiological saline instead of drugs. The acute phase of LAM was analyzed on day21after the first immunization.
2.Echocardiography
Lchocardiographie studies were performed in40mice. After determination of body weight, transthoracie echocardiography was recorded under anaesthesia. Left ventricular internal dimensions at end-systole and end-diastole (LVLDs and LVLDd) were measured digitally on the M-mode tracings. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVF'S) were then both calculated.
3.Histologic examination and assessment of severity of myocarditis
All mice were sacrificed under pentobarbital sodium anesthesia on day21. Heart and body weights were measured, and the ratio of heart weight to body weight (HW/BW) was calculated. The heart was removed, fixed in formalin for24h, embedded in paraffin, and stained with hematoxylin and eosin. Myocarditis was determined by identifying both infiltrating mononuclear cells and myocyte necrosis. The percentage of myocardial inflammation was determined by semi-quantitative image analysis.
4. Enzyme-linked immunosorbent assay of inflammatory cytokines
ELISA assay was performed to determine serum levels of IL-6and TNF-a in mice. Serum IL-6and TNF-α levels were quantified with the use of IL-6and TNF-α kits.
5. Serum lipids levels
On day21, the levels of serum total cholesterol (TC) and triglycerides (TGs) were determined with an automated enzymatic technique, and low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol levels were detected with an automated chemically modified technique.
6. TUNEL assay
Apoptotic cells in myocardium were detected by the terminal transferase-mediated DNA nick end labeling (TUNEL) assay following the manufacturer's protocol (Roche Diagnostics GmbH, Germany).
7. Immunohistochemisty staining
Immunohistochemistry were performed for detecting the expressions of active caspase3, according to the instructions of the manufacturer.
8. Western blotting analysis
Western blotting was performed to detect activation of caspase3,8, and9.
Results
1. Effects of rosuvastatin on heart weight/body weight ratio and serum lipids levels
The heart weight/body weight ratio was significantly increased in group N, compared with that in group C. The heart weight/body weight ratio of group H and group L was lower than that of group N. Rosuvastatin treatment suppressed the increase in that value in a dose-dependent manner. On day21, there was no significant difference in the serum lipids level among the four groups.
2.Improvement of cardiac function by rosuvastatin
After immunization on day21, the echocardiographic analyses exhibited the left ventricular dysfunction following myocarditis, and that the rosuvastatin treatment significantly prevented the progression of heart failure.
3.Effects of rosuvastatin on myocarditis-affected areas and histoscore
On day21after immunization, Histopathological scores of the myocarditis revealed that treatment with rosuvastatin suppressed the prevalence and severity on inilammation and that the effect of rosuvastatin tended to be dose-dependent.
4.Suppression of expression of inflammatory cytokines
KLISA was performed for serum cytokines of the four groups. Rosuvastatin suppressed the increased inflammatory cytokine production on myocarditis mice in a dose-dependent manner. These results suggest that treatment with rosuvastatin on myocarditis mice inhibits pro-inflammatory cytokine production.
5. Apoptosis of cardiomyocyte and active caspase3in myocardium
The number of apoptotic cells was determined by TUNKL immunolluorescence assay, and the apoptotic cells were stained green. The positive staining cells with active Caspase-3protein were mostly distributed in the myocardium around the areas of inilammation. In addition, some infiltration lymphocytes were stained positively. Similarly, large amounts of apoptotic cardiomyocytes and the positive staining cells with active Caspase-3protein were detected in group N, compared with that of other groups. A few apoptotic cardiomyocytes and the positive staining cells with active Caspase-3protein were seen in the mice receiving low-dose rosuvastatin. Only very few apoptotic cardiomyocytes and the positive staining cells with active C'aspase-3protein were found in group H. No TUNEL-positive cardiomyocytes and no activation of caspase-3were found in group C. These results showed that rosuvastatin treatment suppressed cardiomyocyte apoptosis
6.Effects of rosuvastatin on Intracellular Molecules
Activated caspase8and activated caspase9were analyzed at the peak of disease on day21, and theirvlevels were significantly up-regulated in group N compared with group C, group L and group H. Treatment with rosuvastatin dose-dependently decreased the myocardial levels of activated caspase8and activated caspase9significantly.
Conclusion
The present study demonstrated for the first time that rosuvastatin administration markedly interfered with the progression of experimental autoimmune myocarditis through inhibiting cardiac inflammatory infiltration, suppressing release of proinflammatory cytokines, and resisting apoptosis of cardiomyocyte, all of which can contribute to the improvement of LV function and the attenuation of progressive LV remodeling in HAM.
Background
Myocarditis is defined as inflammation of the myocardium with consequent myocardial injury. Myocarditis often progresses to dilated cardiomyopathy (DCM), a major cause of heart failure. This condition is characterized hy infiltration of inflammatory cells into the myocardium with consequent loss of myocytes and development of fibrosis and necrosis. Clinical observations and animal experiments suggest that autoimmunity plays an important role in myocarditis.
Experimental autoimmune myocarditis is a mouse model of postinfectious myocarditis that can be induced in susceptible mouse strains by immunization with cardiac myosin or a myocardiotogenic peptide derived from the cardiac α-myosin heavy chain or cardiac troponin I. It has been demonstrated to progress into the clinieopathological state similar to DCM in the chronic phase, and has been found to be characterized by the enlargement of the heart, dilatation of ventricles, diffuse and extensive myocardial fibrosis, and hypertrophie and atrophic changes of myocardial fibers, resembling human cardiomyopathy. We constructed an HAM derived from BALB/c mice to investigate the pathogenesis of myocarditis induced by autoimmune mechanism.
The3-hydroxy-3-methylgutaryl coenzyme A (HMG-CoA) reduetase inhibitors, commonly referred to as "statins", are well-known potent lipid lowering agents. In addition to their primary effects, the statins have been shown to have pleiotropic effects on the cardiovascular system, including immunomodulation, antiinflammatory, antioxidative, endothelial protective effects, cellular senescence and cardiac remodeling. Based on these beneficial effects of statins, this study was designed to examine whether rosuvastatin in an EAM BALB/c mouse model can affect the myocarditis progression and cardiac remodeling in vivo, and to elucidate the probable mechanisms.
Aims
1. To assess the effect of rosuvastatin on the chronic phase of EAM.
2. To investigate the effect of rosuvastatin on cardiac function.
3. To elucidate the molecular mechanisms of rosuvastatin therapentic effects on cardiac remodeling, especially cardiac myocyte hypertrophy, and interstitial fibrosis in the EAM.
Methods
1. Model of EAM
BALB/c mice were immunized at6weeks of age with a peptide derived from murine cardiac α-myosin heavy chain (MyHc-α614-629[Ac-SLKLMATLFSTYASAD-OH]), as described previously. Mice with EAM were divided into three groups:non-treated EAM group (group N); low-dose rosuvastatin group (1mg/kg/day, group L), and high-dose rosuvastatin group (10mg/kg/day, group H). The other BALB/c mice, received neither immunization nor statins therapy, were used as normal controls (group C).
2. Rosuvastatin treatment
Rosuvastatin therapy started at the same time of immunization. They were administered orally by gastric gavage for9weeks from day0to day63after immunization. Group N and group C received physiological saline instead of drugs.
3. Echocardiography
Echocardiography was performed on days21and63after the first immunization. Echocardiographic images were taken from2D M-mode, parasternal long axis views, and short axis views at the mitral valve and mid-papillary muscle levels, the left ventricular (LV) end diastolic dimension (LVEDD), LV end systolic dimension (LVESD), LV septal wall thickness at end diastole (IVSED) and LV posterior wall thickness at end diastole (PWTED) were measured digitally on the M-mode tracings and averaged from at least three cardiac cycles. FS and EF were then calculated.
4. Histological assessment of severity of myocarditis
Mice were evaluated for the development of EAM at the peak of disease on day 21. The heart sections were stained by haematoxylin and eosin. The percentage of myocardial inflammation was determined by semi-quantitative image analysis.
5. Enzyme-linked immunosorbent assay of inflammatory cytokines
Serum concentrations of IL-6and TNF-α were quantified with the use of IL-6and TNF-α ELISA kits on days21and63, according to the manufacturer's instructions.
6. Histological assessment of cardiac myocyte hypertrophy and fibrosis
Mice were euthanized in chronic stage of KAM on day63after immunization. Histological analysis was performed on deparaffinized5-μm-thick tissue sections, which were stained with H&E to assess cardiomyocyte hypertrophy, with Masson trichrome for evaluation of fibrosis and with picrosirius red for evaluation of collagen deposition and orientation.
7. Western blotting analysis
The protein levels of p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38and t-p38were analyzed by Western blot measurement at the peak of disease on day21. TGF-β and p-Smad2/3protein were analyzed by Western blot measurement In chronic stage of KAM on day63.
Results
1.Effects of rosuvastatin on cardiac structure and function
The echocardiographic analyses exhibited the left ventricular dysfunction and remodeling following myocarditis. Treatment with high-dose rosuvastatin which of effectiveness was better than low-dose rosuvastatin prevented the left ventricular remodeling, dysfunction and the progression of heart failure. And rosuvastatin administration from day0to day63further improved cardiac function and suppressed cardiac remodeling compared with that administration from day0to day21in group H and group L
2.Rosuvastatin ameliorated myocarditis progression in EAM mice
Severe inflammatory lesions were observed in the hearts of group N mice. In contrast, area of cellular infiltration into the myocardium was significantly decreased in both group L and group H, compared with group N. Histopathological scores of the myocarditis also revealed that treatment with rosuvastatin suppressed the prevalence and severity on inflammation and that the effect of rosuvastatin tended to be dose-dependent.
3.Effects of rosuvastatin on serum inflammatory cytokines
ELISA was performed for serum cytokines of the four groups on day21and63. The serum levels of IL-6and TNF-a were significantly lower in the two rosuvastatin-treated groups than that in group N, and rosuvastatin also suppressed the increased inflammatory cytokine production on myocarditis mice in a dose-dependent manner.
4. Effect of rosuvastatin on myocardial Fibrosis and remodeling
Marked interstitial fibrosis and collagen deposition were detected in the hearts of mice in each group on day63. Rosuvastatin treatment significantly reduced the areas of fibrosis and collagen deposition in a dose-dependent manner compared with those in group N. The myocyte size was significantly increased in group N. and dose-dependently reduced in the treatment with rosuvastatin groups compared with those in group N.
5. Effects of rosuvastatin on Intracellular Signaling Kinases and Molecules
Treatment with rosuvastatin dose-dependently decreased the myocardial levels of pERK1/2, pJNK, p-p38, TGF-β1and pSmad2/3protein significantly. These results showed that rosuvastatin treatment down-regulated Mitogen-activated protein kinase, and TGF-β1-Smad2/3signaling pathways in the hearts of EAM mice.
Conclusion
This investigation demonstrates for the first time that rosuvastatin attenuates cardiac remodeling, especially cardiac myocyte hypertrophy, and interstitial fibrosis in the EAM mice, all of which can contribute to the improvement of LV function. These beneficial effects of rosuvastatin treatment are related partially to down-regulated Mitogen-activated protein kinase, TGF-β1-Smad2/3and caspase-mediated signaling pathways. Further clinical studies are needed to clarify whether this beneficial effect can be translated into clinical practice.
引文
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