【作者】 孙琳；

【导师】 苑海涛；

【作者基本信息】 山东大学 ， 内科学（心血管病）（专业学位）， 2021， 硕士

【摘要】 背景:病毒性心肌炎(Viral myocarditis,VMC)是指由于感染各类嗜心肌性病毒所导致的心肌非特异性疾病,VMC是导致青年人发生心力衰竭和心源性猝死的重要原因。临床表现为乏力、胸痛、心力衰竭、恶性心律失常、休克,甚至发生心脏骤停。虽然大多数病毒性心肌炎患者能够自愈或经治疗后好转,但有部分患者进展为扩张型心肌病、心律失常和严重的心力衰竭,极大地影响了患者的生活质量和寿命。现阶段仍然没有特异性的针对病毒性心肌炎的有效治疗方法,目前临床上应用的抗病毒治疗及对症支持治疗,效果并不显著。一直以来,国内外学者长期致力于探究病毒性心肌炎的发病机制,目前广为接受的机制包括病毒对心肌的直接损伤、免疫反应过度激活及各种细胞因子介导的心肌损伤,然而其具体机制仍不完全清楚。焦亡(Pyroptosis)是一种新型的炎症性细胞程序性死亡,由炎症小体介导,并且伴有包括IL-1β和IL-18在内的多种炎性细胞因子释放,与凋亡不同。炎症小体是一种胞内蛋白复合体,由三部分组成:胞浆内模式识别受体、适配蛋白ASC和半胱氨酸蛋白酶原-1(Caspase-1),炎症小体激活GSDMD,导致细胞膜孔形成,细胞破裂,释放IL-1β和IL-18。炎症小体与许多炎症性疾病有关,如心肌梗死和缺血再灌注损伤。最近的研究表明,在VMC患者和VMC小鼠模型中存在炎性小体。因此,心肌细胞焦亡在VMC发病进程中可能具有重要作用,而阻断这一过程有希望成为治疗VMC的新靶点。白介素-37(IL-37)属于IL-1家族,是一种新型的潜在的炎症抑制因子。在自身免疫性疾病、风湿性疾病、恶性肿瘤等疾病中发挥抗炎作用,而小鼠体内不表达IL-37。我们先前的动物实验研究表明,IL-37能够减轻小鼠病毒性心肌炎的心脏内炎症反应,降低小鼠的死亡率。然而,IL-37对病毒性心肌炎心肌细胞焦亡的作用及其机制尚不清楚。因此,本研究通过建立小鼠病毒性心肌炎模型,应用IL-37对模型小鼠进行干预,研究IL-37对病毒性心肌炎发病过程中的具体作用,并进一步探讨IL-37参与心肌细胞焦亡的影响及其机制,为病毒性心肌炎的治疗提供崭新的治疗靶点。我们的实验主要围绕以下两个部分展开:第一部分:小鼠病毒性心肌炎动物模型的构建研究背景及意义心肌炎是指心肌组织内的非特异性炎性疾病,全球每年约有150万人患心肌炎,可以表现为急性、亚急性或者慢性心肌炎。任何年龄阶段的人群均可能患心肌炎,但以青少年最为多见。病毒是其最常见的病原体,肠道病毒属的柯萨奇病毒最为多见。细菌、真菌、寄生虫、立克次体等也可以引起心肌炎,但临床上相对比较少见。病毒性心肌炎的患者症状表现差异较大,主要取决于心肌的病变所累及的范围,轻症患者可以没有任何自觉症状,部分患者发病前存在7-20天左右的前驱期,表现为发热、乏力,肌肉疼痛等感染症状。随后临床表现为胸痛、心律失常、充血性心力衰竭、心源性休克甚至心脏骤停。一般认为,造成VMC患者心肌损害的原因主要如下:病毒复制的直接损伤;免疫反应被触发后过度激活;炎症反应过程中细胞因子的作用以及各种原因诱导的心肌细胞凋亡。病毒入侵后在心肌内复制,并导致免疫系统激活,其病理生理学进展过程通常有三个阶段:急性阶段、亚急性阶段和慢性阶段。急性阶段是指病毒对心肌的直接毒性作用,亚急性阶段主要表现为T细胞及B细胞的活化及其引发的自身免疫反应,慢性阶段表现为心肌弥漫纤维化与心脏泵功能障碍,如扩张性心肌病。大量病理学证据表明,病毒性心肌炎表现为心肌炎性细胞浸润,主要包括单核细胞,包括淋巴细胞,巨噬细胞,树突状细胞(DC)和自然杀伤(NK)细胞。病毒性心肌炎患者的预后取决于发病原因、临床症状的严重程度和起始治疗是否及时。其预后不良的因素包括射血分数降低和左束支传导阻滞等。轻度心肌炎患者通常预后良好,约有50%的VMC患者可自行缓解。然而,仍有25%的VMC患者可发展为心功能不全,严重者发展为扩张性心肌病。重症心肌炎最常见的死亡原因是心源性休克,其1年内死亡率为20%,5年死亡率为50%。然而现阶段仍无特异性的治疗方法,单纯抗病毒治疗及对症支持治疗,效果并不明显。在本实验的第一部分中,为了模拟病毒性心肌炎的发病进程,我们对balb/c小鼠腹腔注射CVB3病毒液,观察病毒性心肌炎的病理学特征,探究其病理机制,为VMC的治疗提供新的思路。研究目的1.通过对balb/c小鼠腹腔注射CVB3病毒液,建立急性病毒性心肌炎模型,观察小鼠心肌损伤的病理学改变。2.通过对小鼠体重、活力、毛发、心脏超声、心肌病理学切片等进行观察与监测,评估使用CVB3 Nancy株构建小鼠病毒性心肌炎模型的效果。研究方法1.建立动物模型将12只雄性6周龄balb/c小鼠饲养于山东省医学科学院SPF类动物房,随机分为对照组和模型组,每组6只。Day 0对对照组小鼠腹腔注射100μl PBS,对模型组小鼠腹腔注射25μl 109TICD50病毒液(加入75μl PBS稀释至100μl),饲养9天后建立急性心肌炎动物模型。2.超声心动图Day 9用3%异氟醚麻醉小鼠,使用Visual Sonics高分辨率Vevo 2100系统(Visualsonics Inc.,加拿大多伦多)采集胸骨旁长轴图像,记录每组小鼠左心室射血分数(LVEF),缩短分数(LVFS),收缩期室间隔厚度(IVSs),舒张期室间隔厚度(IVSd),收缩期左室内径(LVIDs),舒张期左室内径(LVIDd),收缩期左室后壁厚度(LVPWs),舒张期左室后壁厚度(LVPWd)。3.心肌组织病理学检测于Day 9心脏超声检查后收集各组小鼠的心脏标本,石蜡包埋切片后进行苏木精和伊红(H&E)染色,Masson染色检测心肌纤维化程度。4.ELISA 检测收集各组小鼠外周血,离心收集上层血清,ELISA检测心肌损伤标志物cTnI与炎症因子IL-1β、IL-18。研究结果1.模型组小鼠一般状态较差对照组小鼠活力良好,毛色鲜亮;与对照组小鼠相比,模型组小鼠活力欠佳,不喜活动,毛发暗淡无光泽,抓取时反应不明显。与对照组相比,VMC模型组小鼠体重自第三天开始明显下降,心脏重量/体重比值升高。2.模型组小鼠左心功能下降与对照组小鼠相比,模型组小鼠心脏超声示IVSs(P<0.01),IVSd(P<0.05)明显减小,LVEF、LVFS测量值下降,表明对小鼠腹腔注射CVB3后室间隔明显变薄,心肌收缩功能存在受损的可能性。3.模型组小鼠心肌炎症损伤明显对照组小鼠切片HE染色显示心肌细胞规律排列,未见明显炎症浸润,模型组小鼠HE染色可见部分心肌细胞坏死及炎症病灶,间质内炎症细胞浸润明显,病理学评分较对照组升高(P<0.0001)。模型组小鼠比对照组小鼠血清心肌损伤标志物 cTnI(P<0.01)、IL-1β(P<0.05)、IL-18(P<0.05)明显升高。4.模型组小鼠心肌纤维化明显Masson染色可见对照组小鼠心肌间质内无明显胶原纤维分布,模型组小鼠心肌可见灶性坏死伴胶原纤维增生,间质内可见较多胶原纤维分布。研究结论1.对balb/c小鼠腹腔注射CVB3病毒液能够成功构建小鼠VMC模型。2.病毒性心肌炎小鼠心肌损伤表现为炎性细胞因子表达升高,炎症浸润增加及心肌纤维化加剧。第二部分:IL-37对VMC小鼠心肌细胞焦亡的作用及机制研究研究背景及意义病毒性心肌炎是指机体感染嗜心肌病毒后,心肌组织内出现的非特异性炎症,其临床症状及体征轻重不一。尽管部分患者临床表现轻微,甚至症状表现呈自限性,病毒性心肌炎仍然能够引起一系列严重的并发症。据统计,VMC能够占到年轻人猝死原因的10%左右。VMC的发病机制尚不完全清楚。近期有研究证实,炎症小体的过度激活可能引起心肌炎、动脉粥样硬化及结肠炎等疾病。炎症小体又称焦亡小体,是一种细胞内的蛋白复合体,由胞质内的模式识别受体(PRR)、适配蛋白ASC与半胱氨酸蛋白酶原-1(Caspase1)构成,其中研究最为广泛的模式识别受体是NOD样受体家族的NLRP3。当NLRP3被细胞毒素、ATP或胆固醇等激活后,进一步募集ASC与Caspase1,炎症小体激活后切割IL-1β与IL-18。同时,NLRP3裂解Gasdermin D,释放其N末端结构域,形成细胞膜孔,释放炎性因子,引起一系列免疫反应。因此,抑制炎症小体表达或为病毒性心肌炎的治疗提供新的思路。IL-37是IL-1家族的一种潜在炎症抑制因子,对机体固有免疫和适应性免疫存在一定抑制作用。IL-37在人外周血单核细胞和树突状细胞中低表达,而小鼠体内不表达IL-37。IL-37已被证实能够改善CVB3引起的小鼠病毒性心肌炎,然而其具体机制并不清楚。因此,本部分实验通过应用重组人IL-37对病毒性心肌炎小鼠干预,观察其对心肌炎小鼠炎症浸润及NLRP3炎症小体表达的影响,探讨IL-37对病毒性心肌炎的作用及具体机制。研究目的:1.构建病毒性心肌炎模型,通过IL-37对病毒性心肌炎小鼠进行干预,观察IL-37对病毒性心肌炎小鼠一般状态、心功能和心肌病理学切片的影响。2.通过检测各组小鼠心肌细胞的炎症小体的表达,探讨IL-37减轻小鼠病毒性心肌炎的具体机制。研究方法:1.IL-37干预病毒性心肌炎小鼠将32只雄性6周龄balb/c小鼠饲养在山东省医学科学院SPF级动物房,随机分为4组:对照组(n=6)、IL-37对照组(n=6)、模型组(n=10)、实验组(n=10)。Day 0对对照组、IL-37对照组小鼠各腹腔注射100μtlPBS,同时对模型组、实验组小鼠腹腔注射25μl 109TICD50病毒液(加入75μl PBS稀释至100μl)。Day3、Day7对对照组、模型组小鼠腹腔注射200μl PBS,同时对IL-37对照组、实验组腹腔注射2μg IL-37(加入200μl PBS溶解),饲养9天后建立急性心肌炎动物模型。2.超声心动图评估小鼠心功能Day 9用异氟醚麻醉小鼠,采集胸骨旁长轴图像,记录每组小鼠左心室射血分数(LVEF),缩短分数(FS),收缩期室间隔厚度(IVSs),舒张期室间隔厚度(IVSd),收缩期左室内径(LVIDs),舒张期左室内径(LVIDd),收缩期左室后壁厚度(LVPWs),舒张期左室后壁厚度(LVPWd)。3.心肌组织病理学检测收集各组心脏标本,石蜡包埋后用苏木精和伊红(H&E)染色、Masson染色分别检测心肌组织的炎症变化及纤维化程度,免疫荧光检测各组大鼠心肌组织GSDMD蛋白表达情况。4.ELISA 检测收集各组小鼠外周血,离心收集上层血清,ELISA检测心肌损伤标志物cTnI与炎症因子IL-1β、IL-18。5.蛋白印迹实验收集各组小鼠心肌组织,提取心肌组织总蛋白后,Western blot检测NLRP3、ASC dimer、cleavaged caspase 1、NFkB P65 及 phospho-P65 在心肌组织中的表达。6.RT-PCR从液氮中取出各组小鼠心肌组织,液氮研磨组织提取总RNA,PCR检测NFκB P65的mRNA表达水平。研究结果1.IL-37改善病毒性心肌炎小鼠一般状态与模型组小鼠相比,实验组小鼠活力较好,毛色光滑,抓取时反应明显。与模型组相比,实验组小鼠体重同样呈现持续下降趋势,心脏重量/体重比值低于模型组小鼠(P<0.05)。2.IL-37改善病毒性心肌炎小鼠心功能与模型组小鼠相比,实验组小鼠LVEF(P<0.05)、LVFS(P<0.05)、IVSs(P<0.01)和IVSd(P<0.01)均有不同程度的改善,表明实验组小鼠心脏室间隔厚度高于模型组,左室收缩功能较模型组有所改善。3.IL-37降低病毒性心肌炎小鼠心肌炎症损伤小鼠心肌切片HE染色显示,与模型组相比,实验组小鼠心肌炎症病灶浸润较少,心肌细胞坏死减少,间质内炎症细胞分布较少。外周血Elisa检测显示,与模型组小鼠相比,实验组小鼠cTnI(P<0.01)与IL-18(P<0.05)表达明显下降。4.NLRP3炎症小体在模型组小鼠表达升高,在实验组中表达降低Western blot检测显示:模型组小鼠NLRP3(P<0.01)、ASC(P<0.05)与cleaved-caspase 1(P<0.05)蛋白比对照组升高,实验组小鼠NLRP3(P<0.05)、ASC dimer(P<0.001)与cleaved-caspase 1(P<0.05)蛋白比模型组表达降低。GSDMD的免疫荧光染色检测显示:模型组中GSDMD的表达水平高于对照组(P<0.01);实验组GSDMD的表达水平低于模型组(P<0.01)。5.IL-37可能通过抑制NFκB降低心肌炎小鼠NLRP3炎症小体表达Western blot检测显示:模型组小鼠比对照组NFκB P65(P<0.05)、phospho P65(P<0.01)的表达明显升高;实验组 P65(P<0.01)、phosphoP65(P<0.01)的表达低于模型组,IL-37对照组中P65(P<0.05)、phospho P65(P<0.05)的表达水平明显低于对照组。RT-PCR检测发现模型组P65 mRNA表达高于对照组(P<0.05),实验组P65 mRNA表达水平明显低于模型组(P<0.05),IL-37对照组P65 mRNA比对照组表达水平明显下降(P<0.05)。研究结论1.IL-37可以减轻病毒性心肌炎小鼠的心肌损害,改善小鼠一般状态。2.NLRP3炎症小体参与CVB3诱导的病毒性心肌炎,IL-37抑制NLRP3炎症小体表达。3.IL-37可能通过抑制NFκB通路改善小鼠病毒性心肌炎。更多还原

【Abstract】 Background:Viral myocarditis(VMC)is a kind of a non-specific inflammatory disease in myocardium,which is an important cause of heart failure and sudden death in young people.It is mainly caused by viral infection.The clinical manifestations including fatigue,chest pain,congestive heart failure,cardiogenic shock and even cardiac arrest.Although most viral myocarditis is self-limited,some patients developed into dilated cardiomyopathy,malignant arrhythmia and heart failure,which seriously affect the quality of life.There is still no specific treatment for VMC.Antiviral and symptomatic supportive treatment is not significant.For a long time,scholars have devoted themselves to the study of the pathogenesis of viral myocarditis,including direct viral damage,immune response and various cytokine-mediated myocardial damage,but the mechanism is still unclear.Pyroptosis refers to inflammatory cell programmed death,which is mediated by inflammasomes.Pyroptosis is accompanied by the release of many inflammatory cytokines,such as IL-1β and IL-18.The inflammasome is an intracellular protein complex,and it is composed of three parts:pattern recognition receptor in cytoplasm,adapted protein ASC and cysteine Caspase-1 inflammasome activates GSDMD,causing the cell membrane to rupture and release IL-1β and IL-18.Inflammasomes are related to many inflammation-related diseases,such as myocardial infarction and ischemia-reperfusion injury.Recent studies have shown that there are over-activated inflammasomes in VMC patients and mice.Therefore,cardiomyocyte pyroptosis may play an important role in the pathogenesis of VMC,and blocking this process may become a new target for the treatment of VMC.Interleukin 37(IL-37)belongs to the IL-1 family and is a potential inhibitor of inflammation.It plays an anti-inflammatory effect in ankylosing spondylitis,ulcerative colitis,small cell lung cancer and other diseases.Previous studies have shown that IL-37 can reduce the inflammatory response of viral myocarditis in mice and reduce mortality.However,the effect of IL-37 on myocardial cell pyroptosis in viral myocarditis and its mechanism are still unclear.Therefore,we established a viral myocarditis model in this study and applied IL-37 to intervene in model mice to uncover the role of IL-37 in the pathological process of viral myocarditis,and further explored the involvement of IL-37 in myocardial cell pyroptosis.This study is divided into two parts:Part I:Establishment of an Animal Model of Viral Myocarditis in MiceBackgroundMyocarditis refers to an inflammatory disease of the myocardium,which is diagnosed by non-invasive imaging techniques such as electrocardiogram,serum virological detection,echocardiography,and endocardial biopsy(EMB).Approximately 1.5 million people worldwide suffer from myocarditis each year,which can manifest as acute,subacute or chronic myocarditis.Myocarditis can affect people of any age,and is most common in adolescents.Viruses are the most common pathogen that causes myocarditis.Among the most common viruses are enteroviruses,including Coxsackie virus.Parvovirus B-19 and human herpesvirus 6 are also common pathogens.Bacteria,fungi,rickettsia,etc.can also cause myocarditis,but it is relatively rare.Patients with viral myocarditis have different symptoms,depending on the extent of the cardiomyopathy.Mild patients may have no conscious symptoms.Some patients have a prodromal period of about 7-20 days before the onset,which manifests as fever,fatigue,and muscles.Pain and other infection symptoms.The subsequent clinical manifestations include chest pain,arrhythmia,cardiogenic shock and sudden cardiac arrest.It is generally believed that myocardial damage in patients with viral myocarditis is mainly caused by four aspects:direct damage of the virus;immune response triggered by the virus;the role of cytokines in the process of inflammation and the apoptosis of myocardial cells induced by various reasons.After the virus invades,it replicates in the myocardium and activates the immune system.Its pathophysiological progress usually has three stages:acute stage,subacute stage and chronic stage.The acute stage refers to the direct toxic effect of the virus on the myocardium.The subacute stage is mainly manifested by the activation of T cells and B cells and the autoimmune response triggered by it.The chronic stage is manifested by diffuse myocardial fibrosis and cardiac dysfunction,such as dilated myocardium.disease.A large amount of pathological evidence indicates that viral myocarditis is manifested by myocardial inflammatory cell infiltration,mainly including monocytes,including lymphocytes,macrophages,dendritic cells(DC)and natural killer(NK)cells.The prognosis in VMC patients depends on the pathogenisis,clinical symptoms,and whether the initial treatment is timely.Poor prognostic factors include decreased ejection fraction and left bundle branch block,etc.Patients with mild myocarditis usually have a good prognosis.However,the most common cause of death in severe myocarditis is cardiogenic shock,with a 1-year mortality rate of 20%and a 5-year mortality rate of 50%.However,there is still no specific treatment at this stage.Only antiviral and supportive treatment is given,and the therapeutic effect is not significant.In the first part of this experiment,we established the model of viral myocarditis by intraperitoneal injection of viral myocarditis into balb/c mice,observed the pathological characteristics of viral myocarditis,explored its pathological mechanism,and provided some new ideas for treatment of viral myocarditis.Objective1.To establish a model of viral myocarditis in mice by intraperitoneal injection of CVB3 virus and observe the pathological changes of myocardial injury in mice.2.To observe the general state of mouse,including vitality,hair,etc.And to detect the cardiac function and cardiac pathological changes.To evaluate the feasibility of establishment of VMC model induced by CVB3.Methods1.Establishment of an animal modelTwelve male 6-week-old balb/c mice were reared in the SPF animal room of Shandong Academy of Medical Sciences.The mice were randomly divided into the control group(n=6)and the VMC group(n=8).On Day 0,mice in the control group had intraperitoneal injection with 100gL PBS,and mice in the VMC group were injected with 25μl 109TICD50(Add 75μl PBS to dilute it to 100μl).An animal model of acute myocarditis was established on day 9.2.EchocardiographyOn Day 9,mice were anesthetized with 3%isoflurane.We collected parasternal long-axis images by Visual Sonics high resolution Vevo 2100 system(Visualsonics Inc.,Toronto,Canada).The left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),systolic septal thickness(IVSs),diastolic septal thickness(IVSd),systolic left ventricular diameter(LVIDs),diastolic left ventricular diameter(LVIDd),the systolic left ventricular posterior wall thickness(LVPWs)and the diastolic left ventricular posterior wall thickness(LVPWd)of each group were recorded.3.Histological examinationAll 12 balb/c mice were sacrificed on the day 9.The heart tissue was immersed in 4%paraformaldehyde for 24 to 48 hours.After being embedded in paraffin,tissues were cut into 5 μm thick sections.Then the sections were stained with hematoxylin and eosin(H&E)and Masson.4.ELISA testCollect peripheral blood in each group.And collect the upper serum after centrifugation.The specific ELISA kits were used to detect the myocardial injury marker cTnI and the inflammatory factors IL-1β and IL-18.Results1.General statusThe mice in the control group were vigorous and had bright hair.Compared with the mice in the control group,the mice in the VMC group had poor vigor,and did not like to move.Mice in VMC group and had obvious hair loss and had no obvious response when grabbing.The weight of the mice in the VMC group were lower than the control group,and the heart weight/body weight ratio was increased in the VMC group.2.Decreased left heart function in VMC groupThe IVSs(P<0.01)and IVSd(P<0.05)of the VMC group mice were significantly lower than the control group.The values of LVEF and LVFS decreased in the VMC group,indicating that the ventricular septum was significantly thinner after intraperitoneal injection of CVB3 in mice.The contractile function of myocardium may be impaired.3.Myocardial inflammatory injury in the VMC group was obviousHE staining showed regular arrangement of myocardial fibers in the control group,without obvious inflammation infiltration.HE staining of mice in the VMC group showed some cell necrosis and inflammatory lesions,with obvious interstitial inflammation infiltration.The levels of serum cTnI(P<0.01),IL-1β(P<0.05)and IL-18(P<0.05)in the VMC group was significantly higher than the control group.4.Myocardial fibrosis was obvious in the VMC groupMasson staining showed obvious collagen fiber distribution in myocardial interstitium in the control group.The VMC group showed much areas of focal necrosis and collagen fibers.Conclusion1.Intraperitoneal injection of CVB3 into balb/c mice can successfully establish a mouse viral myocarditis model.2.Myocardial damage in mice with viral myocarditis includes increased levels of serum inflammatory cytokines,inflammatory infiltration and myocardial fibrosis in myocardial tissue.Part II:Study on the role and mechanism of IL-37 in viral myocarditis myocardial cell pyrolysisBackgroundViral myocarditis(VMC)refers to the non-specific inflammation of myocardium caused by virus infection,including CVB3.Its clinical manifestations vary from person to person.Although some patients have mild clinical manifestations and even self-limiting symptoms,viral myocarditis can still cause a series of serious complications,including arrhythmia,dilated cardiomyopathy,and heart failure.It is reported that VMC can account for about 10%of sudden deaths among young people.The pathogenesis of VMC is not fully clear.Recent studies have confirmed that the excessive activation of inflammasome plays an important role in diseases such as myocarditis,colitis and atherosclerosis.Inflammasome is a kind of intracellular protein complex,which is composed of cytoplasmic pattern recognition receptor,aptamer protein ASC and cysteine protease-1.Among them,the most widely studied pattern recognition receptor is NOD-like receptor.NLRP3 of the body family.When NLRP3 is activated by cytotoxin,ATP or cholesterol,ASC and Caspasel are further recruited,the inflammasome is activated and further cut IL-1β and IL-18.At the same time,NLRP3 inflammasome activates the lysis of Gasdermin D,releases its N-terminal domain,forms cell membrane pores,releases inflammatory factors,and causes a series of immune responses.Therefore,inhibiting the expression of inflammasomes may provide new ideas for the treatment of viral myocarditis.IL-37 is a potential inflammation inhibitor of the IL-1 family,which can inhibit the body’s innate immunity and adaptive immunity.IL-37 is low expressed in human peripheral blood monocytes and dendritic cells,while IL-37 is not expressed in mice.IL-37 has been proven to ameliorate viral myocarditis in mice caused by CVB3,but its specific mechanism is not clear.Therefore,this part of the experiment uses IL-37 to intervene in viral myocarditis mice to observe its effect on inflammatory infiltration and NLRP3 inflammasome expression in myocarditis mice,and to explore the effect of IL-37 on viral myocarditis and the specific mechanism.Objective1.To establish a viral myocarditis model,and use IL-37 to intervene in mice with viral myocarditis,and observe the effects of IL-37 on the general state,cardiac function and myocardial pathology of mice with viral myocarditis.2.To explore the specific mechanism of IL-37 to alleviate viral myocarditis in mice by detecting the expression of pyroptosis in the cardiomyocytes of each group of mice.Methods1.Intervention of IL-37 in mice with viral myocarditisThirty-two male 6-week-old balb/c mice were divided into 4 groups randomly:control group(n=6),IL-37 control group(n=6),VMC group(n=10),and IL-37 group(n=10).On Day 0,mice in the control group and IL-37 control group were injected with 100μl PBS,and mice in the VMC group and IL-37 group undergone intraperitoneal injection of 25μl 109TICD50(Add 75μl PBS to dilute it to 100μl).On Day3 and Day7,mice in the control group and VMC group were injected with 200μl of PBS,and the IL-37 control group and IL-37 group were intraperitoneally injected with 2μg IL-37(with 200μl PBS diluted).After 9 days,an animal model of acute myocarditis was established.2.Measurement of cardiac functionThe left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),systolic septal thickness(IVSs),diastolic septal thickness(IVSd),systolic left ventricular diameter(LVIDs),diastolic left ventricular diameter(LVIDd),the systolic left ventricular posterior wall thickness(LVPWs)and the diastolic left ventricular posterior wall thickness(LVPWd)of each group were recorded.3.Histology and immunohistochemistryWe collect heart specimens of each group after all mice were sacrificed on Day 10,and the myocardial tissue was fixed and embedded in paraffin.5μm-thick sections were then stained with hematoxylin and eosin(H&E)and Masson.And GSDMD was detected by immunofluorescence staining.4.ELISA5.Collect peripheral blood in each group.And collect the upper serum aftercentrifugation.The specific ELISA kits were used to detect the myocardial injury marker cTnI and the inflammatory factors IL-1β and IL-18.5.Western blotWe collected the myocardial tissue of each group and extracted the total protein.NLRP3,ASC dimer,cleavaged caspase 1,NFκB P65 and phospho-P65 were detected by Western blo.5.RT-PCRThe myocardial tissue of each group of mice was collected,and total RNA of myocardial tissue was extracted.Real-time fluorescent quantitative PCR technology was used to detect the mRNA expression levels of NFκB P65.Results1.IL-37 improves the general state of mice in VMC groupCompared with the mice in VMC group,mice in IL-37 group were more vigorous with smooth hair,and had obvious response when grasped.Compared with the VMC group,mice in the IL-37 group lost less weight,and the heart weight/body weight ratio was lower(P<0.05).2.IL-37 improves heart function in mice with viral myocarditisCompared with the VMC group mice,the LVEF(P<0.05),LVFS(P<0.05),IVSs(P<0.01)and IVSd(P<0.01)in experimental group mice were all improved to varying degrees,indicating that the ventricular septum was thicker in the experimental group,and the left ventricular systolic function was improved.3.IL-37 reduces myocardial damage in VMC groupHE staining showed that compared with VMC group,IL-37 group had less infiltration of myocardial inflammation,less necrosis of myocardial cells,and less interstitial inflammation infiltration.Elisa examination showed that the expression of cTnI(P<0.01)and IL-18(P<0.05)in serum of IL-37 group was lower.4.The expression of NLRP3 inflammasome was increased in the VMC group of mice and decreased in the IL-37 groupWestern blot detection showed that the protein expression levels of NLRP3(P<0.01),ASC(P<0.05)and cleaved-caspase 1(P<0.05)in the VMC group were higher than control group.In the IL-37 group,the expressions of NLRP3(P<0.05),ASC dimer(P<0.001)and cleaved-caspase 1(P<0.05)were lower.The expression of GSDMD in the VMC group was higher the control group in immunofluorescence staining(P<0.01).The expression of GSDMD in the IL-37 group was lower(P<0.01)than VMC group.5.IL-37 reduces NLRP3 inflammasome expression in mice with myocarditis by inhibiting NFκBWestern blot detection showed the expression of NFκB P65(P<0.05)and phospho-P65(P<0.01)were significantly increased in the VMC group.NFκB P65(P<0.01)and phospho-P65(P<0.01)was lower in the experimental group than the VMC group.The expression levels of P65(P<0.05)and phospho-P65(P<0.05)were significantly lower in the IL-37 control group than the control group(P<0.05).RT-PCR detection showed that the expression of P65 in the VMC group was higher than that in the control group(P<0.05),and higher than the experimental group(P<0.05).And the expression level of P65 mRNA in the IL-37 control group was lower than the control mice(P<0.05).Conclusion1.IL-37 reduced myocardial damage and improved the general state of mice in VMC group.2.NLRP3 inflammasome is involved in CVB3-induced viral myocarditis,and IL-37 inhibits the expression of NLRP3 inflammasome.3.IL-37 may alleviate viral myocarditis in mice by inhibiting NLRP3 inflammasome via NFκB pathway.更多还原