【作者】 陈亮；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 心血管内科， 2018， 博士

【摘要】 研究背景和目的病毒性心肌炎(viral myocarditis)表现为心肌组织不同程度炎症细胞浸润,人群发病率约22/10,000,多数病人表现为亚临床症状,少数病患呈爆发性心肌炎,心功能及血流动力学紊乱严重,约20%病患病情迁延反复发展为慢性心肌炎及扩张性心肌病。目前,心肌炎发病最主要致病因素为病毒感染,最为常见致病原为肠病毒属的柯萨奇病毒B组3型病毒(CVB3)。在急性爆发性病毒性心肌炎及慢性心肌炎患者血中检测CVB3抗体升高。病毒性心肌炎主要病理生理学表现为心脏局限性或弥漫性炎症细胞浸润,心肌细胞凋亡坏死,心肌间质反应性纤维化形成,病毒高水平复制。通常病毒性心肌炎经历三个阶段:1,急性期,病毒进入心肌细胞直接引起心肌裂解坏死;2,第二阶段为亚急性期,免疫反应(自然免疫、体液免疫和细胞免疫)激活;第三阶段为慢性期,病毒持续低滴度复制,进展为慢性扩张性心肌病。形成“病毒侵入—免疫反应激活—慢性病毒性心肌炎—扩张性心肌病”病理发展过程。细胞色素P450表氧化酶2J2(CYP2J2)将花生四烯酸(arachidonic acid,AA)代谢生成4种环氧-二十碳三烯酸(Epoxyeicosatrienoic Acids,EETs),分别为5,6-,8,9-,11,12-和14,15-EET。EET经可溶性表氧化物水解酶(s EH)代谢为生物活性弱的DHET。大量研究证实,CYP2J2/EET有扩张血管降低血压、改善内皮功能、抑制平滑肌细胞增殖和迁移、缓解心肌细胞肥大等生理保护效应。CYP2J2/EET对急性病毒性心肌炎发生发展是否有缓解作用,是本实验研究重点。实验方法和结果1,病毒性心肌炎模型建立:选取4~6周龄Balb/c雄鼠,分为con、GFP、2J2、CVB3、CVB3+GFP、CVB3+2J2、CVB3+TPPU七组,其中con、GFP、2J2三组每组10只,CVB3、CVB3+GFP、CVB3+2J2、CVB3+TPPU四组每组15只。重组腺相关病毒r AAV-GFP和r AAV-2J2,尾静脉注射1Χ1011pfu,2周后腹腔注射100ul 105TCID50CVB3病毒液。CVB3+TPPU组提前TPPU灌胃,3 mg/kg/d,连续灌胃10天,之后腹腔注射100ul 105TCID50 CVB3病毒液。病毒注射10天,观察并记录小鼠体重及生存状况,第10天行心脏超声及血流动力学检测,留取心脏、肝脏、脾脏组织,行HE染色、天狼猩红染色、免疫组化及TUNEL染色。结果显示CVB3病毒成功诱导急性病毒性心肌炎的形成,心肌炎小鼠心脏/体重比例下降,10天生存率约25~30%。心脏超声中左室射血分数(EF)和短轴缩短分数(FS)显著降低,而左室收缩内径(LVID-s)和舒张末内径(LVID-d)显著增高,心功能降低。血流动力学检测显示心肌炎左室收缩功能的左室压力最大上升速率(dp/dt max)和左室压力最大下降速率(dp/dt min)显著降低。心脏HE染色发现,心肌炎组弥漫性炎症细胞浸润和局部组织坏死,钙质沉积,心肌炎病理评分平均2.3分,免疫组化显示心肌炎心脏组织巨噬细胞、中性粒细胞、T细胞浸润增多,天狼猩红显示心肌炎心脏组织纤维化面积约20%,TUNEL显示病毒性心肌炎心肌细胞凋亡比例增高。而过表达CYP2J2或TPPU灌胃后,可改善心功能和血流动力学情况,缓解心肌炎炎症细胞浸润,降低病理评分,降低局部巨噬细胞、中性粒细胞和T细胞比例,减少心肌细胞凋亡比例。2,心脏炎症因子表达水平检测。选取各组4~6只小鼠心脏组织,real-time PCR检测心脏组织炎症因子相对表达水平。利用各组小鼠血浆和组织匀浆,ELISA检测血液及心脏组织炎症因子水平。Real-time PCR检测显示病毒性心肌炎心脏组织促炎因子TNF-α、IL-1β、IL-12A、IL-23、IL-21和IL-17A m RNA水平显著增高,而抑炎因子IL-4、IL-10、IL-13 m RNA和抑制病毒复制IFN-α和IFN-βm RNA水平均上升,过表达2J2或TPPU灌胃后,缓解心脏促炎因子TNF-α、IL-1β、IL-12A、IL-23、IL-21和IL-17A表达水平的增高,而抑炎因子IL-4、IL-10、IL-13和抑制病毒复制的IFN-α/IFN-β表达水平较心肌炎组显著升高,发挥抗炎及抗病毒复制作用。心脏和血浆ELISA示,病毒性心肌炎组TNF-α、IL-1β、IL-17A和IL-10炎症因子水平增高,过表达2J2或TPPU灌胃后,与CVB3组相比,促炎因子TNF-α、IL-1β和IL-17A水平降低,抑炎因子IL-10升高。3,心脏组织Western Blot检测病毒性心肌炎炎症通路激活、转录因子NF-κB入核、氧化应激、纤维化及凋亡通路激活情况。CVB3成功诱导炎症通路激活,P65入核增多,磷酸化IκBα、磷酸化STAT1、磷酸化STAT3表达上调;纤维化通路中1型胶原蛋白、TGF-beta、磷酸化smad表达上调;凋亡激活,活化的cleaved caspase3和Bax表达上调,抗凋亡蛋白Bcl-2表达下调;氧化应激相关重要蛋白gp91、gp67、gp40和gp22均显著上调;同时,心肌炎组AMPK和AKT1磷酸化水平增高。过表达2J2或TPPU灌胃后,炎症通路中重要蛋白分子磷酸化水平降低,抑制P65入核,氧化应激、纤维化及凋亡通路激活均受到不同程度抑制。4,血浆心肌酶谱肌钙蛋白c Tn I和CK-MB检测。心肌炎组血浆c Tn I和CK-MB水平均显著升高,提示心肌组织受损严重。过表达2J2或TPPU灌胃缓解心肌炎导致的心肌酶升高。5,心脏组织和肝脏组织病毒滴度检测,采用组织匀浆,运用Reed&Muench梯度稀释法,检测各组小鼠心脏和肝脏病毒负荷。过表达2J2或TPPU灌胃,降低心脏和肝脏CVB3病毒滴度。6,再次建立小鼠病毒性心肌炎模型,留取心脏和脾脏,流式细胞术检测心脏巨噬细胞、脾脏Th1/Th2/Th17/Treg和CD4—CD8+T细胞比例。心肌炎组心脏巨噬细胞增多,其中M1型巨噬细胞增多明显;脾脏T细胞亚群中促进炎症发生发展的Th1/Th17/CD8+T细胞比例增高,抑制炎症反应的Th2细胞和调节性T细胞反应性增高。过表达2J2或TPPU灌胃缓解CVB3诱导的心脏巨噬细胞和脾脏Th1/Th17/CD8+T细胞比例增高,提高抑制反应的Th2细胞和调节性T细胞的比例。7,原代大鼠心肌细胞和成纤维细胞CVB3病毒复制水平检测。原代大鼠心肌细胞和成纤维细胞提取种板后,11,12-EET和14,15-EEZE提前干预1小时,1CVB3(MOI=1)分别感染原代心肌和成纤维细胞,12小时后,real time PCR检测两种细胞CVB3病毒扩增水平。研究发现11,12-EET缓解CVB3病毒复制,11,12-EET抑制CVB3病毒复制能力可被14,15-EEZE阻断。结论高表达CYP2J2或口服s EH抑制剂TPPU,有效缓解CVB3诱导的病毒性心肌炎心肌损伤,主要通过缓解心脏炎症细胞浸润、心肌细胞凋亡、病毒复制及脾脏炎症细胞分化,为急性病毒性心肌炎心功能障碍提供新的治疗靶点。更多还原

【Abstract】 Background and objective Viral myocarditis occurs mainly in the population of young adults.According to American epidemiological investigation,incidence of viral myocarditis was approximately22 in 100,000 persons,death rate was up to 4.8 in 100,000 people.Clinical symptoms vary from subtle chest pain to sudden death due to many factors such as age,gender,region,race,virus strain and genetic susceptibility.Vast majority of myocarditis has self-limitation and good prognosis.However,about 9~12 % myocarditis evolved to acute fulminant myocarditis with high mortality.Through three years’ follow-up,21 % myocarditis patients were found to progress to chronic myocarditis and finally dilated cardiomyopathy.The common etiology of myocarditis is viral infection,including enterovirus,adenovirus,parvovirus B19(PVB 19),herpes virus type 6(HPV6)and cytomegalovirus,of which coxsackievirus B 3(Coxsackievirus B3 CVB3)was the most dominant causative agent.The pathogenesis of viral myocarditis has three sequential stages: the first stage is acute phase,characterized by the procession that CVB3 virus binds to respective viral receptor embed in cellular surface,enters into myocardial cells via endocytosis and leads to direct myocardial cell toxicity and necrosis;the second stage is subacute phase with activation of immune system(innate immunity,humoral immunity and cell immunity)and infiltration of great deal of inflammatory cells like macrophages and T cells in heart.Inflammatory cells release various cytokines,aggravate the apoptosis and necrosis of cardiomyocytes and secrete different extracellular matrix hydrolases contributing to degradation of interstitial extracellular matrix,which finally leading to cardiac dysfunction and ventricular dilation;the third stage is chronic phase with persistent low titer viral replication and high susceptibility to DCM.Some studies verified that the patients of severe myocarditis with significant hemodynamic abnormalities and inflammatory cell infiltration recovered within a short period and have good prognosis owing to clearing of virus by overwhelming inflammation.But patients with persistent viral replication and few inflammatory cells infiltration had higher risk in progressing to chronic viral myocarditis and dilated cardiomyopathy.Above all,persistent viral replication,inflammatory cell infiltration and apoptosis and necrosis of cardiomyocyte are prominent mechanism of CVB3-induced acute viral myocarditis,responsible for the development of CVB3-induced myocarditis: viral invasion—activation of immunity—chronic myocarditis—dilated cardiomyopathy—heart failure.At present,the pathogenesis of viral myocarditis has not yet been elucidated.Cytochrome P450 oxidase 2J2(CYP2J2)is highly expressed in the human heart and vascular endothelial cells and exert the function of metabolizing arachidonic acid(arachidonic,acid,AA)to four kinds of epoxy-twenty carbon three acid(Epoxyeicosatrienoic Acids,EETs),respectively 5,6-,8,9-,11,12-and 14,15-EET.EETs could be hydrolyzed by soluble epoxide hydrolase(s EH)to dihydmxyeicosatrienoicacacik(DHET),which harbors weak biological activity.Studies have shown that overexpression of CYP2J2 or inhibition of s EH activity increase EETs’ level and exert protective effect on heart: 1,EETs can activate potassium channels to promote hyperpolarization of vascular smooth muscle cells,dilating blood vessels and lower blood pressure;2,EETs inhibit the translocation of NF-kappa B into the nucleus and reduce the migration and adhesion of inflammatory cells;3,EETs can inhibit smooth muscle cell proliferation and migration;4,EETs promotes phosphorylation of endothelial nitric oxide synthase(e NOS)at site Ser1177 and Thr495,thus improving endothelial function in vascular endothelial cells;5,EETs enhanced heart function by reducing macrophage infiltration and phenotype change in lipopolysaccharide-induced septic cardiomyopathy;6,The expression of 2J2 relieved the vascular tension of abdominal aortic aneurysm induced by Ang II infusion;7,EETs can activate AMPK alpha,promote the nuclear translocation of AKT1 and exert anti-hypertrophic effect;8,EETs can inhibit cardiac fibroblasts TGF-beta /smad pathway,alleviate the formation of ROS and ameliorate the deterioration of cardiac fibrosis caused by Ang II infusion.s EH is a negative regulator of tissue and serum concentration of EETs,if inhibited,the degradation of EETs is impaired and concentration of EETs is upregulated.Study confirmed that spontaneously hypertensive rats were administrated with AUDA(25mg/L/d)for 6 weeks,AUDA can reduce the systolic blood pressure and reduce the infarction area.When s EH gene were knocked out,PI3K/AKT/NOS3 and AMPK were activated and apoptosis of renal tubular epithelial cells was decreased,which in turn alleviated STZ-induced diabetic nephropathy.Gavage of TPPU(3mg/kg/d)for 7 days can inhibit bombesin or arginine-induced acute pancreatitis by decreasing the infiltration of inflammatory cells.In our study,we accomplished the overexpression of CYP2J2 or gavage of TPPU in order to determine whether it could reduce CVB3-induced viral myocarditis by decreasing inflammatory cells infiltration and activation of inflammatory factor expression,inhibiting the replication of Coxsackie virus,alleviating the injury of myocardial cells,cardiac troponin release,and impaired heart function.Experimental methods and results1,To establish the model of viral myocarditis: 4~6 week old Balb/c male mice were divided into seven groups: con,GFP,2J2,CVB3,CVB3+GFP,CVB3+2J2,CVB3+TPPU,including which n=10 in each group in con,GFP,2J2 three groups,n=15 in each group within CVB3,CVB3+GFP,CVB3+2J2,CVB3+TPPU four groups.Intravenous injection of r AAV-GFP and r AAV-2J2 recombinant adeno-associated at the concentration of 1x1011 PFU was done 2 weeks before the intraperitoneal injection of 100 ul DMEM in containing105TCID50 CVB3.In group CVB3+TPPU,TPPU(3mg/kg/d)was given orally for 10 days in advance.Then CVB3 was injected into the abdominal cavity with 100 ul 105TCID50CVB3.In the duration of 10 days after CVB3 injection,we observed and recorded the body weight and survival of mice,performed echocardiography and hemodynamic measurements and finally harvested heart,liver and spleen tissue for HE staining,Sirius red staining,immunohistochemistry and TUNEL staining.The results showed that CVB3 successfully induced the formation of acute viral myocarditis.Heart / body weight ratio in myocarditis was decreased.Survival rate of CVB3 myocarditis group was about 25~30%.Echocardiographic left ventricular ejection fraction(EF)and Short axis shortening fraction(FS)were significantly reduced,while the left ventricular systolic diameter(LVID-s)and end diastolic diameter(LVID-d)were significantly increased,and cardiac function was impaired.Hemodynamic tests showed that the maximal rate of left ventricular pressure(dp/dt max)and the maximum rate of left ventricular pressure(dp/dt min)decreased significantly.In HE,diffuse infiltration of inflammatory cells,local tissue necrosis and calcium deposition were found in myocarditis groups,of which pathological score was 2.3.Immunohistochemistry showed macrophages,neutrophils,T cell infiltration increased in myocarditis cardiac tissue.In picrosirius red,we found the myocardial fibrosis area of about 20% in myocarditis group.TUNEL staining showed a higher ratio of apoptosis in myocarditis.The overexpression of CYP2J2 or gavage of TPPU can improve the cardiac function,alleviate the inflammatory cell infiltration of myocarditis and reduce pathological score and lower myocardial apoptosis.2,Detection of inflammatory cytokines in cardiac tissues.4~6 mice in each group were selected and the relative expression levels of inflammatory cytokines were detected by realtime PCR.Plasma and tissue homogenates of each group were used to detect the levels of inflammatory cytokines in blood and heart tissues by ELISA.Real-time PCR showed that pro-inflammatory factor TNF-alpha,IL-1 beta,IL-12 A,IL-23,IL-21 and IL-17 A m RNA levels were significantly increased in heart,while the level of anti-inflammatory cytokines such as IL-4,IL-10,IL-13,IFN-alpha and IFN-beta were increased.Overexpression of 2J2 or gavage of TPPU ameliorated the increase of pro-inflammatory cytokines(TNF-alpha,IL-1 beta,IL-12 A,IL-23,IL-21 and IL-17A)and increased the level of anti-inflammatory cytokines(IL-4,IL-10 and IL-13)and IFN-alpha /IFN-beta inhibiting replication of CVB3.By ELISA,we verified inflammatory cytokines(TNF-alpha,IL-1beta,IL-17 A and IL-10)of heart homogenates and plasma elevated.Overexpression of 2J2 or administration of TPPU,compared with the CVB3 group,reduced the level of inflammatory cytokines and upregulated anti-inflammatory cytokines.3,Western Blot was used to detect the activation of inflammatory pathway,translocation of transcription factor NF-kappa B,oxidative stress,fibrosis and apoptosis in viral myocarditis.Inflammatory pathway is activated with increased nuclear translocation of P65,phosphorylation of IκBα,STAT1 and STAT3.Besides,fibrosis related molecules such as collagen I,TGF-beta and phosphorylated Smad expression also elevated.Pro-apoptotic proteins-activated cleaved Caspase3 and Bax increased,while anti-apoptotic protein Bcl-2expression was decreased.Representative oxidative stress protein gp91,gp67,gp40 and gp22 were significantly increased.Meanwhile,both AMPK and AKT1 were activated.After overexpression of 2J2 or administration of TPPU,the phosphorylation level of remarkable molecules in the inflammatory pathway was decreased and activation of oxidative stress,fibrosis and apoptosis pathway was inhibited to some degree.4,Detection of troponin c Tn I and CK-MB in plasma.The levels of plasma c Tn I and CK-MB were significantly increased in myocarditis group,which suggested severe damage on cardiomyocyte.Overexpression of 2J2 or TPPU alleviated the elevation of myocardial enzymes in viral myocarditis.5,Test of viral titer in heart and liver tissue homogenate with Reed and Muench gradient dilution method.Overexpression of 2J2 or gavage of TPPU reduced burden of CVB3 in heart and liver.6,Flow cytometry was used to detect percentage of Th1/Th2/Th17/Treg and CD4—CD8+ T cells in spleen and macrophage in heart.Macrophage accumulated in myocarditis group,particularly M1 macrophages.Pro-inflammatory subsets of T cell(Th1/Th17/CD8+ T cell)were promoted in the progression of myocarditis.The percentage of regulatory T cells also increased in response to CVB3 infection.Overexpression of 2J2 or TPPU gavage reduced the proportions of Th1/Th17/CD8+ T cells in spleen and macrophages in heart and increased the proportions of Th2 cells and regulatory T cells in spleen.7,Measurements of CVB3 virus replication in primary rat cardiomyocytes and fibroblasts.Primary rat myocardial cells and fibroblasts were cultured on plate,11,12-EET and14,15-EEZE intervention were done one hour before CVB3(MOI=1)infection.After 12 hours,realtime PCR was used to test viral titer in two primary cells.It was found that11,12-EET could alleviate the replication of CVB3 virus and the protective effect of11,12-EET on the replication of CVB3 could be blocked by 14,15-EEZE.Conclusion Overexpression of CYP2J2 or administration of oral s EH inhibitor effectively alleviates cardiac injury in viral myocarditis,mainly through ameliorating inflammatory cell infiltration,apoptosis of myocardial cell,viral replication and differentiation of splenic T cells,which provides a new therapeutic target for acute viral myocarditis.更多还原