缬沙坦对实验性自身免疫性心肌炎的作用及其机制研究
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摘要
背景：
心肌炎以心肌细胞的变性、坏死及单个核细胞浸润为特征,伴或不伴有心肌纤维化,是一种特殊的心肌炎症疾病。临床可表现为不同程度的心功能障碍和全身症状。部分患者可以康复,而部分患者转化为扩张型心肌病(dilated cardiomyopathy, DCM),出现进行性心力衰竭,成为40岁以内人群患心血管疾病和死亡的主要原因之一。
迄今为止,心肌炎的发生发展,以及演变为扩张型心肌病的病理生理机制仍然不清楚。心肌炎的发病机制很复杂,最常见的是病毒感染,除此之外,还包括特发的、细菌和寄生虫等感染、药物或毒素的诱发以及自身免疫反应机制等。其中越来越多的研究表明,自身免疫机制或免疫介导机制对包括心肌炎在内的很多心血管疾病的发生发展起着关键性的作用,成为目前国内外的研究焦点。有证据显示,病毒感染后机体对心脏自身抗原,尤其是心脏肌凝蛋白所产生的自身免疫反应加速了心肌炎的发生发展过程。国外学者用纯化的心肌肌凝蛋白免疫易感鼠种建立了实验性自身免疫性心肌炎模型(experimental model of autoimmune myocarditis, EAM),这种心肌炎模型与人类心肌炎极为相似并可发展为扩张型心肌病,为探讨心肌炎及扩张型心肌病的发病机制、病理生理以及探索新的防治措施提供了一个理想的实验动物模型,然而在此领域的研究仍然不多见。
因为心肌炎及扩张型心肌病的发病机理仍然不清楚,临床上对心肌炎或扩张型心肌病主要是以减少或消除可能的诱发因素以及针对其相关症状和并发症的对症治疗,例如充血性心力衰竭、心源性休克及心律失常等,而并不是针对疾病本身。随着研究的不断深入,近年来自身免疫损伤在心肌炎的发生发展中的所起的重要作用成为众多学者研究的焦点。已有研究表明EAM是CD4+T细胞介导的自身免疫性疾病,并且EAM的急性期与Th1细胞介导的免疫反应息息相关。促炎细胞因子以及Th1／Th2平衡的偏移对EAM的诱导产生以及心肌损伤的发生发展发挥了极其关键的作用。
由于近年来有证据表明自身免疫反应对心肌炎起重要的作用,使免疫调节剂在心肌炎的防治中越来越受到重视,但是其有效性仍然存在很大的争议。肾素-血管紧张素系统(RAS)在调节血管张力的作用中扮演着重要的角色,并且作为血管扩张药物作用的一个重要的治疗靶点。阻断这种通路的药物,比如血管紧张素Ⅱ受体(AT1)阻滞剂很成功的应用于高血压、心功能不全等多种心血管疾病的有效治疗。然而近来,此类药物的非血流动力学作用成为了一个新的研究热点。有些研究报道了一些血管紧张素Ⅱ受体阻滞剂(ARBs)有调节炎症、纤维化及影响粘附分子表达的作用。此类药物发挥其心脏保护作用可能部分归因于通过减少过度表达的炎性细胞因子调节炎症来实现的。尽管如此,此类药物对自身免疫性疾病例如自身免疫性心肌炎的作用的研究并不多见。目的：
本研究中,我们分两次皮下注射猪的纯化的心肌肌凝蛋白来免疫易感大鼠建立实验性自身免疫性心肌炎模型,并且用缬沙坦,一种有效的ARB,来干预EAM大鼠。本实验旨在探讨缬沙坦对EAM发生发展所起的作用,并且深入分析其中的相关免疫机制,以期为心肌炎及扩张型心肌病的治疗提供新的治疗策略,同时也为其他由免疫介导的疾病提供一个免疫调节治疗的典范。方法：
32只健康6周龄雄性Lewis大鼠用于实验,其中24只用纯化的猪的心肌肌凝蛋白注射后脚垫免疫建立EAM模型。所有的大鼠随机分为四组：1)正常对照组(Group C, n=8):同样的方式皮下注射不含肌凝蛋白的完全弗氏佐剂,并且用生理盐水灌胃代替药物治疗；2)未治疗组(Group N, n=8):将蛋白浓度为10mg／ml的纯化的猪的心肌肌凝蛋白与等体积的含有热灭活的结核杆菌的完全弗氏佐剂充分混合成乳浊液,于第0天和第7天大鼠后脚垫皮下注射乳浊液(含有1mg肌凝蛋白)建立EAM模型,采用生理盐水灌胃代替药物治疗；3)：低剂量缬沙坦治疗组(3mg／kg／day,Group L,n=8)：同样方式用猪心肌肌凝蛋白分别免疫两次Lewis大鼠建立EAM模型,并且在免疫的同时给予低剂量缬沙坦(3mg／kg／day)灌胃治疗21天；4)：高剂量缬沙坦治疗组(10mg／kg／day,Group H,n=8)：同样方式用猪心肌肌凝蛋白分别免疫两次Lewis大鼠建立EAM模型,并且在免疫的同时给予高剂量缬沙坦(10mg／kg／day)灌胃治疗21天。
免疫的同时,记录大鼠体重变化以及心率、收缩压和舒张压(tail-cuff)的变化。所有的大鼠在初次免疫后21天处死收集标本行进一步的实验和分析。
初次免疫后21天,将大鼠麻醉、固定,应用Agilent sonos 5500超声检测仪,11-13L探头经胸进行心脏超声检测大鼠心脏结构及心功能变化,并于左室短轴切面腱索水平获得清晰的M-型超声图像；超声检查结束后,处死大鼠,立即取出心脏,称重,计算心脏重量与体重的比值；心肌组织用4%中性甲醛固定,心肌切片行H-E染色及Masson's三联染色,光镜下观察炎细胞浸润及心肌纤维化改变,并计算病理积分；处死大鼠后,无菌取脾研磨得到细胞悬液,MTT比色法测定淋巴细胞增值反应,观察淋巴细胞对心肌肌凝蛋白的增殖反应；更进一步,我们用ELISA检测血清Th1型细胞因子IFN-γ、IL-2以及Th2型细胞因子IL-4、IL-10的水平变化。结果：
1.未治疗组的收缩压和舒张压比正常对照组略微降低,但是没有统计学差异。血压和心率在各组之间没有显著差异。该结果说明所使用的缬沙坦的剂量并没有影响血压。
2.未治疗组大鼠的体重明显降低,而经过缬沙坦治疗后的EAM大鼠体重并没有显著降低；初次免疫后3周,相比较于正常对照组及高剂量缬沙坦治疗组,未治疗组大鼠的心脏重量明显增加；就心脏重量与体重的比值的变化而言,未治疗组明显高于正常对照组,而缬沙坦了显著降低了此比值的增加,并且呈剂量依赖性。
3.心脏超声检查结果显示：相对于正常对照组,未治疗组大鼠的左室收缩末内径(LVEDs)和左室舒张末内经(LVEDd)都明显增大；左室短轴缩短率(LVFS)显著降低。而高剂量缬沙坦阻止了EAM大鼠的心功能损伤。并且,未治疗组EAM大鼠的室间隔厚度(IVS)及左室后壁厚度(LVPW)明显增厚,而低、高剂量的缬沙坦治疗后IVS及LVPW厚度均明显降低。心脏超声结果提示EAM大鼠心功能显著受损,心脏结构发生明显改变,缬沙坦治疗后显著的改善了心肌炎大鼠心功能的降低及心室重构的改变。
4.初次免疫后21天,肉眼观察,未治疗组大鼠的心脏明显增大,心脏表面广泛苍白病变区域；镜下观察,EAM大鼠心肌组织呈现典型的病理改变,表现为心肌变性、坏死以及大量的炎细胞浸润。Masson's三联染色显示未治疗组大鼠心肌存在广泛的心肌纤维化。低、高剂量的缬沙坦均能显著减轻了EAM的这些肉眼及病理改变。此外,我们用组织学评分进行定量评估,结果显示缬沙坦治疗组的病理积分均显著低于未治疗组且呈剂量依赖性。
5.淋巴细胞增值实验结果显示：相对于正常大鼠,未治疗组大鼠脾淋巴细胞在猪心肌肌凝蛋白刺激下产生了高水平的增值反应,而在两缬沙坦治疗组,淋巴细胞对猪心肌肌凝蛋白刺激所产生的增值反应受到显著的抑制,且成剂量依赖性。而各组脾淋巴细胞对非特异性丝裂原ConA的刺激所产生的增值反应没有统计学差异。说明给予缬沙坦治疗有效的抑制了肌凝蛋白诱导的抗原特异性淋巴细胞增值反应。
6. ELISA测定血清Th1及Th2细胞因子的变化反应Th1／Th2平衡的偏移。结果显示,相比较于正常对照组,未治疗组大鼠的血清IFN-γ与IL-2水平显著增高,缬沙坦治疗后显著的下调了这两种细胞因子的浓度。而Th2型细胞因子包括IL-4及IL-10,在未治疗组均明显低于正常对照组,缬沙坦显著上调了IL-4及IL-10的水平。结果表明了缬沙坦调节EAM大鼠血清中辅助性T淋巴细胞亚群的平衡由Th1型向Th2型转变。
结论：
我们的研究结果显示,缬沙坦可以阻止EAM所导致的心功能损害、心室重构以及形态学的改变,明显减轻了EAM,并且呈剂量依赖性。独立于其降压作用之外,缬沙坦改善EAM的机制很可能跟缬沙坦减少了促炎细胞因子的产生以及修正了Th1／Th2的失衡有关。
背景：
心肌炎是引起扩张型心肌病及严重充血性心力衰竭的一个很重要的原因。尽管习惯上人们认为心肌炎是由好几个不同的病理阶段连续发展而构成,其确切的机制仍然不清楚。迄今为止,并没有一致公认的有效的治疗措施。临床上也仅限于对心肌炎的症状和并发症的治疗,而并非针对疾病本身。心肌炎是一种独特的心肌炎症病症,一般认为其发病由多种复杂的机制参与,包括特发性的、感染性的以及自身免疫因素等。越来越多的证据表明,心肌炎是一种跟免疫功能障碍和炎症反应有关的疾病,并可以进展成为扩张型心肌病。如今人们了解到,促炎细胞因子对心肌炎等有害的自身免疫反应的激活起着极为关键的作用。心肌肌凝蛋白诱导的实验性自身免疫性心肌炎(experimental autoimmune myocarditis, EAM)可以作为人们研究由免疫反应启动的人类心脏炎症疾病的代表模型。
Janus激酶-信号转导子与转录激活子(Janus kinase-signal transducers and activators of transcription, JAK-STAT)最初发现是作为细胞因子超家族的一种重要的细胞内信号转导通路。多种细胞因子和生长因子都可以激活JAK-STAT信号通路。当这些配体与受体结合后,激活JAKs家族,激活的JAKs经过一系列的作用,导致STATs转录因子磷酸化而被激活,激活的STATs与受体分离,二聚体化转位到细胞核,一旦移位到细胞核,活化的STAT二聚体能与相应靶基因的启动子反应元件结合,从而启动相应基因的转录。不同的细胞因子或生长因子可以激活不同的JAK激酶和STAT转录因子,从而引起不同基因的转录。JAK-STAT信号通路在多种炎症反应中发挥着十分重要的作用。近年来,有研究表明JAK-STAT通路在多种心肌损伤中发挥作用,包括心肌梗死、氧化性损伤、心肌炎、心肌肥厚以及心室重构等。就目前而言,对心脏中STATs的研究多是对STAT以及STAT3的探讨。然而,迄今为止对STAT蛋白的作用仍然知之甚少,有待于进一步的研究来阐明。
肾素-血管紧张素系统(RAS)是血管扩张剂治疗心血管疾病的重要作用靶点。血管紧张素Ⅱ(AngⅡ)是一种具有多种功能的激素,可以通过AT1及AT2受体作用于一系列复杂的细胞内信号通路,从而影响心脏、血管细胞的功能。既往的研究表明,血管紧张素Ⅱ通过作用于AT1受体在肾损伤、慢性同种异体移植排斥反应、冠状动脉的移植以及病毒性心肌炎等急性炎症反应的免疫病理过程起着极其重要的作用。有证据表明,肾素-血管紧张素系统与多种炎症反应的过程有关。关于炎性疾病方面的研究表明血管紧张素Ⅱ参与了免疫及炎症反应,RAS拮抗剂通过阻断RAS通路而发挥对抗炎症及免疫反应的作用。然而,RAS拮抗剂对EAM的作用近年来才见报道,对其作用及机制仍然知之甚少。另外,近些年来的研究揭示,血管紧张素Ⅱ可以通过作用于AT1受体激活JAK-STAT通路。有相关的研究发现,在大鼠的心脏及心肌细胞中,血管紧张素Ⅱ与3AK-STAT的激活有很大的关系。尽管如此,血管紧张素Ⅱ受体阻滞剂缬沙坦对心肌自身抗原肌凝蛋白诱导的实验性自身免疫性心肌炎JAK-STAT信号通路的作用研究国内外未见报道。
目的：
本研究中,我们用纯化的猪的心肌肌凝蛋白免疫Lewis大鼠建立自身免疫性心肌炎(EAM)模型,该模型是代表人类心肌炎的理想模型。并且在免疫的同时给予EAM大鼠血管紧张素Ⅱ受体阻滞剂缬沙坦治疗3周。探讨EAM的发生发展中是否有JAK-STAT信号通路的激活,以及应用缬沙坦阻断RAS系统对EAM大鼠心肌中的JAK-STAT信号转导通路的影响。
方法：
6周龄雄性Lewis大鼠,SPF级,以猪的心肌肌凝蛋白免疫大鼠建立自身免疫性心肌炎模型(EAM)。所有的Lewis大鼠(n=24)随机分为3组,分别命名为未治疗组(Group N, n=8)、正常对照组(Group C, n=8)、缬沙坦治疗组(GroupH,10mg／kg／day,n=8)。将纯化的猪的心肌肌凝蛋白(浓度为10.0 mg／ml)与含有10.0 mg／ml灭活结核杆菌的等体积的完全弗氏佐剂充分混合成乳浊液用来免疫大鼠。1)Group N:将总体积0.2ml的乳浊液(含有肌凝蛋白1mg)分别于第0、7天皮下注射大鼠后脚垫建立EAM模型；2)Group C:对照组大鼠只给予等体积的不含肌凝蛋白的完全弗氏佐剂免疫；3)Group H:采用跟Group N组一样的方式对大鼠进行两次皮下注射免疫。
免疫的同时,缬沙坦治疗组的大鼠给予缬沙坦(10mg／kg／day)灌胃治疗三周。未治疗组及正常对照组大鼠给予生理盐水灌胃代替缬沙坦治疗。
在实验过程中,记录大鼠体重变化；测量心率、收缩压及舒张压的变化。初次免疫后21天,采用Agilent sonos 5500超声检测仪进行心脏超声检查,具体操作如第一部分所示。心脏超声检查结束后,处死老鼠,取出脾脏及心脏称重。计算心脏重量／体重比值,行病理学检查及淋巴细胞增值实验,如第一部分操作所述。
除此之外,免疫后三周,在处死之前取大鼠血清。用含有10μg／mL肌凝蛋白的磷酸盐缓冲液包被平底96孔板,采用间接ELISA法测定抗心肌肌凝蛋白自身抗体水平,结果以酶标仪读取405 nm波长处的吸光度值表示。进一步,处死大鼠后,立刻无菌取出心脏,一部分立刻放入液氮罐中,后转入-80℃保存备用。采用western blot测定大鼠心肌中JAK-STAT信号通道家族中p-JAK2, p-STAT1以及p-STAT3磷酸化蛋白表达水平,并以β-actin作为内参。
结果：
1.三组实验大鼠之间的心率、收缩压及舒张压并没有统计学差异,10mg／kg／day缬沙坦并没有影响大鼠的心率及血压。
2.心脏超声检查、病理学检查及淋巴细胞增值实验结果显示缬沙坦显著的改善了EAM。简言之,缬沙坦治疗显著的阻止了EAM大鼠心功能的损伤及心室重构；减轻了由心肌炎引发的病理损害；抑制了淋巴细胞对心肌肌凝蛋白刺激产生的抗原特异性增值反应。
3.缬沙坦对大鼠血清抗心肌肌凝蛋白自身抗体的影响结果显示：Group N组大鼠产生了大量的抗心肌肌凝蛋白自身抗体,抗体滴度显著高于Group C组及Group H组。该结果提示,心肌肌凝蛋白引发的自身免疫反应诱导体内产生了高水平的抗心肌肌凝蛋白自身抗体,缬沙坦抑制了此类抗体的产生。
4.免疫印迹(western blot)结果显示：与Group C组及Group H组相比较,Group N组大鼠心肌组织中磷酸化的JAK2(p-JAK2)表达显著的增高。该结果说明在JAK2在由心肌肌凝蛋白诱导的心肌损伤中被显著激活,而缬沙坦治疗抑制了JAK2的激活。
5. Western blot检测STATs家族磷酸化水平结果显示：Group N组大鼠心肌组织中p-STAT1及p-STAT3表达水平明显高于Group C组。更加有趣的是,缬沙坦同样减少了两种p-STATs的表达。该结果说明肌凝蛋白诱导的自身免疫性心肌损伤引起了STAT1及STAT3的活化,而缬沙坦可以抑制其活化程度。
结论：
以上的研究结果说明,JAK-STAT信号转导通路在心肌肌凝蛋白引起的自身免疫性心肌炎心肌损伤中显著激活。缬沙坦可以抑制磷酸化的JAK2、STAT1及STAT3的过度表达。缬沙坦可以不依赖于降压作用改善心肌炎,相关机制跟缬沙坦阻止JAK-STAT信号转导通路的激活有关。
Backgroud:
Myocarditis, a heterogeneous myocardial inflammatory disease, is characterized by myocyte necrosis and degeneration with mononuclear cell infiltration in the presence or absence of fibrosis. The clinical manifestation ranges from the asymptomatic state due to the focal inflammation to the fatal congestive heart failure due to diffuse myocarditis. Most patients with myocarditis can completely recover, while some may develop dilated cardiomyopathy (DCM) and manifest cardiac dilation and heart failure. Myocarditis is the major cause of sudden unexpected death in patients less than 40 years of age.
To date, the pathogenesis of myocarditis and the pathogenesis of myocarditis developing into DCM remains unclear. The etiology of myocarditis appears to be rather complicated involving idiopathic, invasion of virus (the major cause), invasion of bacterium and parasite, infectious exposure to drugs and toxins and autoimmune factors. Autoimmunity and immune mediators are known to play a critical role in the pathogenesis of an array of cardiovascular diseases including myocarditis. So the autoimmunity factor becomes a worldwide focus. There is substantial evidence suggesting that autoimmune responses to heart antigens, particularly cardiac myosin, following viral infection may contribute to the disease process. An experimental model of autoimmune myocarditis (EAM) has been induced in susceptible strains of rats produced by immunization with cardiac myosin, which resembles myocarditis in humans and can develop into DCM. So EAM is an excellent experimental model for studying the pathogenesis of myocarditis and the effective therapy. Domestic reports in this field are still relatively rare.
Because the pathogenesis of myocarditis and DCM remains unclear, treatments for myocarditis are directed toward reducing or eliminating the inciting agent when possible and tailoring therapy toward the associated the symptoms and complications, such as congestive heart failure, cardiogenic shock, conduction abnormalities and arrhythmia. Recent focus has been on the point that autoimmune response plays an important role in the development of myocarditis. It has been reported that EAM in rats is a CD4+T cell-mediated disease and is thought to be related to Thl responses in the acute phase. Proinflammatory cytokines and Thl/Th2 imbalance play crucial roles in the induction of EAM and in the progression of myocardial injury in this disease.
Recently, because evidences suggest that autoimmune response plays important role in the development of myocarditis, immune therapies are being taken more and more attention for the treatment of myocarditis. However, their effects are still controversial. The renin-angiotensin system (RAS) is primarily responsible for regulating vascular tone, which is a key target of vasodilator therapy. The effects of angiotensin II type 1 receptor blockers (ARBs) on the treatment of hypertension, heart failure, and other cardiovascular diseases have been confirmed extensively. However, recent studies have emphasized the nonhemodynamic effects of these drugs. Recently, interest has focused on the nonhemodynamic effects of these drugs. Some Ang-Ⅱreceptor type 1 (AT1) antagonists have been reported to be effective agents in modulating inflammation, adhesion molecule expression, and fibrosis. The cardioprotection of these drugs may be partly due to their regulation of inflammation as a result of removal of overproduced cytokines. Nevertheless, the effects of the ARBs on autoimmune diseases such as autoimmune myocarditis have not been well investigated.
Objective:
In the present study, the EAM rat model was established by injecting cardiac myosin twice and valsartan, a novel ARB, has been tested in the experimental model of autoimmune myocarditis. It was aimed at investigating the effects of valsartan on the development of EAM and the immune mechanisms involved, In addition, the present study might provide a promising new strategy for myocarditis and DCM. It represents a paradigm of immunosuppressive therapy on immune-mediated diseases.
Methods:
Thirty-two male Lewis rats (6 weeks old) were maintained in the study. Twenty-four susceptible rats were immunized with porcine cardiac myosin to establish EAM. All Lewis rats were randomly divided into four groups:1) Normal control group (Group C, n=8):Rats were immunized with Freund's complete adjuvant alone, and they received physiological saline instead of durgs administration for three weeks; 2) Non-treated group (Group N, n=8):Purified porcine cardiac myosin (1 mg) was injected subcutaneously in the rear foot pads mixed with an equal volume of Freund's complete adjuvant supplemented with Mycobacterium tuberculosis on days 0 and 7 respectively to establish EAM, and they also received physiological saline instead of durgs administration; 3) Low-dose valsartan (3 mg/kg/day, Group L, n=8): Rats were immunized twice with porcine cardiac myosin to establish EAM and they were administrated orally by valsartan (3mg/kg/day) at the same time of immunization from day 0 to day 21; 4) High-dose valsartan (10 mg/kg/day, Group H, n=8):Rats were immunized twice with porcine cardiac myosin to establish EAM and they received valsartan therapy (10 mg/kg/day) for three weeks.
At the time of immunization, we noted the changes of body weight. Heart rate, systolic and diastolic blood pressure were measured by tail-cuff method using a photoelectric tail-cuff detection system and recorded regularly. All rats were killed 21 days after the first immunization.
21 days after the after the first immunization, Lewis rats were anesthetized and secured to a warming table. To evaluate the cardiac function and heart structure of the rats, transthoracic echocardiographic analysis was performed by the accurate method using Agilent sonos 5500 echocardiograph with 11-13 L transducer. We obtained the M-mode echocardiogram in the two-dimensional short-axis view of the left ventricular at the chordae tendineae level. After the echocardiography examination, all the rats were sacrificed and their hearts were immediately removed and weighted, then the ratio of heart weight/body weight (g/kg) was calculated. The heart specimens were fixed in 4.0%formaldehyde. Heart sections were stained were stained by the hematoxylin-eosin or Masson's trichrome. Inflammation and fibrosis was examined by light microscopy. Pathologic score was calculated. In order to observe lymphocyte proliferation to cardiac myosin, spleens were collected and cell suspensions were prepared on day 21 after the first immunization and used to preform lymphocyte proliferation assay by MTT colorimetric technique immediately. In addition, serum samples were obtained and the serum concentrations of IFN-y, interleukin (IL)-2 as representative Thl cytokines and IL-4, IL-10 as representative Th2 cytokines were analyzed by ELISA.
Results:
1. There was no statistically significant difference in blood pressure or heart rate values between the groups. Systolic blood pressure and diastolic blood pressure in Group N were slightly lower than Group C. However, the valsartan dosages did not affect blood pressure.
2. Body weights were significantly lower in Group N compared with the groups, the effect of which was ablated by valsartan treatment. Following 3 weeks of autoimmune disease, hearts were significantly enlarged in rats from Group N compared with Group C or Group H. With regard to the heart weight/body weight ratio, it was increased in Group N compared with that in Group C. Valsartan treatment suppressed the increase in that value in a dose-dependent manner.
3. Transthoracic echocardiography was performed to assess heart function in all three rat groups. Compared with Group C, LVEDs and LVEDd were significantly enhanced, left ventricular fractional shortening (LVFS) was greatly impaired in Group N, and treatment with high-dose valsartan prevented the left ventricular dysfunction. Moreover, the experimental autoimmune myocarditis significantly enhanced IVS and LVPW, while treatments with both low and high-dose valsartan inhibited the increase. Echocardiography analysis demonstrated that heart structure and cardiac function were greatly impaired in EAM rats, and the treatment with valsartan significantly prevented the progression of autoimmune myocarditis-induced left ventricular dysfunction and ventricular remodeling.
4. On day 21 after the first immunization, hearts from Group N were markedly enlarged and contained large grayish areas. Autoimmune myocarditis elicited typical cardiac histopathological changes in myocardium which were manifested as presence of fragments of necrotic myocardial fibers, mononuclear cells, polymorphonuclear neutrophils, eosinophils and giant cells. Masson's trichrome staining showed extensive myocardial fibrosis under autoimmune myocarditis. Treatment with 3 and 10 mg·kg-1·day-1 valsartan significantly reduced the severity of the disease as assessed by detecting the typical signs of myocarditis. Our further quantitative evaluation revealed that the severity of EAM and affected area was significantly suppressed by valsartan in a dose-dependent manner.
5. Our data from lymphocyte proliferation assay suggested that lymphocytes obtained from rats in Group N had remarkable proliferative response to porcine cardiac myosin. And this antigen-specific proliferative response to myosin stimulation was significantly suppressed by valsartan treatment in a dose-dependent manner. However, the difference did not reach statistical significance in lymphocytes proliferative reaction to the non-specific mitogen ConA among the four groups.
6. Serum analysis revealed significantly elevated serum levels of the Thl cytokines IFN-y and IL-2 in rats from Group N compared with groups, the effect of which was abrogated by valsartan treatment in a dose-dependent manner. In contrast, production of Th2-type cytokines, including IL-4 and IL-10 was significantly higher in the two valsartan-treated groups compared with Group N. These results suggested that valsartan treatment may shift the balance of helper T cells from Thl to Th2.
Conclusions:
The collective results suggest that valsartan inhibites the progression of myocardial function, remodeling and morphological alteration in a rat model of experimental autoimmune myocarditis in a dose-dependent manner. These data favor the notion that the mechanism of amelioration of the disease by this inhibitory agent can be at least partly explained by the removal of proinflammatory cytokines and the Th1/Th2 imbalance independent of BP-lowering effects.
Backgroud:
Myocarditis is one of the important causes of dilated cardiomyopathy and severe congestive heart failure. Although myocarditis is habitually viewed as a chronological sequence of several pathologically distinct phases, its precise pathogenesis remains unclear. To date, there is no universally accepted effective therapy for myocarditis. Treatments for myocarditis are not directed toward the disease itself but instead of managing the symptoms and complications. It is a heterogeneous myocardial inflammatory disease, the etiology of myocarditis appears to be rather complicated involving idiopathic, infectious or autoimmune factors. Myocarditis is a disease associated with immune dysfunction and inflammatory reaction that frequently precedes the development of dilated cardiomyopathy. It is now known that proinflammatory cytokines are important for the activation of the detrimental autoimmune responses, such as myocarditis. Cardiac myosin-induced experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by immune response.
The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway was initially discovered as a major intracellular signal transduction pathway of the cytokine superfamilies. It is activated by many cytokines and growth factors. Binding of ligands to receptors leads to the activation of the JAK tyrosine kinase family, and activated JAK kinases phosphorylate various combinations of STAT transcription factors, which form dimers, translocate into the nucleus, and bind response elements in the promoters of target genes to stimulate transcription. Different cytokines and growth factors activate different combinations of JAK kinases and STAT transcription factors. JAK-STAT plays a crucial role in various inflammatory reaction. The JAK/STAT pathway has recently been shown to be an integral part of the response of the myocardium to various cardiac insults, including myocardial infarction, oxidative damage, myocarditis, hypertrophy and remodeling. The majority of data involving STAT activity in the heart is confined to STAT1 and STAT3. However, these two STAT proteins have not been well have not been well investigated.
The renin-angiotensin system (RAS) is a key target of vasodilator therapy. AngiotensinⅡ(AngⅡ) is a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of AngⅡwith AT1 and AT2 receptors. Previous studies have reported that Ang II, acting through the AT1 receptor, played an important role in the immunopathology involved in renal injury, chronic allograft rejection and graft coronary artery disease, as well as acute inflammation, such as viral myocarditis. Evidence suggests that the renin-angiotensin system(RAS)Contributes to several steps of the inflammatory process. Studies in inflammatory diseases suggested that Ang II was involved in immune and inflammatory responses and RAS antagonists produced effects by blocking the action markedly. The effects of RAS antagonists on EAM have only recently been addressed. In addition, recent studies have revealed that Ang II activates the JAK-STAT pathway via the AT1 receptor. It has been reported that the JAK/STAT pathway is activated in the rat heart or cardiomyocytes and angiotensinⅡis involved in. However, the effects of angiotensinⅡreceptor blocker valsartan on JAK-STAT signal transduction pathway of experimental models of antigen-induced autoimmune myocarditis have not been addressed.
Objective:
In our study, Lewis rats were immunized with porcine cardiac myosin to induce experimental autoimmune myocarditis, which is an ideal animal model of human myocarditis. Valsartan was administered orally for 3 weeks to rats with EAM at the same time of immunization. We investigated whether activation of the JAK/STAT pathway was involved in the process of EAM and whether blockade of the renin-angiotensin system by an AT1 receptor antagonist (valsartan) inhibited the JAK/STAT pathway in the rat heart of EAM.
Methods:
Inbred 6-week-old male Lewis rats (SPF) were immunized with purified porcine cardiac myosin to induce experimental autoimmune myocarditis (EAM). All Lewis rats (n=24) were divided into three groups at random, namely non-treated group (Group N, n=8), normal control group (Group C, n=8), and valsartan-treated group (Group H, n=8). The purified porcine cardiac myosin (10.0 mg/ml) was mixed with an equal volume of Freund's complete adjuvant supplemented with 10.0 mg/ml of Mycobacterium tuberculosis.1) Group N:A total volume of 0.2 ml of the emulsion (lmg porcine cardiac myosin) was injected subcutaneously in each rear footpad of rats on days 0 and 7 respectively; 2) Group C:Rats in control group were given an equal volume of Freund's complete adjuvant alone; 3) Group H:Rats were immunized twice on days 0 and 7 with 0.2 ml of the emulsion.
At the same time of immunization. Rats in Group H were received valsartan (10 mg/kg/day) orally by gastric gavage for 3 weeks from day 0 to day 21 prior to sacrifice. Rats from Group C and Group N received physiological saline instead of valsartan.
During the study, the changes of body weight were noted. We measured heart rate, systolic and diastolic blood pressure regularly by tail-cuff method using a photoelectric tail-cuff detection system. Twenty-one days after the first immunization, transthoracic echocardiography was performed on animals using Agilent sonos 5500 echocardiograph with 11-13 L transducer. The examination of transthoracic echocardiography was the same as described in part 1. After the transthoracic echocardiographic analysis, the rats were sacrificed and their hearts were immediately removed. Histologic evaluation and lymphocyte proliferation assay were performed also the same as described in part 1.
In addition, rat serum samples were obtained 3 weeks postimmunization before sacrificed. Cardiac myosin (10μg/mL) was coated in flat-bottom 96-well ELISA plates, and anti-cardiac myosin autoantibody assay were performed. Absorbance was evaluated as indicator for the levels of anti-cardiac myosin autoantibody using a test wavelength of 405 nm. Furthermore, after the animals were sacrificed, the rat hearts samples were immediately harvested, frozen in liquid nitrogen and stored at-80℃until analysis. We detected tyrosine-phosphorylated protein members of the JAK-STAT family (p-JAK2, p-STAT1 and p-STAT3) in rat myocardial tissue by western blot. To normalize protein loading, the membranes were cut and the lower molecular weight portion was analyzed with the primary antibody forβ-actin.
Results:
1. There were no statistically significant differences between the three groups with regard to systolic blood pressure, diastolic blood pressure and heart rate values between the groups.
2. The results of transthoracic echocardiography, histologic evaluation and lymphocyte proliferation assay showed that valsartan improved EAM significantly. In summary, valsartan treatment significantly prevented the left ventricular dysfunction and severely altered heart structure following myocarditis, reduced the myocarditis-affected histopathologic areas, and suppressed antigen-specific proliferative response to cardiac myosin stimulation.
3. Effect of valsartan treatment on serum anti-cardiac myosin autoantibody was examined. Rats in Group N produced large numbers of anti-cardiac myosin autoantibody and the titer of the autoantibody was significantly higher in Group N than in Group C and Group H. These data indicated that autoimmunity triggered by porcine cardiac myosin significantly facilitated large numbers of anti-cardiac myosin autoantibody, the effect of which was nullified by valsartan treatment.
4. Western blot showed that expression of phosphorylation JAK2 (p-JAK2) in myocardium of EAM rats were much higher than Group C and Group H. The date suggested that JAK2 was activated in myosin-induced cardiac injury, and the activation of p-JAK2 was abrogated by valsartan treatment.
5. Tyrosine-phosphorylated members of the STAT family (p-STAT1 and p-STAT3) were also detected by western blot. The expression levels of p-STAT1 and p-STAT3 were significantly increased in Group N compared with Group C. Interestingly, this expression was attenuated by valsartan administration. This suggests that the activation of STATs is induced by autoimmune myocarditis and valsartan might block the induction.
Conclusions:
As is refered in our article, the JAK-STAT signal transduction pathway was significantly activated in cardiac myosin-induced autoimmune myocarditis, and valsartan treatment suppressed the over-expression of phosphorylated JAK2, STAT1 and STAT3. Valsartan could ameliorate EAM independent of BP-lowering effects. The mechanism involved might be the effects of valsartan by which blocking the activity of JAK-STAT signal transduction pathway.
Key words:
Valsartan; experimental autoimmune myocarditis; JAK-STAT signal transduction pathway.
心肌炎以心肌细胞的变性、坏死及单个核细胞浸润为特征,伴或不伴有心肌纤维化,是一种特殊的心肌炎症疾病。临床可表现为不同程度的心功能障碍和全身症状。部分患者可以康复,而部分患者转化为扩张型心肌病(dilated cardiomyopathy, DCM),出现进行性心力衰竭,成为40岁以内人群患心血管疾病和死亡的主要原因之一。
迄今为止,心肌炎的发生发展,以及演变为扩张型心肌病的病理生理机制仍然不清楚。心肌炎的发病机制很复杂,最常见的是病毒感染,除此之外,还包括特发的、细菌和寄生虫等感染、药物或毒素的诱发以及自身免疫反应机制等。其中越来越多的研究表明,自身免疫机制或免疫介导机制对包括心肌炎在内的很多心血管疾病的发生发展起着关键性的作用,成为目前国内外的研究焦点。有证据显示,病毒感染后机体对心脏自身抗原,尤其是心脏肌凝蛋白所产生的自身免疫反应加速了心肌炎的发生发展过程。国外学者用纯化的心肌肌凝蛋白免疫易感鼠种建立了实验性自身免疫性心肌炎模型(experimental model of autoimmune myocarditis, EAM),这种心肌炎模型与人类心肌炎极为相似并可发展为扩张型心肌病,为探讨心肌炎及扩张型心肌病的发病机制、病理生理以及探索新的防治措施提供了一个理想的实验动物模型,然而在此领域的研究仍然不多见。
因为心肌炎及扩张型心肌病的发病机理仍然不清楚,临床上对心肌炎或扩张型心肌病主要是以减少或消除可能的诱发因素以及针对其相关症状和并发症的对症治疗,例如充血性心力衰竭、心源性休克及心律失常等,而并不是针对疾病本身。随着研究的不断深入,近年来自身免疫损伤在心肌炎的发生发展中的所起的重要作用成为众多学者研究的焦点。已有研究表明EAM是CD4+T细胞介导的自身免疫性疾病,并且EAM的急性期与Th1细胞介导的免疫反应息息相关。促炎细胞因子以及Th1／Th2平衡的偏移对EAM的诱导产生以及心肌损伤的发生发展发挥了极其关键的作用。
由于近年来有证据表明自身免疫反应对心肌炎起重要的作用,使免疫调节剂在心肌炎的防治中越来越受到重视,但是其有效性仍然存在很大的争议。肾素-血管紧张素系统(RAS)在调节血管张力的作用中扮演着重要的角色,并且作为血管扩张药物作用的一个重要的治疗靶点。阻断这种通路的药物,比如血管紧张素Ⅱ受体(AT1)阻滞剂很成功的应用于高血压、心功能不全等多种心血管疾病的有效治疗。然而近来,此类药物的非血流动力学作用成为了一个新的研究热点。有些研究报道了一些血管紧张素Ⅱ受体阻滞剂(ARBs)有调节炎症、纤维化及影响粘附分子表达的作用。此类药物发挥其心脏保护作用可能部分归因于通过减少过度表达的炎性细胞因子调节炎症来实现的。尽管如此,此类药物对自身免疫性疾病例如自身免疫性心肌炎的作用的研究并不多见。目的：
本研究中,我们分两次皮下注射猪的纯化的心肌肌凝蛋白来免疫易感大鼠建立实验性自身免疫性心肌炎模型,并且用缬沙坦,一种有效的ARB,来干预EAM大鼠。本实验旨在探讨缬沙坦对EAM发生发展所起的作用,并且深入分析其中的相关免疫机制,以期为心肌炎及扩张型心肌病的治疗提供新的治疗策略,同时也为其他由免疫介导的疾病提供一个免疫调节治疗的典范。方法：
32只健康6周龄雄性Lewis大鼠用于实验,其中24只用纯化的猪的心肌肌凝蛋白注射后脚垫免疫建立EAM模型。所有的大鼠随机分为四组：1)正常对照组(Group C, n=8):同样的方式皮下注射不含肌凝蛋白的完全弗氏佐剂,并且用生理盐水灌胃代替药物治疗；2)未治疗组(Group N, n=8):将蛋白浓度为10mg／ml的纯化的猪的心肌肌凝蛋白与等体积的含有热灭活的结核杆菌的完全弗氏佐剂充分混合成乳浊液,于第0天和第7天大鼠后脚垫皮下注射乳浊液(含有1mg肌凝蛋白)建立EAM模型,采用生理盐水灌胃代替药物治疗；3)：低剂量缬沙坦治疗组(3mg／kg／day,Group L,n=8)：同样方式用猪心肌肌凝蛋白分别免疫两次Lewis大鼠建立EAM模型,并且在免疫的同时给予低剂量缬沙坦(3mg／kg／day)灌胃治疗21天；4)：高剂量缬沙坦治疗组(10mg／kg／day,Group H,n=8)：同样方式用猪心肌肌凝蛋白分别免疫两次Lewis大鼠建立EAM模型,并且在免疫的同时给予高剂量缬沙坦(10mg／kg／day)灌胃治疗21天。
免疫的同时,记录大鼠体重变化以及心率、收缩压和舒张压(tail-cuff)的变化。所有的大鼠在初次免疫后21天处死收集标本行进一步的实验和分析。
初次免疫后21天,将大鼠麻醉、固定,应用Agilent sonos 5500超声检测仪,11-13L探头经胸进行心脏超声检测大鼠心脏结构及心功能变化,并于左室短轴切面腱索水平获得清晰的M-型超声图像；超声检查结束后,处死大鼠,立即取出心脏,称重,计算心脏重量与体重的比值；心肌组织用4%中性甲醛固定,心肌切片行H-E染色及Masson's三联染色,光镜下观察炎细胞浸润及心肌纤维化改变,并计算病理积分；处死大鼠后,无菌取脾研磨得到细胞悬液,MTT比色法测定淋巴细胞增值反应,观察淋巴细胞对心肌肌凝蛋白的增殖反应；更进一步,我们用ELISA检测血清Th1型细胞因子IFN-γ、IL-2以及Th2型细胞因子IL-4、IL-10的水平变化。结果：
1.未治疗组的收缩压和舒张压比正常对照组略微降低,但是没有统计学差异。血压和心率在各组之间没有显著差异。该结果说明所使用的缬沙坦的剂量并没有影响血压。
2.未治疗组大鼠的体重明显降低,而经过缬沙坦治疗后的EAM大鼠体重并没有显著降低；初次免疫后3周,相比较于正常对照组及高剂量缬沙坦治疗组,未治疗组大鼠的心脏重量明显增加；就心脏重量与体重的比值的变化而言,未治疗组明显高于正常对照组,而缬沙坦了显著降低了此比值的增加,并且呈剂量依赖性。
3.心脏超声检查结果显示：相对于正常对照组,未治疗组大鼠的左室收缩末内径(LVEDs)和左室舒张末内经(LVEDd)都明显增大；左室短轴缩短率(LVFS)显著降低。而高剂量缬沙坦阻止了EAM大鼠的心功能损伤。并且,未治疗组EAM大鼠的室间隔厚度(IVS)及左室后壁厚度(LVPW)明显增厚,而低、高剂量的缬沙坦治疗后IVS及LVPW厚度均明显降低。心脏超声结果提示EAM大鼠心功能显著受损,心脏结构发生明显改变,缬沙坦治疗后显著的改善了心肌炎大鼠心功能的降低及心室重构的改变。
4.初次免疫后21天,肉眼观察,未治疗组大鼠的心脏明显增大,心脏表面广泛苍白病变区域；镜下观察,EAM大鼠心肌组织呈现典型的病理改变,表现为心肌变性、坏死以及大量的炎细胞浸润。Masson's三联染色显示未治疗组大鼠心肌存在广泛的心肌纤维化。低、高剂量的缬沙坦均能显著减轻了EAM的这些肉眼及病理改变。此外,我们用组织学评分进行定量评估,结果显示缬沙坦治疗组的病理积分均显著低于未治疗组且呈剂量依赖性。
5.淋巴细胞增值实验结果显示：相对于正常大鼠,未治疗组大鼠脾淋巴细胞在猪心肌肌凝蛋白刺激下产生了高水平的增值反应,而在两缬沙坦治疗组,淋巴细胞对猪心肌肌凝蛋白刺激所产生的增值反应受到显著的抑制,且成剂量依赖性。而各组脾淋巴细胞对非特异性丝裂原ConA的刺激所产生的增值反应没有统计学差异。说明给予缬沙坦治疗有效的抑制了肌凝蛋白诱导的抗原特异性淋巴细胞增值反应。
6. ELISA测定血清Th1及Th2细胞因子的变化反应Th1／Th2平衡的偏移。结果显示,相比较于正常对照组,未治疗组大鼠的血清IFN-γ与IL-2水平显著增高,缬沙坦治疗后显著的下调了这两种细胞因子的浓度。而Th2型细胞因子包括IL-4及IL-10,在未治疗组均明显低于正常对照组,缬沙坦显著上调了IL-4及IL-10的水平。结果表明了缬沙坦调节EAM大鼠血清中辅助性T淋巴细胞亚群的平衡由Th1型向Th2型转变。
结论：
我们的研究结果显示,缬沙坦可以阻止EAM所导致的心功能损害、心室重构以及形态学的改变,明显减轻了EAM,并且呈剂量依赖性。独立于其降压作用之外,缬沙坦改善EAM的机制很可能跟缬沙坦减少了促炎细胞因子的产生以及修正了Th1／Th2的失衡有关。
背景：
心肌炎是引起扩张型心肌病及严重充血性心力衰竭的一个很重要的原因。尽管习惯上人们认为心肌炎是由好几个不同的病理阶段连续发展而构成,其确切的机制仍然不清楚。迄今为止,并没有一致公认的有效的治疗措施。临床上也仅限于对心肌炎的症状和并发症的治疗,而并非针对疾病本身。心肌炎是一种独特的心肌炎症病症,一般认为其发病由多种复杂的机制参与,包括特发性的、感染性的以及自身免疫因素等。越来越多的证据表明,心肌炎是一种跟免疫功能障碍和炎症反应有关的疾病,并可以进展成为扩张型心肌病。如今人们了解到,促炎细胞因子对心肌炎等有害的自身免疫反应的激活起着极为关键的作用。心肌肌凝蛋白诱导的实验性自身免疫性心肌炎(experimental autoimmune myocarditis, EAM)可以作为人们研究由免疫反应启动的人类心脏炎症疾病的代表模型。
Janus激酶-信号转导子与转录激活子(Janus kinase-signal transducers and activators of transcription, JAK-STAT)最初发现是作为细胞因子超家族的一种重要的细胞内信号转导通路。多种细胞因子和生长因子都可以激活JAK-STAT信号通路。当这些配体与受体结合后,激活JAKs家族,激活的JAKs经过一系列的作用,导致STATs转录因子磷酸化而被激活,激活的STATs与受体分离,二聚体化转位到细胞核,一旦移位到细胞核,活化的STAT二聚体能与相应靶基因的启动子反应元件结合,从而启动相应基因的转录。不同的细胞因子或生长因子可以激活不同的JAK激酶和STAT转录因子,从而引起不同基因的转录。JAK-STAT信号通路在多种炎症反应中发挥着十分重要的作用。近年来,有研究表明JAK-STAT通路在多种心肌损伤中发挥作用,包括心肌梗死、氧化性损伤、心肌炎、心肌肥厚以及心室重构等。就目前而言,对心脏中STATs的研究多是对STAT以及STAT3的探讨。然而,迄今为止对STAT蛋白的作用仍然知之甚少,有待于进一步的研究来阐明。
肾素-血管紧张素系统(RAS)是血管扩张剂治疗心血管疾病的重要作用靶点。血管紧张素Ⅱ(AngⅡ)是一种具有多种功能的激素,可以通过AT1及AT2受体作用于一系列复杂的细胞内信号通路,从而影响心脏、血管细胞的功能。既往的研究表明,血管紧张素Ⅱ通过作用于AT1受体在肾损伤、慢性同种异体移植排斥反应、冠状动脉的移植以及病毒性心肌炎等急性炎症反应的免疫病理过程起着极其重要的作用。有证据表明,肾素-血管紧张素系统与多种炎症反应的过程有关。关于炎性疾病方面的研究表明血管紧张素Ⅱ参与了免疫及炎症反应,RAS拮抗剂通过阻断RAS通路而发挥对抗炎症及免疫反应的作用。然而,RAS拮抗剂对EAM的作用近年来才见报道,对其作用及机制仍然知之甚少。另外,近些年来的研究揭示,血管紧张素Ⅱ可以通过作用于AT1受体激活JAK-STAT通路。有相关的研究发现,在大鼠的心脏及心肌细胞中,血管紧张素Ⅱ与3AK-STAT的激活有很大的关系。尽管如此,血管紧张素Ⅱ受体阻滞剂缬沙坦对心肌自身抗原肌凝蛋白诱导的实验性自身免疫性心肌炎JAK-STAT信号通路的作用研究国内外未见报道。
目的：
本研究中,我们用纯化的猪的心肌肌凝蛋白免疫Lewis大鼠建立自身免疫性心肌炎(EAM)模型,该模型是代表人类心肌炎的理想模型。并且在免疫的同时给予EAM大鼠血管紧张素Ⅱ受体阻滞剂缬沙坦治疗3周。探讨EAM的发生发展中是否有JAK-STAT信号通路的激活,以及应用缬沙坦阻断RAS系统对EAM大鼠心肌中的JAK-STAT信号转导通路的影响。
方法：
6周龄雄性Lewis大鼠,SPF级,以猪的心肌肌凝蛋白免疫大鼠建立自身免疫性心肌炎模型(EAM)。所有的Lewis大鼠(n=24)随机分为3组,分别命名为未治疗组(Group N, n=8)、正常对照组(Group C, n=8)、缬沙坦治疗组(GroupH,10mg／kg／day,n=8)。将纯化的猪的心肌肌凝蛋白(浓度为10.0 mg／ml)与含有10.0 mg／ml灭活结核杆菌的等体积的完全弗氏佐剂充分混合成乳浊液用来免疫大鼠。1)Group N:将总体积0.2ml的乳浊液(含有肌凝蛋白1mg)分别于第0、7天皮下注射大鼠后脚垫建立EAM模型；2)Group C:对照组大鼠只给予等体积的不含肌凝蛋白的完全弗氏佐剂免疫；3)Group H:采用跟Group N组一样的方式对大鼠进行两次皮下注射免疫。
免疫的同时,缬沙坦治疗组的大鼠给予缬沙坦(10mg／kg／day)灌胃治疗三周。未治疗组及正常对照组大鼠给予生理盐水灌胃代替缬沙坦治疗。
在实验过程中,记录大鼠体重变化；测量心率、收缩压及舒张压的变化。初次免疫后21天,采用Agilent sonos 5500超声检测仪进行心脏超声检查,具体操作如第一部分所示。心脏超声检查结束后,处死老鼠,取出脾脏及心脏称重。计算心脏重量／体重比值,行病理学检查及淋巴细胞增值实验,如第一部分操作所述。
除此之外,免疫后三周,在处死之前取大鼠血清。用含有10μg／mL肌凝蛋白的磷酸盐缓冲液包被平底96孔板,采用间接ELISA法测定抗心肌肌凝蛋白自身抗体水平,结果以酶标仪读取405 nm波长处的吸光度值表示。进一步,处死大鼠后,立刻无菌取出心脏,一部分立刻放入液氮罐中,后转入-80℃保存备用。采用western blot测定大鼠心肌中JAK-STAT信号通道家族中p-JAK2, p-STAT1以及p-STAT3磷酸化蛋白表达水平,并以β-actin作为内参。
结果：
1.三组实验大鼠之间的心率、收缩压及舒张压并没有统计学差异,10mg／kg／day缬沙坦并没有影响大鼠的心率及血压。
2.心脏超声检查、病理学检查及淋巴细胞增值实验结果显示缬沙坦显著的改善了EAM。简言之,缬沙坦治疗显著的阻止了EAM大鼠心功能的损伤及心室重构；减轻了由心肌炎引发的病理损害；抑制了淋巴细胞对心肌肌凝蛋白刺激产生的抗原特异性增值反应。
3.缬沙坦对大鼠血清抗心肌肌凝蛋白自身抗体的影响结果显示：Group N组大鼠产生了大量的抗心肌肌凝蛋白自身抗体,抗体滴度显著高于Group C组及Group H组。该结果提示,心肌肌凝蛋白引发的自身免疫反应诱导体内产生了高水平的抗心肌肌凝蛋白自身抗体,缬沙坦抑制了此类抗体的产生。
4.免疫印迹(western blot)结果显示：与Group C组及Group H组相比较,Group N组大鼠心肌组织中磷酸化的JAK2(p-JAK2)表达显著的增高。该结果说明在JAK2在由心肌肌凝蛋白诱导的心肌损伤中被显著激活,而缬沙坦治疗抑制了JAK2的激活。
5. Western blot检测STATs家族磷酸化水平结果显示：Group N组大鼠心肌组织中p-STAT1及p-STAT3表达水平明显高于Group C组。更加有趣的是,缬沙坦同样减少了两种p-STATs的表达。该结果说明肌凝蛋白诱导的自身免疫性心肌损伤引起了STAT1及STAT3的活化,而缬沙坦可以抑制其活化程度。
结论：
以上的研究结果说明,JAK-STAT信号转导通路在心肌肌凝蛋白引起的自身免疫性心肌炎心肌损伤中显著激活。缬沙坦可以抑制磷酸化的JAK2、STAT1及STAT3的过度表达。缬沙坦可以不依赖于降压作用改善心肌炎,相关机制跟缬沙坦阻止JAK-STAT信号转导通路的激活有关。
Backgroud:
Myocarditis, a heterogeneous myocardial inflammatory disease, is characterized by myocyte necrosis and degeneration with mononuclear cell infiltration in the presence or absence of fibrosis. The clinical manifestation ranges from the asymptomatic state due to the focal inflammation to the fatal congestive heart failure due to diffuse myocarditis. Most patients with myocarditis can completely recover, while some may develop dilated cardiomyopathy (DCM) and manifest cardiac dilation and heart failure. Myocarditis is the major cause of sudden unexpected death in patients less than 40 years of age.
To date, the pathogenesis of myocarditis and the pathogenesis of myocarditis developing into DCM remains unclear. The etiology of myocarditis appears to be rather complicated involving idiopathic, invasion of virus (the major cause), invasion of bacterium and parasite, infectious exposure to drugs and toxins and autoimmune factors. Autoimmunity and immune mediators are known to play a critical role in the pathogenesis of an array of cardiovascular diseases including myocarditis. So the autoimmunity factor becomes a worldwide focus. There is substantial evidence suggesting that autoimmune responses to heart antigens, particularly cardiac myosin, following viral infection may contribute to the disease process. An experimental model of autoimmune myocarditis (EAM) has been induced in susceptible strains of rats produced by immunization with cardiac myosin, which resembles myocarditis in humans and can develop into DCM. So EAM is an excellent experimental model for studying the pathogenesis of myocarditis and the effective therapy. Domestic reports in this field are still relatively rare.
Because the pathogenesis of myocarditis and DCM remains unclear, treatments for myocarditis are directed toward reducing or eliminating the inciting agent when possible and tailoring therapy toward the associated the symptoms and complications, such as congestive heart failure, cardiogenic shock, conduction abnormalities and arrhythmia. Recent focus has been on the point that autoimmune response plays an important role in the development of myocarditis. It has been reported that EAM in rats is a CD4+T cell-mediated disease and is thought to be related to Thl responses in the acute phase. Proinflammatory cytokines and Thl/Th2 imbalance play crucial roles in the induction of EAM and in the progression of myocardial injury in this disease.
Recently, because evidences suggest that autoimmune response plays important role in the development of myocarditis, immune therapies are being taken more and more attention for the treatment of myocarditis. However, their effects are still controversial. The renin-angiotensin system (RAS) is primarily responsible for regulating vascular tone, which is a key target of vasodilator therapy. The effects of angiotensin II type 1 receptor blockers (ARBs) on the treatment of hypertension, heart failure, and other cardiovascular diseases have been confirmed extensively. However, recent studies have emphasized the nonhemodynamic effects of these drugs. Recently, interest has focused on the nonhemodynamic effects of these drugs. Some Ang-Ⅱreceptor type 1 (AT1) antagonists have been reported to be effective agents in modulating inflammation, adhesion molecule expression, and fibrosis. The cardioprotection of these drugs may be partly due to their regulation of inflammation as a result of removal of overproduced cytokines. Nevertheless, the effects of the ARBs on autoimmune diseases such as autoimmune myocarditis have not been well investigated.
Objective:
In the present study, the EAM rat model was established by injecting cardiac myosin twice and valsartan, a novel ARB, has been tested in the experimental model of autoimmune myocarditis. It was aimed at investigating the effects of valsartan on the development of EAM and the immune mechanisms involved, In addition, the present study might provide a promising new strategy for myocarditis and DCM. It represents a paradigm of immunosuppressive therapy on immune-mediated diseases.
Methods:
Thirty-two male Lewis rats (6 weeks old) were maintained in the study. Twenty-four susceptible rats were immunized with porcine cardiac myosin to establish EAM. All Lewis rats were randomly divided into four groups:1) Normal control group (Group C, n=8):Rats were immunized with Freund's complete adjuvant alone, and they received physiological saline instead of durgs administration for three weeks; 2) Non-treated group (Group N, n=8):Purified porcine cardiac myosin (1 mg) was injected subcutaneously in the rear foot pads mixed with an equal volume of Freund's complete adjuvant supplemented with Mycobacterium tuberculosis on days 0 and 7 respectively to establish EAM, and they also received physiological saline instead of durgs administration; 3) Low-dose valsartan (3 mg/kg/day, Group L, n=8): Rats were immunized twice with porcine cardiac myosin to establish EAM and they were administrated orally by valsartan (3mg/kg/day) at the same time of immunization from day 0 to day 21; 4) High-dose valsartan (10 mg/kg/day, Group H, n=8):Rats were immunized twice with porcine cardiac myosin to establish EAM and they received valsartan therapy (10 mg/kg/day) for three weeks.
At the time of immunization, we noted the changes of body weight. Heart rate, systolic and diastolic blood pressure were measured by tail-cuff method using a photoelectric tail-cuff detection system and recorded regularly. All rats were killed 21 days after the first immunization.
21 days after the after the first immunization, Lewis rats were anesthetized and secured to a warming table. To evaluate the cardiac function and heart structure of the rats, transthoracic echocardiographic analysis was performed by the accurate method using Agilent sonos 5500 echocardiograph with 11-13 L transducer. We obtained the M-mode echocardiogram in the two-dimensional short-axis view of the left ventricular at the chordae tendineae level. After the echocardiography examination, all the rats were sacrificed and their hearts were immediately removed and weighted, then the ratio of heart weight/body weight (g/kg) was calculated. The heart specimens were fixed in 4.0%formaldehyde. Heart sections were stained were stained by the hematoxylin-eosin or Masson's trichrome. Inflammation and fibrosis was examined by light microscopy. Pathologic score was calculated. In order to observe lymphocyte proliferation to cardiac myosin, spleens were collected and cell suspensions were prepared on day 21 after the first immunization and used to preform lymphocyte proliferation assay by MTT colorimetric technique immediately. In addition, serum samples were obtained and the serum concentrations of IFN-y, interleukin (IL)-2 as representative Thl cytokines and IL-4, IL-10 as representative Th2 cytokines were analyzed by ELISA.
Results:
1. There was no statistically significant difference in blood pressure or heart rate values between the groups. Systolic blood pressure and diastolic blood pressure in Group N were slightly lower than Group C. However, the valsartan dosages did not affect blood pressure.
2. Body weights were significantly lower in Group N compared with the groups, the effect of which was ablated by valsartan treatment. Following 3 weeks of autoimmune disease, hearts were significantly enlarged in rats from Group N compared with Group C or Group H. With regard to the heart weight/body weight ratio, it was increased in Group N compared with that in Group C. Valsartan treatment suppressed the increase in that value in a dose-dependent manner.
3. Transthoracic echocardiography was performed to assess heart function in all three rat groups. Compared with Group C, LVEDs and LVEDd were significantly enhanced, left ventricular fractional shortening (LVFS) was greatly impaired in Group N, and treatment with high-dose valsartan prevented the left ventricular dysfunction. Moreover, the experimental autoimmune myocarditis significantly enhanced IVS and LVPW, while treatments with both low and high-dose valsartan inhibited the increase. Echocardiography analysis demonstrated that heart structure and cardiac function were greatly impaired in EAM rats, and the treatment with valsartan significantly prevented the progression of autoimmune myocarditis-induced left ventricular dysfunction and ventricular remodeling.
4. On day 21 after the first immunization, hearts from Group N were markedly enlarged and contained large grayish areas. Autoimmune myocarditis elicited typical cardiac histopathological changes in myocardium which were manifested as presence of fragments of necrotic myocardial fibers, mononuclear cells, polymorphonuclear neutrophils, eosinophils and giant cells. Masson's trichrome staining showed extensive myocardial fibrosis under autoimmune myocarditis. Treatment with 3 and 10 mg·kg-1·day-1 valsartan significantly reduced the severity of the disease as assessed by detecting the typical signs of myocarditis. Our further quantitative evaluation revealed that the severity of EAM and affected area was significantly suppressed by valsartan in a dose-dependent manner.
5. Our data from lymphocyte proliferation assay suggested that lymphocytes obtained from rats in Group N had remarkable proliferative response to porcine cardiac myosin. And this antigen-specific proliferative response to myosin stimulation was significantly suppressed by valsartan treatment in a dose-dependent manner. However, the difference did not reach statistical significance in lymphocytes proliferative reaction to the non-specific mitogen ConA among the four groups.
6. Serum analysis revealed significantly elevated serum levels of the Thl cytokines IFN-y and IL-2 in rats from Group N compared with groups, the effect of which was abrogated by valsartan treatment in a dose-dependent manner. In contrast, production of Th2-type cytokines, including IL-4 and IL-10 was significantly higher in the two valsartan-treated groups compared with Group N. These results suggested that valsartan treatment may shift the balance of helper T cells from Thl to Th2.
Conclusions:
The collective results suggest that valsartan inhibites the progression of myocardial function, remodeling and morphological alteration in a rat model of experimental autoimmune myocarditis in a dose-dependent manner. These data favor the notion that the mechanism of amelioration of the disease by this inhibitory agent can be at least partly explained by the removal of proinflammatory cytokines and the Th1/Th2 imbalance independent of BP-lowering effects.
Backgroud:
Myocarditis is one of the important causes of dilated cardiomyopathy and severe congestive heart failure. Although myocarditis is habitually viewed as a chronological sequence of several pathologically distinct phases, its precise pathogenesis remains unclear. To date, there is no universally accepted effective therapy for myocarditis. Treatments for myocarditis are not directed toward the disease itself but instead of managing the symptoms and complications. It is a heterogeneous myocardial inflammatory disease, the etiology of myocarditis appears to be rather complicated involving idiopathic, infectious or autoimmune factors. Myocarditis is a disease associated with immune dysfunction and inflammatory reaction that frequently precedes the development of dilated cardiomyopathy. It is now known that proinflammatory cytokines are important for the activation of the detrimental autoimmune responses, such as myocarditis. Cardiac myosin-induced experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by immune response.
The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway was initially discovered as a major intracellular signal transduction pathway of the cytokine superfamilies. It is activated by many cytokines and growth factors. Binding of ligands to receptors leads to the activation of the JAK tyrosine kinase family, and activated JAK kinases phosphorylate various combinations of STAT transcription factors, which form dimers, translocate into the nucleus, and bind response elements in the promoters of target genes to stimulate transcription. Different cytokines and growth factors activate different combinations of JAK kinases and STAT transcription factors. JAK-STAT plays a crucial role in various inflammatory reaction. The JAK/STAT pathway has recently been shown to be an integral part of the response of the myocardium to various cardiac insults, including myocardial infarction, oxidative damage, myocarditis, hypertrophy and remodeling. The majority of data involving STAT activity in the heart is confined to STAT1 and STAT3. However, these two STAT proteins have not been well have not been well investigated.
The renin-angiotensin system (RAS) is a key target of vasodilator therapy. AngiotensinⅡ(AngⅡ) is a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of AngⅡwith AT1 and AT2 receptors. Previous studies have reported that Ang II, acting through the AT1 receptor, played an important role in the immunopathology involved in renal injury, chronic allograft rejection and graft coronary artery disease, as well as acute inflammation, such as viral myocarditis. Evidence suggests that the renin-angiotensin system(RAS)Contributes to several steps of the inflammatory process. Studies in inflammatory diseases suggested that Ang II was involved in immune and inflammatory responses and RAS antagonists produced effects by blocking the action markedly. The effects of RAS antagonists on EAM have only recently been addressed. In addition, recent studies have revealed that Ang II activates the JAK-STAT pathway via the AT1 receptor. It has been reported that the JAK/STAT pathway is activated in the rat heart or cardiomyocytes and angiotensinⅡis involved in. However, the effects of angiotensinⅡreceptor blocker valsartan on JAK-STAT signal transduction pathway of experimental models of antigen-induced autoimmune myocarditis have not been addressed.
Objective:
In our study, Lewis rats were immunized with porcine cardiac myosin to induce experimental autoimmune myocarditis, which is an ideal animal model of human myocarditis. Valsartan was administered orally for 3 weeks to rats with EAM at the same time of immunization. We investigated whether activation of the JAK/STAT pathway was involved in the process of EAM and whether blockade of the renin-angiotensin system by an AT1 receptor antagonist (valsartan) inhibited the JAK/STAT pathway in the rat heart of EAM.
Methods:
Inbred 6-week-old male Lewis rats (SPF) were immunized with purified porcine cardiac myosin to induce experimental autoimmune myocarditis (EAM). All Lewis rats (n=24) were divided into three groups at random, namely non-treated group (Group N, n=8), normal control group (Group C, n=8), and valsartan-treated group (Group H, n=8). The purified porcine cardiac myosin (10.0 mg/ml) was mixed with an equal volume of Freund's complete adjuvant supplemented with 10.0 mg/ml of Mycobacterium tuberculosis.1) Group N:A total volume of 0.2 ml of the emulsion (lmg porcine cardiac myosin) was injected subcutaneously in each rear footpad of rats on days 0 and 7 respectively; 2) Group C:Rats in control group were given an equal volume of Freund's complete adjuvant alone; 3) Group H:Rats were immunized twice on days 0 and 7 with 0.2 ml of the emulsion.
At the same time of immunization. Rats in Group H were received valsartan (10 mg/kg/day) orally by gastric gavage for 3 weeks from day 0 to day 21 prior to sacrifice. Rats from Group C and Group N received physiological saline instead of valsartan.
During the study, the changes of body weight were noted. We measured heart rate, systolic and diastolic blood pressure regularly by tail-cuff method using a photoelectric tail-cuff detection system. Twenty-one days after the first immunization, transthoracic echocardiography was performed on animals using Agilent sonos 5500 echocardiograph with 11-13 L transducer. The examination of transthoracic echocardiography was the same as described in part 1. After the transthoracic echocardiographic analysis, the rats were sacrificed and their hearts were immediately removed. Histologic evaluation and lymphocyte proliferation assay were performed also the same as described in part 1.
In addition, rat serum samples were obtained 3 weeks postimmunization before sacrificed. Cardiac myosin (10μg/mL) was coated in flat-bottom 96-well ELISA plates, and anti-cardiac myosin autoantibody assay were performed. Absorbance was evaluated as indicator for the levels of anti-cardiac myosin autoantibody using a test wavelength of 405 nm. Furthermore, after the animals were sacrificed, the rat hearts samples were immediately harvested, frozen in liquid nitrogen and stored at-80℃until analysis. We detected tyrosine-phosphorylated protein members of the JAK-STAT family (p-JAK2, p-STAT1 and p-STAT3) in rat myocardial tissue by western blot. To normalize protein loading, the membranes were cut and the lower molecular weight portion was analyzed with the primary antibody forβ-actin.
Results:
1. There were no statistically significant differences between the three groups with regard to systolic blood pressure, diastolic blood pressure and heart rate values between the groups.
2. The results of transthoracic echocardiography, histologic evaluation and lymphocyte proliferation assay showed that valsartan improved EAM significantly. In summary, valsartan treatment significantly prevented the left ventricular dysfunction and severely altered heart structure following myocarditis, reduced the myocarditis-affected histopathologic areas, and suppressed antigen-specific proliferative response to cardiac myosin stimulation.
3. Effect of valsartan treatment on serum anti-cardiac myosin autoantibody was examined. Rats in Group N produced large numbers of anti-cardiac myosin autoantibody and the titer of the autoantibody was significantly higher in Group N than in Group C and Group H. These data indicated that autoimmunity triggered by porcine cardiac myosin significantly facilitated large numbers of anti-cardiac myosin autoantibody, the effect of which was nullified by valsartan treatment.
4. Western blot showed that expression of phosphorylation JAK2 (p-JAK2) in myocardium of EAM rats were much higher than Group C and Group H. The date suggested that JAK2 was activated in myosin-induced cardiac injury, and the activation of p-JAK2 was abrogated by valsartan treatment.
5. Tyrosine-phosphorylated members of the STAT family (p-STAT1 and p-STAT3) were also detected by western blot. The expression levels of p-STAT1 and p-STAT3 were significantly increased in Group N compared with Group C. Interestingly, this expression was attenuated by valsartan administration. This suggests that the activation of STATs is induced by autoimmune myocarditis and valsartan might block the induction.
Conclusions:
As is refered in our article, the JAK-STAT signal transduction pathway was significantly activated in cardiac myosin-induced autoimmune myocarditis, and valsartan treatment suppressed the over-expression of phosphorylated JAK2, STAT1 and STAT3. Valsartan could ameliorate EAM independent of BP-lowering effects. The mechanism involved might be the effects of valsartan by which blocking the activity of JAK-STAT signal transduction pathway.
Key words:
Valsartan; experimental autoimmune myocarditis; JAK-STAT signal transduction pathway.
引文
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