【作者】 王文珂；

【导师】 张运； 张铭湘；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 研究背景糖尿病心肌病(diabetic cardiomyopathy, DCM)是由糖尿病状态所导致的一种心脏疾病,与高血压、冠心病和心脏瓣膜病的病理改变无关,早期表现为心室顺应性下降和无症状的舒张功能障碍；后期将进一步发展为收缩功能受损和有症状的充血性心力衰竭。心肌病理结构的异常改变主要表现为心肌细胞肥厚、凋亡和心肌间质纤维化,其中心肌间质纤维化是糖尿病心肌病的主要病理特征。糖代谢异常是糖尿病心肌病的关键始动因素,但引起糖尿病心肌病的具体分子机制仍然不明确,因此研究糖尿病心肌病的病理新机制,寻找作用靶点,成为临床治疗中亟待解决的重要问题。高迁移率族蛋白(HMGB1)是一种高度保守的DNA结合蛋白,广泛存在于哺乳动物体内。在核内,HMGB1参与核小体的构建和稳定,调节基因的转录；在核外,HMGB1可由免疫细胞(如单核细胞、巨噬细胞等)在受到刺激活化后主动释放到细胞质或由坏死细胞破裂被动释放至胞外,引起炎症反应和介导相关信号通路的传导。近来发现,除外免疫细胞外,HMGB1也可被某些非免疫细胞主动分泌,如肿瘤细胞、垂体细胞、肠上皮细胞、心肌细胞、脂肪细胞等,参与炎症反应、肿瘤发生、细胞增殖及分化等病理过程。以往研究发现,HMGB1在胞外可通过与晚期糖基化终产物受体(RAGE)、套样受体(TLR2、TLR4)等受体结合,通过激活丝裂原活化蛋白激酶(MAPK)途径,介导炎症反应以及促进细胞因子活化。近年,越来越多的研究表明,HMGB1在多种心血管疾病的发生与发展中发挥重要作用,包括心肌缺血/再灌损伤、心肌梗死、动脉粥样硬化、肺动脉高压等。目前研究发现,1型糖尿病患者血清中HMGB1水平较健康人群显著增高,提示我们HMGB1可能在糖尿病心肌病的发病过程中起重要作用。因此,本研究利用链脲佐菌素(STZ)诱导小鼠1型糖尿病模型,探讨HMGB1在糖尿病诱导心肌重构中的作用及其机制。研究目的1.研究高糖是否能诱导心肌细胞和成纤维细胞HMGB1的核转位,并进一步分泌至胞外；2.研究心肌组织中HMGB1在糖尿病诱导心肌纤维化中的作用及其机制；3.探讨重组HMGB1蛋白刺激对心肌成纤维细胞增殖、迁移及合成胶原能力的影响。研究方法1.慢病毒shRNA-HMGB1载体构建：设计3对HMGB1基因的shRNA质粒,根据每个序列的干扰效率,选择效率最高的序列构建含有绿色荧光蛋白(GFP)报告基因的慢病毒载体。针对HMGB1的干扰序列为5’-GGCTCGTTATGAAAGAGAAAT-3’,同时应用5’-GTTCTCCGAACGTGTCACGT-3’作为阴性对照序列。2.动物模型建立及HMGB1shRNA慢病毒体内转染将8周龄雄性C57BL/6J小鼠(约25-30g)随机分为模型组(60只)和对照组(15只)。模型组小鼠连续5天腹腔内注射链脲佐菌素(STZ)溶液,剂量为50mg/kg.d。对照组小鼠注射相应体积的0.1mmol/l无菌柠檬酸缓冲液(pH4.5)。建模当天禁食4h,不禁水。STZ注射1周后取尾静脉血测随机血糖,将随机血糖＞16.7mmol/l的小鼠纳入糖尿病模型组。于糖尿病12周末将模型组小鼠随机分为3组：模型组、shHMGB1慢病毒干预组、shN.C慢病毒对照组。小鼠体内慢病毒转染采用心肌注射的局部转染方式,每只小鼠转染病毒量为1×107UT／30μ1,并选择多点注射入左室壁中。病毒转染4周后取材,并于显微镜下观察心肌组织中GFP的阳性率分析病毒转染效率。3.超声心动图检测心功能采用二维、M超、脉冲多普勒和组织多普勒超声成像技术评价小鼠左室功能。4.组织学染色收集各组小鼠心脏,制备石蜡切片,进行H&E、Masson、天狼星红染色,分别显示心脏的大体形态结构和心肌内胶原的含量。应用免疫组织化学染色,观察心肌组织内HMGB1、胶原I、胶原III、转化生长因子β1(TGF-β1)的分布及表达情况。5.细胞培养本实验采用新生1-3天的Wistar乳鼠心脏分离和培养心肌原代细胞和心肌成纤维细胞作为研究对象,以正常糖浓度培养的细胞为对照组,应用不同浓度及不同时间的高糖分别刺激细胞,观察其对细胞的影响。6.激光共聚焦显微镜检测应用细胞免疫荧光法检测细胞中HMGB1和Ki67的分布与表达水平。7.实时定量逆转录PCR提取各组细胞及组织RNA,进行逆转录和实时定量PCR,检测细胞及组织中HMGB1的mRNA表达水平。8.蛋白印迹收集各组细胞及组织,提取蛋白,分别检测HMGB1、乳酸脱氢酶(LDH)、I型胶原、III型胶原、TGF-β1、MMP-2、MMP-9、ERK、p-ERK、JNK、p-JNK、p38和p-p38等蛋白的表达水平。9.明胶酶谱法采用明胶酶谱法检测心肌成纤维细胞中MMP-2和MMP-9的活性。10.免疫酶联吸附法(ELISA)收集各组细胞上清,检测细胞HMGB1的分泌水平。11.细胞的增殖和迁移实验采用细胞计数试剂盒CCK8检测心肌成纤维细胞的增殖情况。利用Transwell小室观察成纤维细胞在不同刺激下的迁移情况。12.碘化丙啶染色检测利用Annexin V/PI试剂盒检测细胞坏死的情况。13.统计学分析应用SPSS18.0软件分析处理数据,并以均数±标准差(Mean±SD)表示。研究结果1.高血糖促进小鼠心肌组织HMGB1表达并向组织间隙分泌糖尿病小鼠心肌组织与正常小鼠相比,HMGB1蛋白和mRNA表达水平明显升高,且能观察到HMGB1由胞核向组织间隙分泌。而在正常小鼠心肌组织中,HMGB1的表达主要分布于细胞核中,未见明显的移位。2.抑制HMGB1改善糖尿病引起的左室重构及心功能异常HMGB1’慢病毒干扰shRNA转染小鼠心肌组织4周后,观察各组小鼠心脏左室重构的情况。与正常小鼠相比,糖尿病小鼠心脏形态明显增大、心尖变圆钝。与糖尿病模型组小鼠相比,抑制HMGB1能够明显改善糖尿病引起的心肌结构及形态的改变,降低了心脏重量(HW)和心身重量之比(HW/BW),减小了心肌细胞的体积,但对小鼠体重没有明显影响。STZ注射16周后,糖尿病小鼠心功能损害主要为舒张功能异常,表现为E/A、 E’/A’的降低,左室后壁舒张期增厚；同时伴有收缩功能障碍,表现为EF、FS值减低。抑制HMGB1能够显著改善糖尿病所引起的心功能舒张和收缩异常。3.抑制HMGB1明显减轻糖尿病小鼠心肌纤维化程度Masson染色和天狼猩红染色显示糖尿病小鼠心肌间质纤维化程度较正常小鼠明显增加；免疫组织化学和western blot检测显示心肌组织Ⅰ、Ⅲ型胶原和TGF-β1蛋白表达水平显著增高。与模型组和慢病毒干预对照组相比,抑制HMGB1明显降低心肌组织胶原沉积以及胶原Ⅰ、Ⅲ和促纤维化因子TGF-β1的水平。4.高糖刺激能增加原代心肌细胞和心肌成纤维细胞中HMGB1的表达及分泌应用不同浓度的高糖(15mM,20mM,25mM)分别刺激原代心肌细胞和心肌成纤维细胞48h后,与对照组(普通培养)相比,培养基上清液中HMGB1含量呈浓度依赖性明显升高,且细胞未发生坏死。细胞免疫荧光染色显示,对照组HMGB1主要定位于细胞核,而高糖刺激48h后,HMGB1由细胞核向细胞浆移位。同时测定HMGB1mRNA表达水平,显示高糖刺激心肌细胞和心肌成纤维细胞8h后,HMGB1mRNA水平开始增加,并于24h达高峰。5. HMGB1能增强心肌成纤维细胞的胶原合成、增殖及迁移能力分别用不同浓度的重组HMGB1蛋白(50ng/ml、100ng/ml、200ng/ml、400ng/ml)刺激心肌成纤维细胞48h,与正常对照组相比,rHMGB1能够显著增加细胞中Ⅰ、Ⅲ型胶原和TGF-β1蛋白的表达量。与对照组相比,应用rHMGB1刺激心肌成纤维细胞后,细胞增殖能力明显增强,核增殖相关抗原Ki67阳性率明显增加。此外,应用rHMGB1刺激心肌成纤维细胞24h能明显增强细胞的迁移能力。在高糖刺激条件下,通过中和抗体或基因沉默方式抑制HMGB1后,心肌成纤维细胞合成胶原及增殖能力明显下降。6.抑制HMGB1能减少心肌成纤维细胞中MMP-2、MMP-9的表达与活性高糖刺激心肌成纤维细胞后,western blot检测发现MMP-2和MMP-9的蛋白表达水平明显增高；而通过中和抗体或基因沉默抑制HMGB1后,心肌成纤维细胞中MMP-2和MMP-9的蛋白表达水平降低。应用明胶酶谱法检测发现,高糖刺激可以增加细胞中MMP-2的活性,抑制HMGB1后MMP-2的活性显著降低,但在该实验中未能检测出MMP-9的活性。此外,应用rHMGB1蛋白刺激心肌成纤维细胞亦能增加细胞中MMP-2、MMP-9的蛋白表达水平及MMP-2的活性。7. HMGB1能促进心肌成纤维细胞中ERK、JNK和Akt的磷酸化水平rHMGB1刺激能明显增加心肌成纤维细胞内ERK、JNK和Akt的磷酸化水平。通过中和抗体或基因沉默方式抑制HMGB1后,能够显著降低高糖刺激后心肌成纤维细胞内ERK、JNK和Akt的磷酸化水平。而各组间p38的磷酸化水平无明显差异。同时,在糖尿病小鼠中,抑制HMGB1也能降低心肌组织中ERK和JNK的磷酸化水平。该实验结果提示ERK和JNK的激活参与了HMGB1介导的糖尿病心肌病。研究结论1.高糖促进小鼠心脏组织中HMGB1表达增加,且向胞外分泌；2.下调心肌组织中HMGB1表达可改善糖尿病小鼠心肌重构、心肌纤维化及心功能障碍；3.下调HMGB1减轻糖尿病心肌病的机制可能与抑制ERK和JNK磷酸化有关。研究背景糖尿病是一种由多种因素引起的以慢性高血糖为特征的全身代谢性疾病,可导致机体多种脏器损害,其中心血管疾病是其最主要的死因。糖尿病的发病率在全世界急剧上升,已成为威胁人类健康最主要的慢性疾病之一。心力衰竭在糖尿病患者中发生的比例是非糖尿病患者的2～3倍,将其中发生不依赖于高血压、冠心病及瓣膜性心脏病等疾病的心衰称为糖尿病心肌病。糖尿病心肌病的主要病理改变表现为心肌细胞肥大、凋亡及间质纤维化,整个病程过程中伴随着心肌重构的发生。细胞凋亡是由基因调控的细胞程序性死亡,心肌细胞凋亡在糖尿病心肌病的发生发展中起着关键性作用,心肌细胞数目改变与心室重构和心脏功能密切相关。研究发现糖尿病患者早期即可出现心肌细胞凋亡,但糖尿病诱导心肌细胞凋亡的机制仍不明确。高迁移率族蛋白1(HMGB1)是调控基因转录和维持核小体稳定的非染色体核蛋白,它可被活化的免疫细胞或坏死细胞由细胞核释放到胞外,发挥致炎作用。HMGB1可通过与TLRs及RAGE受体激活MAPK信号通路介导炎症因子产生和细胞凋亡,导致炎性反应及组织损伤。以往研究发现,抑制HMGB1可减轻化疗药物引起的心肌细胞凋亡。我们前期实验研究发现,高糖可刺激心肌细胞主动分泌HMGB1,并参与糖尿病心肌病心肌纤维化的发生。基于以上研究结果,提示HMGB1可能参与了高糖诱导的心肌细胞凋亡过程,并促进糖尿病心肌病的发生与发展。研究目的1.研究HMGB1对高糖诱导心肌细胞凋亡的影响；2.探讨HMGB1介导高糖环境下心肌细胞凋亡的信号分子机制。研究方法1.细胞培养及模型建立：本实验采用新生的Wistar乳鼠心脏分离和培养心肌原代细胞,应用正常糖浓度(5.5mM) DMEM培养大乳鼠原代心肌细胞,用高糖(33mM) DMEM刺激细胞；以5.5mM葡糖糖+27.5mM甘露醇作为高渗对照。2.基因表达的干预：体外实验,采用慢病毒转染HMGB1shRNA的方式抑制HMGB1的基因表达；应用瞬时转染Ets-1siRNA的方式抑制Ets-1的基因表达。3.激光共聚焦显微镜：采用细胞免疫荧光染色法检测原代心肌细胞内p-Ets-1的含量及分布。4.TUNEL检测凋亡：检测各刺激组心肌细胞及组织的凋亡水平。5.蛋白印迹(western blot analysis):收集各组细胞及组织,提取蛋白,分别检测HMGB1、caspase-3、Bcl-2、Bax、 Ets-1、p-Ets-1、ERK、p-ERK蛋白水平。6.实时定量逆转录PCR (Real-time RT-PCR):收集各组细胞,提取RNA,进行逆转录和实时定量PCR,检测HMGB1的mRNA表达水平。7.糖尿病动物模型建立及HMGB1shRNA慢病毒体内转染：模型组小鼠采用腹腔注射链脲佐菌素(STZ)建立1型糖尿病模型。1周后测量血糖,将随机血糖＞16.7mmol/l的小鼠纳入实验模型组。在8周末将模型组小鼠随机分为3组：模型组(15只)、shHMGB1漫病毒干预组(15只)、shN.C慢病毒对照组(15只)。小鼠体内慢病毒转染采用心肌注射的局部转染方法,选择多点注射入左室壁中。病毒转染4周后取材。8.组织学检测：应用免疫组织化学染色,观察心肌组织内p-Ets-1的蛋白表达水平。9.统计学分析：应用SPSS18.0软件分析处理数据,并以均数±标准差表示。研究结果1.抑制HMGB1能够减少高糖诱导的心肌细胞凋亡应用慢病毒HMGB1shRNA转染心肌细胞,检测各组心肌细胞的凋亡情况。结果显示,与正常对照组和高渗对照组相比,高糖能够增加细胞的凋亡水平；抑制HMGB1能够降低高糖诱导增加的cleaved caspase-3、Bax/Bcl-2水平及细胞凋亡率。2. HMGB1介导高糖诱导的Ets-1活化在体外实验中,我们进一步研究了高糖增加细胞凋亡的可能机制。应用高糖刺激心肌细胞6h、8h、12h、24h, western blot结果显示,Ets-1的表达在6h较正常对照有所增加,其余时间点Ets-1水平均无明显变化；与正常对照组相比,Ets-1磷酸化(p-Ets-1)水平在6h、8h、12h和24h均明显升高；细胞免疫荧光检测结果发现,高糖刺激主要增加细胞核中p-Ets-1的表达。高糖刺激心肌细胞也能够增加细胞核中p-Ets-1水平；而抑制HMGB1可降低高糖诱导增加的Ets-1磷酸化水平。3.抑制Ets-1能减少高糖诱导的心肌细胞凋亡应用Ets-1siRNA转染心肌细胞抑制Ets-1基因表达,检测Ets-1对细胞凋亡的影响。Western blot及TUNEL检测结果显示,高糖能增加心肌细胞凋亡,而抑制Ets-1可降低高糖刺激增加的cleaved caspase-3、Bax/Bcl-2水平及细胞凋亡率。4. HMGB1通过磷酸化ERK促进Ets-1的活化高糖刺激心肌细胞能促进ERK磷酸化,在60min时效果最显著。抑制HMGB1能够降低高糖诱导的ERK磷酸化。为进一步研究ERK磷酸化在高糖诱导Ets-1活化中的作用,我们应用U0126抑制ERK磷酸化,western blot及细胞免疫荧光染色检测结果发现,抑制ERK磷酸化能降低高糖诱导的Ets-1活化。5.抑制HMGB1能够减少糖尿病小鼠心肌细胞凋亡心肌组织western blot及TUNEL检测结果显示,糖尿病小鼠心肌组织中cleave caspase-3、Bax/Bcl-2及细胞凋亡数目较正常小鼠明显增加；然而,与模型组和慢病毒干预对照组相比,抑制]3MGB1明显降低糖尿病心肌组织中cleave caspase-3、Bax/Bcl-2及细胞凋亡数目。6.抑制HMGB1能够抑制糖尿病小鼠心肌组织中ERK及Ets-1的磷酸化水平Western blot检测心肌中p-ERK及t-ERK水平,结果显示抑制HMGB1能够显著降低糖尿病小鼠心肌组织中ERK磷酸化水平；免疫组织化学检测心肌中p-Ets-1水平发现,抑制HMGB1能够降低糖尿病小鼠心肌组织中Ets-1的活化。研究结论1.抑制HMGB1能够减少高糖诱导的心肌细胞凋亡；2.抑制HMGB1减少心肌细胞凋亡的作用是通过阻断ERK/Ets-1信号通路实现的。更多还原

【Abstract】 BackgroundDiabetes mellitus is associated with increased risk of heart failure, independent of hypertension and underlying coronary artery disease, called diabetic cardiomyopathy (DCM). DCM is characterized by functional and structural cardiac changes, including myocardial cell death and accumulation of extracellular matrix (ECM) protein. In particular, myocardial fibrosis is the most frequently proposed mechanism responsible for the cardiac changes in DCM. Although chronic hyperglycemia plays an important role in the pathogenesis of diabetic complications, the molecular mechanisms underlying cardiac fibrosis are not clear, and factors contributing to the cardiac dysfunction remain to be elucidated.High-mobility group box1(HMGB1) is a nuclear protein existing widely in almost all eukaryotic cells. In addition to its nuclear role, HMGB1is also released from activated macrophages, monocytes and necrotic cells but not apoptotic cells, which results in an inflammatory reaction and signal transduction pathway. In addition to its secretion by macrophages and monocytes, HMGB1can be secreted by other viable non-immune cells, including cancer cells, pituicytes, enterocytes, and cardiomyocytes. Therefore, it may participate in other pathological processes such as oncogenesis, proliferation and differentiation, which challenges the limited role of HMGB1as an important mediator in inflammatory cellular processes. Increasing evidence has demonstrated the multiple functions of HMGB1in various heart diseases.Serum HMGB1levels in patients with type1diabetes are significantly higher than in healthy controls so HMGB1may be a newly identified cytokine associated with DCM.Objectives1. To investigate whether high glucose could induce HMGB1translocation and secretion in mice heart.2. To investigate the role and mechanism of cardiac HMGB1in diabetic cardiomyopathy.3. To study the effect of recombinant HMGB1on cardiac fibroblasts collagen synthesis, proliferation and migration.Methods1. Lentivirus vector RNA interferenceDesigning and synthesizing3pieces of short-hairpin RNAs (shRNAs) against HMGB1according to RNAi principle. We used a lentivirus vector containing a green fluorescent protein (GFP) reporter and a U6promoter upstream of the cloning site for the most effective shRNA. The target sequence for HMGB1was5’-GGCTCGTTATGAAAGAGAAAT-3’ and negative control sequence5’-GTTCTCCGAACGTGTCACGT-3’.2. Animal model and RNA interferenceC57BL/6J wild-type (WT) mice8weeks-old (25～30g) were divided into2groups:control (n=60) and diabetes (n=15). Diabetes was induced by injecting streptozotocin dissolved in0.1ml citrate buffer (pH4.5) intraperitoneally (i.p.) at50mg/kg per mouse for5consecutive days. Control mice were injected with citrate buffer only. After7days, whole blood was obtained from the tail vein, and random glucose levels were measured. Diabetes was determined as blood glucose at least16.7mmol/1. After the induction of diabetes (12weeks), mice were divided into3groups for treatment:control, shRNA-HMGBl and shRNA-N.C for the following experiments. An amount of1×107UT/30μl of lentivector with HMGB1shRNA or the same volume of lenti-vehicle was injected into various sites of the left ventricle.3. Cardiac function measurementCardiac diameter and function was measured by use of the Vevo770imaging system.2D echocardiography, M-mode echocardiography, pulsed-wave Doppler echocardiography and tissue Doppler imaging were used to evaluate cardiac function.4. Histology and immunohistochemistryTissue was paraffin-embedded and sectioned (4μm) for staining with hematoxylin and eosin (H&E), Masson’s trichome and Picrosirius red to examine heart size and extracellular matrix (ECM) deposition. Immunohistochemistry was used to determine the levels of HMGB1, collagen Ⅰ, collagen Ⅲ and transforming growth factor-β1(TGF-β1) in myocardial tissues.5. Cell cultureCardiomyocytes and cardiac fibroblasts (CFs) were isolated from neonatal rat ventricular tissues. When cell populations reached60%confluence, cultures were exposed to high glucose (HG;15-25mM). Some cell cultures were exposed to normal glucose (NG;5.5mM) as controls.6. Laser scanning confocal microscopyImmunofluorescence analysis was used to evaluate HMGB1and Ki67expression levels and disposition.7. Real-time RT-PCRTotal RNA was extracted from cardiomyocytes and CFs. In the experiment, the mRNA expression of HMGB1in cardiomyocytes and CFs was analyzed.8. Western blottingProteins were extracted from cardiomyocytes and CFs. The protein expression of HMGB1, lactate dehydrogenase (LDH), collagen I, collagen Ⅲ, TGF-β1, matrix metalloproteinase (MMP-2), MMP-9, ERK, p-ERK, JNK, p-JNK, p38and p-p38was analyzed in our experiment.9. Gelatin zymographyThe activity of MMP-2and MMP-9in CFs was determined by zymography.10. Enzyme-linked immuno sorbent assay (ELISA)HMGB1released into cell culture supernatants was evaluated by Elisa in CFs in vitro.11. Assessment of cell proliferation and migration assay:CFs proliferation assays were determined by use of the Cell Counting Kit-8(CCK-8). CFs migration assays were performed in Transwell chambers.12. Propidium iodide staining for necrosis:Necrotic cells were stained positive for propidium iodide and negative for Annexin-V.13. Statistical analysis:All statistical analyses involved use of SPSS18.0. Data are reported as mean±standard deviation.Results1. Hyperglycemia increased the level of HMGB1and induced HMGB1translocation and secretion in mice heartDiabetic mice showed significantly increased myocardial HMGB1mRNA level and protein level. Immunohistochemistry analysis revealed HMGB1expression in nuclei of normal heart, while hyperglycemia induced HMGB1to diffuse from the nucleus to the myocardial interstitium in diabetic heart. 2. HMGB1inhibition prevented diabetes-induced myocardial remodeling and cardiac dysfunctionAfter shRNA-HMGB1treatment for4weeks, we measured cardiac structure and function. Inhibition of HMGB1mitigated heart structural alterations in type1diabetic mice. Heart weight and ratio of heart weight to body weight were lower with shRNA-HMGB1than vehicle treatment. HMGB1gene silencing attenuated the enlarged cardiomyocytes as compared with vehicle treatment. shRNA-HMGB1and vehicle-transfected diabetic mice did not differ in body weight.After16weeks of diabetes, cardiac function features were mainly diastolic dysfunction, which showed deceased E/A and E’/A’as well as thickened LVPWd. Cardiac systolic function lightly lower in diabetic than control mice, showed that deceased EF and FS. ShRNA-HMGB1treatment attenuated diabetes-induced cardiac dysfunction.3. HMGB1inhibition limited diabetes-induced myocardial fibrosisMasson’s trichome and Picrosirius red staining of heart sections revealed greater ECM in the interstitial regions of the diabetic than control myocardium. Diabetes enhanced the expression of the fibrotic markers collagen Ⅰ, Ⅲ and fibrotic TGF-β1as compared with the control. While ShRNA-HMGB1treatment reduced collagen deposition, the expression of collagen Ⅰ, Ⅲ and TGF-β1as compared with vehicle treatment4. HG induced HMGB1translocation and secretion in viable primary cardiomyocytes and CFsCardiomyocytes and CFs were exposed to various concentrations (15mM,20mM,25mM) of high glucose for48h. High glucose significantly increased levels of extracellular HMGB1in both cardiomyocytes and CFs and these effects were not due to cell necrosis. Immunofluorescence analysis revealed HMGB1expression in nuclei of both cardiomyocytes and CFs cultured under NG. As compared with NG, HG induced HMGB1to diffuse from the nucleus to the cytoplasm. HMGB1mRNA activity began to increase in cardiomyocytes after8h, peaking at24h with HG.5. HMGB1increased cardiac fibroblasts collagen systhesis, proliferation and migrationWith physiological glucose concentration, rHMGB1dose-dependent increased the protein level of collagen I, III and TGF-β1in as compared with NG with maximum effect at200ng/ml. CFs showed fast growth rate with rHMGB1treatment, which was simliar to CFs with HG. We used Transwell migration assay to evaluate CFs migration. After24h, we found prominent dose-dependent HMGB1chemotaxis of CFs. Meanwhile, pharmacological or genetic inhibition of HMGB1inhibited the HG-induced increase in cell collagen systhesis, proliferation.6. HMGB1inhibition reduced the MMP-2and MMP-9expression and activity in CFsHG increased the expression of MMP-2and MMP-9. These effects were significantly downregulated by Ab-HMGB1or shRNA-HMGB1treatment. HG had a similar effect of HMGB1as compared with NG. Gelatin zymography showed that HG increased the activity of MMP-2, and inhibition of HMGB1downregulated this effect. However, MMP-9activity was not detected. rHMGB1treatment had a similar effect of HG on MMP-2and MMP-9.7. HMGB1increased activation of ERK, JNK and Akt in CFsrHMGBl treatment increased ERK, JNK and Akt phosphorylation in CFs as compared with NG alone. While HMGB1inhibition reduced the HG-induced phosphorylation of ERK, JNK and Akt. HMGB1had no effect on the expression of p-p38. In vivo, HMGB1inhibition reduced diabetes-induced myocardium phosphorylation of ERK and JNK.Conclusion1. Hyperglycemia induced HMGB1translocation and secretion in type1diabetes mice heart.2. Silencing HMGB1gene could attenuate myocardial remodeling, fibrosis and cardiac dysfunction.3. Inhibition of HMGB1improved diabetic cardiomyopathy via ERK and JNK signal pathway. BackgroundDiabetes mellitus is an increasing worldwide systemic metabolic disease. Hyperglycemia is the major feature of diabetes mellitus and can induce organ damage such as cardiovascular disease, the most frequent cause of death in the diabetic population. The sharp rise in the incidence of diabetes in the world has become one of the major threats to human health. The rate of heart failure in diabetic patients is2-3times as large as it in non-diabetic patient. Heart failure in diabetes, which occurs independent of changes in blood pressure and coronary artery disease, is called diabetic cardiomyopathy.The process of diabetic cardiomyopathy consists of a series of sequential and interrelated steps, including myocardial apoptosis, hypertrophy and fibrosis. Apoptosis is programmed cell death. Cardiomyocyte apoptosis is the keystone in the process. The loss of cardiomyocytes has been implicated in the development of myocardial remodeling and heart dysfunction. Increased cardiomyocyte apoptosis has been detected in hearts of diabetic patient. However, little is known about the molecular mechanisms that regulate cardiomyocyte apoptosis under hyperglycemia. High mobility group box1protein (HMGB1) is a non-chromosomal nuclear protein that regulates gene transcription and maintains the nucleosome structure; it can be released from necrotic or activated immune cells. Released HMGB1together with its receptor TLRs or RAGE activates mitogen-activated protein kinases (MAPKs), which could mediate inflammatory response and apoptosis. Previous studies have found that inhibition of HMGB1could reduce cardiomyocyte apoptosis induced by chemotherapeutic drugs. Our previous study found that high glucose stimulated cardiomyocyte actively secreted HMGB1, which participated in diabetes-induced myocardial fibrosis and heart dysfunction. We suggested that HMGB1might involve in hyperglycemia-induced cardiomyocyte apoptosis.Objectives1. To investigate the effect of HMGB1in high glucose-induced cardiomyocyte apoptosis.2. To study the signaling pathway involved in HMGB1-mediated cardiomyocyte apoptosis.Methods1. Cell culture and cell model:Cardiomyocytes were isolated from neonatal rat ventricular tissues. Cardiomyocytes were cultured in normal glucose (5.5mM, NG) DMEM and stimulated by high glucose (33mM, HG) in our experiments. High mannose (33mM, OC) was served as osmolarity controls.2. Interference of gene expression:In vitro, lentivector with HMGB1shRNA transfection was performed to inhibit HMGB1expression; transient transfection with Ets-1siRNA was performed to inhibit Ets-1expression.3. Laser scanning confocal microscopy:Immunofluorescence analysis was used to evaluate p-Ets-1expression levels and disposition. 4. TUNEL assay:Cardiomyocyte apoptosis is determined by TUNEL assay.5. Western blotting:Proteins were extracted from cardiomyocytes. The protein expression of HMGB1, cleaved caspase-3, Bax, Bcl-2, Ets-1, p-Ets-1, ERK and p-ERK was analyzed in our experiment.6. Real-time RT-PCR:Total RNA was extracted from cardiomyocytes and CFs. In the experiment, the mRNA expression of HMGB1in cardiomyocytes and CFs was analyzed.7. Animal model:Type1diabetes was induced by injecting streptozotocin. Control mice were injected with citrate buffer only. After7days, random glucose＞16.7mmol/1was determined as diabetes. After the induction of diabetes (8weeks), mice were divided into3groups for treatment:control (n=8), shRNA-HMGBl (n=8) and shRNA-N.C (n=8) for the following experiments. An amount of1×107UT/30μl of lentivector with HMGB1shRNA or the same volume of lenti-vehicle was injected into various sites of the left ventricle.8. Immunohistochemistry:Immunohistochemistry was used to determine the levels of p-Ets-1in myocardial tissues.9. Statistical analysis:All statistical analyses involved use of SPSS18.0. Data are reported as mean±standard deviation.Results1. Inhibition of HMGB1reduced high glucose-induced cardiomyocytes apoptosisHMGB1shRNA was used to inhibit HMGB1expression. The cell apoptosis was determined by western blot and TUNEL assay. Results showed that HG increased the apoptosis of cardiomyocyte compared with the control; while HMGB1inhibition could significantly reduce the cleaved caspase-3, Bax/Bcl-2and apoptotic cells induced by HG.3. HMGB1mediated high glucose-induced activation of Ets-1in neonatal cardiomyocytesTo investigate the underlying mechanism of HG-induced apoptosis, we cultured cardiomyocytes in HG medium for6h,8h,12h,24h. After HG treatment for6h, the protein level of total Ets-1protein expression was slightly increased and p-Ets-1was significantly elevated at6h up to24h. Our results demonstrated that HG but not high media osmolarity enhanced the activation of Ets-1and accumulation of p-Ets-1in the nucleus; inhibition of HMGB1effectively reversed HG-induced increase in the level of p-Ets-1and its accumulation in the nucleus HMGB1.3. Inhibition of Ets-1reduced high glucose-induced cardiomyocytes apoptosisTo confirm whether Ets-1activation was involved in HG-induced cardiomyocyte apoptosis, we used Ets-1siRNA to knock down its protein level. Ets-1siRNA significantly reduced the activation of caspase-3protein, Bax/Bcl-2ratio and TUNEL-positive cells as compared with HG alone.4. ERK pathway was involved in high glucose-induced activation of Ets-1Stimulation of HG increased the phospho-ERK1/2level with a peak at60min. HG but not high osmolarity increased the level of p-ERK1/2in cardiomyocytes as compared with NG. Inhibition of HMGB1with HMGB1-specific shRNA significantly attenuated HG-induced ERK1/2activation in cardiomyocytes. Furthermore, we examined whether HG enhanced Ets-1activation via an ERK pathway. U0126was applied to inhibit p-ERK1/2. HG significantly increased the nuclear level of p-Ets-1, which was reduced by U0126. These observations supported that HMGB1mediated Ets-1activation via an ERK1/2pathway under HG treatment.5. Inhibition of HMGB1reduced diabetes-induced myocardial apoptosis in vivoHyperglycemia significantly increased myocardial caspase-3activity, the ratio of Bax/Bcl-2and apoptotic cells was significantly increased in diabetic hearts. HMGB1inhibition effectively ameliorated hyperglycemia-activated caspase-3and decreased Bax/Bcl-2ratio. Additionally, HMGB1inhibition decreased the proportion of TUNEL-positive cells in the diabetic mouse.6. Inhibition of HMGB1gene prevented ERK and Ets-1activation in the diabetic mouse heartWestern blot analysis demonstrated that inhibition of HMGBl by shRNA significantly reduced hyperglycemia-induced ERK phosphorylation. Immunohistochemistry showed that silencing HMGB1gene significantly decreased the level of p-Ets-1in the diabetic myocardium.Conclusion1. Inhibition of HMGB1reduced high glucose-induced cardiomyocyte apoptosis.2. HMGB1induced apoptosis via the ERK-dependent activation of Ets-1in HG-treated cardiomyocytes.更多还原