TRB3与糖尿病性心肌病关系的实验研究
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摘要
背景
糖尿病作为冠心病的等危症已经得到广泛共识,糖尿病患者的心血管风险显著增加。糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的以左室舒张功能受损和心功能不全为主要表现的心肌病变,是糖尿病患者心力衰竭发生率高和死亡率高的主要原因。然而糖尿病性心肌病发生、发展的具体机制目前仍然不详,因而缺少以机制为基础的靶向性治疗。寻找糖尿病性心肌病发生、发展的具体机制并探索可能的有效治疗方法成为目前国内外研究的热点。
开展相应研究的首要问题是建立适宜开展糖尿病性心肌病研究的动物模型,而2型糖尿病动物模型的建立是前提。目前,建立2型糖尿病动物模型的方法主要有五种：一是遗传相关模型,即自发性糖尿病模型；二是通过敲除与代谢有关的基因联合高脂饮食喂养诱发2型糖尿病；三是化学药物损伤联合高脂饮食所诱发的2型糖尿病；四是胰腺部分切除；五是通过长期高脂饮食喂养诱发2型糖尿病。然而,这些建立动物模型的方法仍然存在部分不足：(1)遗传相关模型,由于遗传因素在其发病过程中起主导作用,不完全与临床相符,使其应用受到限制；(2)由于人类的2型糖尿病是多基因遗传病,现有的基因敲除模型不能完全模拟人类2型糖尿病,而且此模型不能排除代谢相关的基因敲除之后对后续基因干预的影响；(3)化学药物剂量过大或胰腺切除过多造成胰腺过度损伤会使血糖过高,而使动物模型倾向于1型糖尿病；(4)长期高脂饮食诱发的2型糖尿病动物模型的造模周期较长,且停用高脂饮食之后,血糖会逐渐恢复正常。
本课题针对2型糖尿病动物模型的上述不足,采用了高脂饮食联合小剂量链脲佐菌素(streptozocin, STZ)诱导的2型糖尿病模型,通过监测体重、血压、血糖、血脂、糖耐量试验及胰岛素耐量试验的动态变化,并采用高分辨率显微超声技术监测糖尿病性心肌病发生和发展过程,探讨建立2型糖尿病性心肌病动物模型的可行性。
目的
1、探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病大鼠动物模型的可行性；
2、探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病性心肌病大鼠动物模型的可行性。
方法
1.选择5周龄健康雄性SD (Sprague Dawley)大鼠60只,体重120g±20g左右,购自山东中医药大学实验动物中心。行腹腔葡萄糖耐量试验(intraperitoneal glucose tolerance test, IPGTT)及胰岛素耐量试验(intraperitoneal insulin tolerance test, IPITT)后随机分为4组：对照组(control组)、普通饮食STZ组(chow+streptozocin组)、高脂饮食组(high fat, HF组)和糖尿病组(diabetes mellitus, DM组)。Control组和chow+STZ组大鼠喂以基础饲料,主要组成为：20%粗蛋白,3%粗脂肪,3%粗纤维,其他74%(包括碳水化合物、微量元素等)。HF组和DM组大鼠喂以高糖高脂高热量饲料(北京华阜康生物有限公司提供),主要成分为：34.5%脂肪、17.5%蛋白质、48%碳水化合物。4周后再次行IPGTT及IPITT, DM组大鼠出现胰岛素抵抗者给予一次性腹腔注射链脲佐菌素(Streptozotocin, STZ)27.5mg/kg,同时chow+STZ组大鼠给予一次性腹腔注射STZ27.5mg/kgo Control组和HF组大鼠给予同等剂量枸橼酸钠缓冲液腹腔注射。各组以原饲料继续喂养1周后,测定空腹血糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),计算胰岛素敏感指数[ISI, ISI=In (FINS×FBG)-1]。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感性减低且有多尿、多饮、多食现象的大鼠纳入实验。糖尿病成模后16周实施动物安乐死。
2.腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT):大鼠禁食12h后行IPGTT,葡萄糖按1g/kg腹腔注射,于0、15、30、60及120分钟采集尾静脉血,使用强生One-Touch血糖仪测量血糖,并计算血糖曲线下面积(area under curve, AUC)。大鼠禁食4h后行IPITT,胰岛素按1unit/kg腹腔注射,余同IPGTT。
3.血液生化指标的测定：大鼠禁食12h后颈静脉取血,检测血清总胆固醇(Total cholesterol, TC)、甘油三酯(triglycercide, TG)、游离脂肪酸(free fatty acid, FFA),血糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),并计算胰岛素敏感指数(insulin sensitivity index, ISI)。
4.血压及心率监测：应用BP-98A智能鼠尾无创血压计测定收缩压、舒张压、平均动脉压及心率；
5.超声心动图和血流动力学监测：采用二维、M超、脉冲多普勒和组织多普勒超声心动图技术以及超声背向散射技术,连续观察DCM发生和发展过程中左心室收缩和舒张功能变化；
6.应用心导管技术测定左室舒张末压(left ventricular end-diastolic pressure, LVEDP):大鼠深麻醉后,经右侧颈动脉插管至左心室,测量左室收缩压及舒张末期压力,监测2型DCM大鼠心室功能的改变。
结果
1、实验末各组大鼠的一般状况比较：
与对照组相比,普通饮食STZ组大鼠心脏重量/体重、饮水量、摄食量及尿量均明显增加(P<0.05)；与对照组相比,高脂组大鼠体重明显增加,心脏重量、饮水量均有所增加(P<0.05)；与对照组相比,糖尿病组大鼠心脏重量、心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均显著增加(P<0.01)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心脏重量、饮水量、摄食量、尿量均明显增加(P<0.001)；与高脂组大鼠相比,糖尿病组大鼠饮水量、摄食量、尿量均显著增加(P<0.001)。
2、高脂4周各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组大鼠在IPGTT试验60min、120min的血糖明显升高(P<0.05)；与对照组相比,高脂组大鼠在IPGTT试验15min、30min、60min、120min的血糖值明显升高(P<0.05)；与普通饮食STZ组大鼠相比,高脂组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(area under curve, AUC)均明显增大(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组血糖曲线下面积(area under curve, AUC)均明显增大。
(2) IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验0min及15min的血糖值均显著升高(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组大鼠在IPITT试验15min及120min的血糖明显升高(P<0.05)；与对照组相比,高脂组大鼠在IPITT试验0mmin及15min的血糖值明显升高(P<0.05)；与普通饮食STZ组大鼠相比,高脂组大鼠在IPITT试验15min及120mmin的血糖明显升高(P<0.05)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)；与普通饮食STZ组大鼠相比,高脂组及糖尿病组血糖曲线下面积(AUC)均明显增大。
以上结果说明,高脂饮食4周后,高脂组及糖尿病组大鼠出现了明显的胰岛素抵抗。
3、糖尿病成模后12周各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPGTT试验30min及60min的血糖值明显升高(P<0.05)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPGTT试验60mmin的血糖明显升高(P<0.05)。与对照组相比,普通饮食STZ组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(2) IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min、30min、60min及120min的血糖值均显著升高(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05～P<0.01)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPITT试验60min及120mmin的血糖明显升高(P<0.05～P<0.01)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组血糖曲线下面积(AUC)明显增大(P<0.05)。
4、实验末各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验Omin、15min、30min、60min、120mmin的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在OGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在OGTT试验60min、120min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(2) IPITT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验15min、60min的血糖值均明显升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验15min的血糖值明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验Omin、15min、30min、60min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
5、生化指标的测定：高脂饮食4周即STZ注射前,与对照组相比,高脂组及糖尿病组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),空腹血糖无显著变化,提示注射STZ前糖尿病组大鼠已存在胰岛素抵抗；STZ注射后一周,与对照组相比,普通饮食STZ组及糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),高脂组及糖尿病组血清TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后6周,与对照组相比,糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),糖尿病组血清TC、TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后12周,与对照组相比,糖尿病组血清TC、TG、FFA明显升高(P<0.05),ISI显著降低(P<0.05),FINS无显著差异；实验末,与对照组相比,糖尿病组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。
以上结果提示,本研究以高脂饮食诱导联合小剂量链脲佐菌素注射建立的2型糖尿病动物模型,具有中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点。
6、血压及心率监测：实验过程中各组大鼠收缩压、舒张压及平均动脉压均无差异；实验末,与对照组及普通饮食STZ组相比,糖尿病组大鼠心率明显增快(P<0.05)。
7、超声心动图监测：
在糖尿病成模后6周,与对照组相比,糖尿病组E’/A’开始下降(P<0.05)；在糖尿病成模后12周,与对照组相比,糖尿病组LVEF和FS也开始受损(P<0.05),且E’/A’下降更为明显。实验末与对照组大鼠相比,普通饮食STZ组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均明显增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均降低(P<0.05)；与对照组大鼠比较,高脂组大鼠舒张末期左室后壁厚度、左室质量有所增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.01),E’/A’降低(P<0.05)；与对照组大鼠相比,糖尿病组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均显著增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均显著降低(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠左室质量明显增加(P<0.05),等容舒张时间、等容收缩时间明显延长(P<0.01),FS有所降低(P<0.05)。超声背向散射积分结果显示,与对照组大鼠比较,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05~P<0.001),CVIB显著降低(P<0.01～P<0.001)；与对照组大鼠相比,普通饮食STZ组大鼠室间隔及左室侧壁IB%明显增加(P<0.01～P<0.001),室间隔、左室后壁及左室侧壁CVIB显著降低(P<0.05～P<0.001)；与高脂组相比,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001), CVIB明显降低(P<0.001)。
8、心电图检查：与对照组相比,糖尿病组大鼠出现了多种心电图异常。
9、血流动力学检测：与对照组相比,普通饮食STZ组大鼠左室舒张末期压力明显增加(P<0.001)；与对照组相比,高脂组大鼠左室舒张末期压力有所增加(P<0.05)；与对照组相比,糖尿病组左室舒张末期压力显著增加(P<0.001)。
结论
(1)高脂高热量饮食诱导联合小剂量链脲佐菌素注射建立的2型糖尿病动物模型,具有糖尿病状态稳定,中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点；
(2)高分辨显微超声技术可以无创性地监测2型糖尿病性心肌病的发生发展,2型糖尿病性心肌病大鼠以左室舒张功能障碍为主要表现,伴随有轻微的收缩功能异常；
(3)2型糖尿病大鼠模型的建模因素中没有不可干预因素,适于开展后续的糖尿病基因干预研究。
背景
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的以左室舒张功能受损和心功能不全为主要表现的心肌病变,是糖尿病患者心力衰竭发生率高和死亡率高的主要原因。自从1972年Ruber等首先提出DCM这一概念后,国内外学者在此领域中进行了大量的基础和临床研究。虽然临床研究显示,严格控制血糖可明显降低糖尿病微血管病变,但并未显著降低心血管事件的发生,糖尿病患者心力衰竭的发生率仍居高不下。因此,有必要对DCM的发生机制进行深入研究。
DCM发生的始动因素和关键环节是胰岛素抵抗(insulin resistance)。胰岛素抵抗通过抑制胰岛素信号传导和葡萄糖转运,降低心肌对葡萄糖的利用,使机体抑制脂肪分解的能力减弱,使血浆游离脂肪酸(FFA)和甘油三酯升高,而FFA可呈剂量依赖性地抑制细胞葡萄糖的转运和磷酸化作用,造成心肌能量代谢的异常,进一步加重胰岛素抵抗,造成恶性循环。胰岛素缺乏,能量代谢异常,酶系活性受抑导致心肌细胞核皱缩,线粒体肿胀及糖原沉积,形成DCM的病理基础。临床实验同时证实胰岛素抵抗与左室病理性重构和左室舒张功能障碍密切相关。但胰岛素抵抗与糖尿病性心肌病之间的深层链接机制尚不清楚,因此缺乏有针对性的治疗靶点。
TRBs基因是Groβhans等于2000年在果蝇体内新发现的一类丝氨酸/苏氨酸蛋白激酶样蛋白基因家族,是抑制有丝分裂,诱导细胞凋亡的核基因,也被称为神经细胞死亡诱导蛋白激酶。人类TRBs家族包括TRB1、TRB2、TRB3,它们含有一个的相同的中心区,以及70-100氨基酸残基的N-末端和25个残基的C-末端区。果蝇TRB3是哺乳动物的同系物,因此,目前有关TRBs基因的研究主要集中在TRB3。TRB3具有广泛的生物活性,与糖脂代谢及胰岛素抵抗密切相关。研究发现,TRB3基因可以通过抑制Akt的功能影响胰岛素信号传导通路；TRB3是MAPK活性重要的调节蛋白,在MAPKK水平调控MAPK的磷酸化作用和活性,少量的TRB3增加即可显著增强ERK1/2和JNK活性,轻度抑制P38活性。但关于TRB3/MAPK信号通路的研究未见到后续的报道。
Akt和MAPK是选择性“胰岛素抵抗”两个关键通路,而且MAPK是调控心肌间质纤维化的关键信号分子。因此,我们推测在2型糖尿病状态下,胰岛素抵抗刺激TRB3过表达,并可能通过调控Akt及MAPK信号途径而影响DCM的发生和进展,但目前尚未见类似报道。据此,本课题拟通过2型DCM大鼠模型,研究TRB3在DCM发生、发展中的作用,并应用TRB3-siRNA特异性抑制2型糖尿病大鼠体内TRB3的表达,进而探讨TRB3基因沉默是否可以通过调控“胰岛素抵抗”相关信号通路从而起到改善DCM的作用。
目的
1.建立2型糖尿病性心肌病大鼠动物模型,探讨TRB3在2型糖尿病性心肌病发生、发展中的作用；
2.2型糖尿病大鼠体内转染TRB3腺病毒,观察2型糖尿病性心肌病的心脏结构和功能的变化；
3.在分子生物学、组织学和整体功能水平研究TRB3基因沉默改善2型糖尿病性心肌病的机制。
方法
1.pAdxsi-TRB3-shRNA病毒载体的构建：根据RNAi干扰技术原则,设计4对针对大鼠的TRB3基因的siRNA,经筛选后,选择沉默效率最高的序列构建pGenesil-1.2-TRB3-shRNA质粒,随后构建pShuttle-Basic-EGFP-TRB3-shRNA重组穿梭载体质粒,最后构建pAdxsi-GFP-TRB3-shRNA腺病毒载体。
2.选择健康雄性SD大鼠60只,体重120g±20g左右,购自山东中医药大学实验动物中心。行腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)后随机分为4组：对照组(control组)、普通饮食+链脲佐菌素组(chow+STZ组)、高脂组(HF组)、糖尿病组(DM组)。Control组和chow+STZ组大鼠喂以基础饲料,主要组成为：20%粗蛋白,3%粗脂肪,3%粗纤维,其他74%(包括碳水化合物、微量元素等)。HF组和DM组大鼠喂以高糖高脂高热量饲料(北京华阜康生物有限公司提供),主要成分为：34.5%脂肪、17.5%蛋白质、48%碳水化合物。4周后再次行IPGTT及IPITT,DM组大鼠出现胰岛素抵抗者给予一次性腹腔注射STZ27.5mg/kg,同时chow+STZ组大鼠给予一次性腹腔注射STZ27.5mg/kg。Control组和HF组大鼠给予同等剂量枸橼酸钠缓冲液腹腔注射。各组以原饲料继续喂养1周后,测定空腹血糖(FBG)和胰岛素(FINS),计算胰岛素敏感指数[ISI, ISI=In (FINS×FBG)-1]。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感性减低且有多尿、多饮、多食现象的大鼠纳入实验。糖尿病成模后16周实施动物安乐死,称取心脏重量,计算心脏重量/体重比值。
3.腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IP ITT):大鼠禁食12h后行IPGTT,葡萄糖按1g/kg腹腔注射,于0、15、30、60及120分钟采集尾静脉血,使用强生One-Touch血糖仪测量血糖,并计算血糖曲线下面积。大鼠禁食4h后行IPITT,胰岛素按1unit/kg腹腔注射,余同IPGTT。
4.血液生化指标的测定：大鼠禁食12h后颈静脉取血,检测血清总胆固醇(Total cholesterol, TC)、甘油三酯(triglycercide, TG)、游离脂肪酸(free fatty acid, FFA),皿糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),并计算胰岛素敏感指数(insulin sensitivity index, ISI)。
5.超声心动图和血流动力学监测：采用二维、M超、脉冲多普勒和组织多普勒超声心动图技术以及超声背向散射技术,连续观察DCM发生和发展过程中左心室收缩和舒张功能变化；
6.应用心导管技术测定左室舒张末压：大鼠深麻醉后,经右侧颈动脉插管至左心室,测量左室收缩压及舒张末期压力,监测2型DCM大鼠心室功能的改变。
7.病理学检测：对左室心肌组织分别进行H&E染色、Masson染色、天狼猩红染色以及油红O染色,应用图象分析仪测定心肌细胞大小、胶原体积分数、管周胶原面积/管腔面积及心肌脂质含量。
8.心肌羟脯氨酸含量测定：将干燥至恒重的左室心肌组织称重后置于6NHCL中110℃水解16h,应用ELISA试剂盒测定水解物中羟脯氨酸的含量。因羟脯氨酸在胶原组织中占13.5%,所以最终结果显示为胶原含量(ug/mg左室干重)。
9.免疫组织化学染色：应用免疫组织化学染色法,光镜下观察心肌间质的Ⅰ型胶原、Ⅲ型胶原、肿瘤坏死因子-α (TNF-α)及白介素6(IL-6)的分布情况。
10.实时定量RT-PCR检测：取新鲜左室心肌组织,Trizol法提取RNA,实时定量荧光RT-PCR技术检测TRB3、脑钠肽(brain natriuretic peptide, BNP)、TNF-α及IL-6的mRNA相对表达量。
11. Western blot检测：取新鲜左室心肌组织,提取蛋白,Western blot检测心肌组织内TRB3、胶原Ⅰ、胶原Ⅲ、TNF-α、IL-6及磷酸化及非磷酸化的ERK1/2、p38、JNK、 Akt的蛋白表达量。
12. TRB3-siRNA腺病毒体内转染实验：选择健康雄性SD大鼠60只(体重120g±20g左右),随机分为HF+空载体组、HF+TRB3siRNA组、DM+空载体组、DM+TRB3siRNA组。糖尿病组造模过程同上。糖尿病成模后12周,HF+TRB3siRNA组、DM+TRB3siRNA组大鼠经颈静脉注射TRB3腺病毒,HF+空载体组及DM+空载体组注射pAdxsi空病毒载体,2周后补充注射一次,TRB3腺病毒注射4周后处死动物留取标本。
结果
1.2型糖尿病性心肌病大鼠模型的建立：
(1)实验末各组大鼠的一般状况比较：
与对照组相比,普通饮食STZ组大鼠心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均明显增加(P<0.05)；与对照组相比,高脂组大鼠体重明显增加,心脏重量、饮水量及左室舒张末期压力均有所增加(P<0.05)；与对照组相比,糖尿病组大鼠心脏重量、心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均显著增加(P<0.01)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心脏重量、饮水量、摄食量、尿量均明显增加(P<0.001)；与高脂组大鼠相比,糖尿病组大鼠饮水量、摄食量、尿量均显著增加(P<0.001)。
(2)腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
①DM组大鼠高脂4周后与实验前IPGTT比较：
IPGTT结果显示：糖尿病组大鼠高脂4周后与基线状态相比,在IPGTT试验0min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积(AUC)明显增大(P<0.05)。
②DM组大鼠高脂4周后与实验前IPITT比较：
IPITT结果显示：糖尿病组大鼠高脂4周后与基线状态相比,在IPGTT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积(AUC)明显增大(P<0.05)。
③糖尿病成模后12周各组大鼠IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPGTT试验30min及60min的血糖值明显升高(P<0.05)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPGTT试验60min的血糖明显升高(P<0.05)。与对照组相比,普通饮食STZ组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
④糖尿病成模后12周各组大鼠IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min、30min、60min及120min的血糖值均显著升高(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05-P<0.01)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPITT试验60min及120min的血糖明显升高(P<0.05-P<0.01)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组血糖曲线下面积(AUC)明显增大(P<0.05)。
⑤实验末IPGTT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在OGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在OGTT试验60min、120min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
⑥实验末IPITT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验15min、60min的血糖值均明显升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验15min的血糖值明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验0min、15min、30min、60min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(3)生化指标的测定：
STZ注射前,与对照组相比,高脂组及糖尿病组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),空腹血糖无显著变化,提示注射STZ前糖尿病组大鼠已存在胰岛素抵抗；STZ注射后一周,与对照组相比,普通饮食STZ组及糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),高脂组及糖尿病组血清TG、FFA明显升高(P<0.05),FINS无显著差异；实验末,与对照组相比,糖尿病组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。
以上结果提示,本研究以高脂饮食诱导联合小剂量链脲佐菌素注射建立的动物模型,具有中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点。在糖尿病成模后12周这些异常已出现,至实验末稳定存在。
(4)超声心动图及心导管结果比较：
在糖尿病成模后6周,与对照组相比,糖尿病组E/A和E’/A’略有下降(P<0.05)；在糖尿病成模后12周,与对照组相比,糖尿病组LVEF和FS也开始受损(P<0.05)。实验末与对照组大鼠相比,普通饮食STZ组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均明显增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均降低(P<0.05)；与对照组大鼠比较,高脂组大鼠舒张末期左室后壁厚度、左室质量有所增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.01),E’/A’降低(P<0.05)；与对照组大鼠相比,糖尿病组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均显著增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均显著降低(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠左室质量明显增加(P<0.05),等容舒张时间、等容收缩时间明显延长(P<0.01),FS有所降低(P<0.05)。超声背向散射积分结果显示,与对照组大鼠比较,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001),CVIB显著降低(P<0.01～P<0.001)；与对照组大鼠相比,普通饮食STZ组大鼠室间隔及左室侧壁IB%明显增加(P<0.01～P<0.001),室间隔、左室后壁及左室侧壁CVIB显著降低(P<0.05～P<0.001)；与高脂组相比,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001),CVIB明显降低(P<0.001)。
与对照组相比,普通饮食STZ组大鼠左室舒张末期压力明显增加(P<0.001)；与对照组相比,高脂组大鼠左室舒张末期压力有所增加(P<0.05)；与对照组相比,糖尿病组左室舒张末期压力显著增加(P<0.001)。
(5)左室心肌病理学分析：
①心脏大体形态及HE染色结果如下：
心脏大体标本显示糖尿病组心脏大小较其他三组明显增加,定量分析心脏重量／体重比值(HW/BW),结果显示：与正常对照组大鼠相比,糖尿病组HW/BW明显增大(P<0.01)；与正常对照组大鼠相比,普通饮食STZ组大鼠HW/BW增加(P<0.05)；
左室乳头肌部位横截面HE染色显示,与正常对照组比较,糖尿病组大鼠心腔明显扩张,室壁厚度明显增加；
左室局部心肌纵切面及横切面HE染色可见正常对照组大鼠心肌细胞排列整齐、致密,结构清晰,细胞核大小均一,胞浆染色均匀；普通饮食STZ组可见心肌细胞间隙增大、排列紊乱,可见少量肌纤维断裂；高脂组大鼠心肌细胞排列较为整齐,间隙略有增大；糖尿病组大鼠心肌细胞肥大、扭曲、排列紊乱,细胞核大小不甚规则,可见大量心肌纤维断裂,心肌细胞间隙明显增大。
选取×400左室心肌横切面图片,采用Image-pro Plus对各组大鼠心肌细胞横截面积进行分析(每组选6只大鼠),结果显示：与正常对照组大鼠相比,普通饮食STZ组大鼠左室心肌细胞横截面积明显增加(P<0.01)；与正常对照组大鼠相比,高脂组大鼠左室心肌细胞横截面积略有增加(P<0.05)；与正常对照组大鼠相比,糖尿病组大鼠左室心肌细胞横截面积显著增加(P<0.001)；与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠左室心肌细胞横截面积显著增加(P<0.05～P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠左室心肌细胞横截面积明显增加(P<0.05)
BNP是反映心肌肥厚的主要指标之一。与对照组相比,普通饮食STZ组大鼠BNPmRNA表达明显升高,较对照组增加了183%(P<0.001)；高脂组BNP mRNA表达有所增加,较对照组增加了39%；糖尿病组大鼠BNP mRNA表达水平显著升高,较对照组增加了323%(P<0.05)。与高脂组相比,普通饮食STZ组和糖尿病组大鼠BNP mRNA表达均有统计学意义(P<0.01,P<0.05)。
②Masson染色及天狼猩红染色结果如下：
Masson染色显示,心肌细胞染色呈红色,间质胶原呈蓝绿色。正常对照组大鼠心肌胶原纤维分布均匀、纤细,相邻细胞的胶原纤维网完好,血管周围纤维化程度很轻；普通饮食STZ组大鼠胶原纤维增多,排列紊乱,管周纤维化较为明显；高脂组大鼠心肌间质及管周纤维化较对照组有所增多,但胶原纤维仍排列较均匀；糖尿病组心肌内胶原组织明显增多,粗大胶原纤维相互连接成网状或团块状,排列紊乱,分布不匀,紧密围绕于心肌细胞周围及小血管周围。
天狼猩红染色：普通显微镜下胶原组织呈红色,心肌组织呈黄色；偏振光显微镜观察显示：Ⅰ型胶原纤维,紧密排列,显示很强的双折光性,呈黄色或红色的纤维；Ⅱ型胶原纤维,显示弱的双折光,呈多种色彩的疏松网状分布；Ⅲ型胶原纤维,显示弱的双折光,呈绿色的细纤维；Ⅳ型胶原纤维,显示弱的双折光的基膜,呈淡黄色。对照组大鼠胶原纤维均匀纤细,血管周围有极少量胶原；普通饮食STZ组大鼠胶原纤维明显增多,排列紊乱,以黄红色的Ⅰ型胶原为主；高脂组胶原纤维较对照组有所增加,排列较为整齐；糖尿病组大鼠心肌间质胶原纤维以红色及黄红色的Ⅰ型胶原为主,胶原纤维断裂明显,大量红色胶原组织呈不规则网状或团块状围绕在小血管周围。
采用Image-pro Plus对各组大鼠Masson染色图片进行分析,半定量分析结果显示,与正常对照组大鼠相比,普通饮食STZ组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有明显增加(P均<0.001)；与正常对照组大鼠相比,高脂组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有增加(P<0.01；P<0.001)；与正常对照组大鼠相比,糖尿病组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有显著增加(P均<0.001)；与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有显著增加(P<0.05～P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠胶原容积分数(CVF%)明显增加(P<0.05)
采用羟脯氨酸法测定心肌胶原含量,结果显示：与正常对照组相比,普通饮食STZ组大鼠心肌胶原含量明显增加(P<0.001)；与正常对照组相比,高脂组大鼠心肌组织胶原含量略有升高(P<0.01)；与正常对照组相比,糖尿病组大鼠心肌组织胶原含量显著增加(P<0.001);与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠心肌胶原含量明显增加(P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心肌组织胶原含量显著增加(P<0.01)。
(6)油红O染色结果分析：
脂滴呈橙红色,胞核显示蓝色。正常对照组大鼠心肌细胞内几无红色脂滴沉积；普通饮食组及高脂组大鼠心肌细胞内可见散在的脂滴沉着；糖尿病组大鼠心肌细胞内可见大量红色脂滴沉着。采用Image-pro Plus对各组大鼠油红O染色图片进行分析,半定量分析结果显示,与正常对照组相比,普通饮食STZ组、高脂组及糖尿病组脂质沉积均有增加(P<0.01～P<0.001),其中糖尿病组增加最为显著(P<0.001)且糖尿病组较普通饮食STZ组及高脂组均有明显差异(P<0.01,P<0.001)。
(7)心肌炎症浸润结果分析：
与对照组相比,普通饮食STZ组大鼠TNF-a mRNA表达明显升高,较对照组增加了139%(P<0.05)；高脂组TNF-a mRNA表达有所增加,较对照组增加了46%(P<0.05)；糖尿病组大鼠TNF-a mRNA表达水平显著升高,较对照组增加了213%(P<0.05)
与对照组相比,普通饮食STZ组大鼠IL-6mRNA表达明显升高,较对照组增加了119%(P<0.05)；高脂组IL-6mRNA表达有所增加,较对照组增加了106%；糖尿病组大鼠IL-6mRNA表达水平显著升高,较对照组增加了206%(P<0.05)
免疫组化结果显示：与对照组相比,普通饮食STZ组大鼠TNF-α、IL-6阳性表达明显增多；高脂组TNF-α、IL-6表达有所增加；糖尿病组大鼠TNF-α、IL-6阳性表达显著增多。
TNF-α及IL-6的Western blot结果显示：与对照组相比,普通饮食STZ组大鼠TNF-α及IL-6蛋白表达明显增加(P<0.01),高脂组TNF-α及IL-6蛋白含量略有增加(P<0.01～P<0.001),糖尿病组大鼠TNF-α及IL-6蛋白表达显著增加(P<0.001)
(8)心肌胶原Ⅰ及胶原Ⅲ的表达：
免疫组化结果显示：与对照组相比,普通饮食STZ组大鼠collagen Ⅰ及collagen Ⅲ阳性表达明显增多；高脂组collagen Ⅰ及collagen Ⅲ表达有所增加；糖尿病组大鼠collagen Ⅰ及collagen Ⅲ阳性表达显著增多。
Collagen Ⅰ及collagen Ⅲ的Western blot结果显示：与对照组相比,普通饮食STZ组大鼠collagen Ⅰ及collagen Ⅲ蛋白表达明显增加(P<0.001),高脂组collagen Ⅰ及collagen Ⅲ蛋白含量略有增加(P<0.01～P<0.001),糖尿病组大鼠collagen Ⅰ及collagenⅢ蛋白表达显著增加(P<0.001),且collagen Ⅰ蛋白含量增加更为显著。定量分析Western blot结果,以对照组collagen Ⅰ/collagen Ⅲ比值为100%,普通饮食STZ组、高脂组及糖尿病组的比值分别为124%、116%和216%,普通饮食STZ组及糖尿病组较对照组有统计学差异(P<0.05,P<0.001),说明其Collagen Ⅰ蛋白含量增加更为显著。
(9)左室心肌TRB3的表达：
与对照组相比,普通饮食STZ组大鼠TRB3mRNA表达明显升高,较对照组增加了47%(P<0.01)；高脂组TRB3mRNA表达有所增加,较对照组增加了24%(P<0.05)；糖尿病组大鼠TRB3mRNA表达水平显著升高,较对照组增加了285%(P<0.01)；且糖尿病组与高脂组、普通饮食STZ组相比均有统计学意义(P均<0.01)
与对照组相比,普通饮食STZ组大鼠TRB3蛋白表达明显升高,较对照组增加了2.26倍；高脂组TRB3蛋白含量有所增加,较对照组增加了92%；糖尿病组大鼠TRB3蛋白表达水平显著升高,较对照组增加了3.13倍；且糖尿病组与高脂组、普通饮食STZ组相比均有统计学意义(P<0.001,P<0.05)
(10) Western blot检测Akt及MAPK通路各关键分子的表达：
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化Akt与总Akt的比值分别为：0.89,0.53,0.71,0.49,普通饮食STZ组、高脂组及糖尿病组Akt的磷酸化水平与对照组相比均明显降低(P<0.01),其中糖尿病组Akt的磷酸化水平最低,为对照组的55%；与高脂组相比,普通饮食STZ组及糖尿病组Akt的磷酸化水平均有下降,糖尿病组降低更为显著,为高脂组的69%；与普通饮食STZ组相比,糖尿病组Akt的磷酸化水平有所下降,但无统计学意义。
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化ERK1/2与总ERK1/2比值分别为：0.11,0.28,0.21,0.54,普通饮食STZ组、高脂组及糖尿病组ERK1/2的磷酸化水平与对照组相比均明显升高(P<0.001)
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化P38与总P38比值分别为：0.77,0.34,0.43,0.22,普通饮食STZ组、高脂组及糖尿病组P38的磷酸化水平与对照组相比均明显降低(P<0.001)
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化JNK与总JNK比值分别为：0.36,0.63,0.44,0.69,普通饮食STZ组、高脂组及糖尿病组JNK的磷酸化水平与对照组相比均明显升高(P<0.001)。
2.TRB3基因沉默改善2型糖尿病性心肌病的机制：
(1)TRB3基因沉默后心肌及肝脏TRB3mRNA及蛋白的表达
①心肌TRB3mRNA及蛋白的表达：RT-PCR结果显示：与高脂空载体组相比,高脂siRNA组心肌TRB3mRNA表达明显降低(P<0.001),糖尿病siRNA组较糖尿病空载体组心肌TRB3mRNA表达显著降低,大约降低60%(P<0.001)。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组心肌TRB3蛋白水平下降至空载体组的56%(P<0.001)；糖尿病siRNA组较糖尿病空载体组TRB3表达降低,其蛋白水平下降了约52%(P<0.001)。
②肝脏TRB3mRNA及蛋白的表达：与高脂空载体组相比,高脂siRNA组肝脏TRB3mRNA表达明显降低(P<0.001),糖尿病siRNA组较糖尿病空载体组肝脏TRB3mRNA表达显著降低(P<0.001)。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组肝脏TRB3蛋白水平下降至空载体组的59%(P<0.001)；糖尿病siRNA组较糖尿病空载体组TRB3表达降低,其蛋白水平下降至空载体组的45%(P<0.001)。
(2)TRB3基因沉默后2型糖尿病大鼠一般状况的改变：
与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠心脏重量／体重、饮水量及尿量均显著降低(P<0.05),其他指标如体重、饮食量、收缩压、舒张压、平均动脉压及心率等未见统计学差异。
(3)TRB3基因沉默后IPGTT及IPITT的改变：
与高脂空载体组大鼠相比,高脂siRNA组大鼠在OGTT试验60min、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=0.043)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠在OGTT试验30min、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)减少(P<0.05)。
与高脂空载体组大鼠相比,高脂siRNA组大鼠在IPITT试验30min的血糖值明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=0.028)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠在IPITT试验Omin、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=-0.029)。
(4)TRB3基因沉默后生化指标的变化：
经TRB3腺病毒干预2周后,与糖尿病空载体组比较,糖尿病siRNA组大鼠血清TG明显降低(P<0.05)；糖尿病siRNA组大鼠TC、FFA及FBG有所降低但未达到统计学差异；糖尿病siRNA组大鼠ISI有所升高但未达到统计学差异。高脂空载体组较高脂siRNA组各指标变化无统计学差异。
经TRB3腺病毒干预4周后,与糖尿病空载体组比较,糖尿病siRNA组大鼠血清TC、TG、FFA及FBG均明显降低(P<0.05),ISI显著升高(P<0.05)。高脂空载体组较高脂siRNA组各指标变化无统计学差异。
(5)TRB3基因沉默后超声心动图及心导管结果的变化：
高脂siRNA组较高脂空载体组,舒张末期左室后壁厚度及左室质量均减少(P<0.05),等容舒张时间及Tei指数均缩短(P<0.01),室间隔及左室后壁IB%有所降低(P<0.05)；糖尿病siRNA组较糖尿病空载体组,舒张末期左室后壁厚度及左室质量均明显减少(P<0.05),等容舒张时间及Tei指数均缩短(P<0.05),FS及E’/A’均有所升高(P<0.05),室间隔及左室侧壁IB%明显降低,左室后壁及侧壁CVIB明显升高。
与高脂空载体组大鼠相比,高脂siRNA组大鼠左室舒张末期压力明显降低(P<0.05)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠左室舒张末期压力均显著降低(P<0.01)。
(6)TRB3基因沉默后组织病理学的改变：
HE染色显示高脂siRNA组较高脂空载体组心肌细胞间隙减小,心肌细胞排列更为整齐、致密；糖尿病siRNA组较糖尿病空载体组心肌细胞体积减小,心肌细胞扭曲、断裂减少,排列较为整齐,细胞间隙减小。RT-PCR检测心肌组织BNP的mRNA表达,与高脂空载体组大鼠相比,高脂siRNA组大鼠心肌组织BNP的mRNA表达明显降低(P<0.001)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠心肌组织BNP的mRNA表达显著降低(P<0.001)。
油红O染色显示高脂siRNA组较高脂空载体组心肌细胞内脂质沉积有所减少；糖尿病siRNA组较糖尿病空载体组心肌细胞内脂质沉积明显减少。采用Image-pro Plus对各组大鼠油红O染色图片进行分析,半定量分析结果显示,高脂siRNA组较高脂空载体组油红O着色面积减少(P<0.01)；糖尿病siRNA组较糖尿病空载体组脂质沉积明显减少(P<0.001)。
Masson染色显示高脂siRNA组较高脂空载体组间质纤维化及管周纤维化均有所减轻,纤维较为纤细、均匀；糖尿病siRNA组较糖尿病空载体组胶原沉积明显减少,排列较为规整,管周纤维化减轻。天狼猩红染色显示高脂siRNA组较高脂空载体组红黄色胶原沉积有所减轻,纤维组织较为纤细均匀；糖尿病siRNA组较糖尿病空载体组胶原沉积明显减少,排列较为规整,管周可见较少量的纤维组织围绕。采用Image-pro Plus对各组大鼠Masson染色图片进行分析,半定量分析结果显示,高脂siRNA组较高脂空载体组CVF%及PVCA/LA均有所降低(P<0.05,P<0.01)；糖尿病siRNA组较糖尿病空载体组CVF%及PVCA/LA均明显降低(P<0.001,P<0.01)。
羟脯氨酸法测定心肌胶原含量(collagen content)结果显示高脂siRNA组较高脂空载体组胶原含量有所降低(P<0.01)；糖尿病siRNA组较糖尿病空载体组胶原含量明显降低(P<0.001)。
(7)TRB3基因沉默后胶原Ⅰ及胶原Ⅲ表达的变化：
免疫组化结果显示：与高脂空载体组相比,高脂siRNA组Collagen Ⅰ、Ⅲ阳性表达有所减少；糖尿病siRNA组较糖尿病空载体组collagen Ⅰ表达显著减少,collagen Ⅲ阳性表达稍有下降。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组collagen Ⅰ、Ⅲ蛋白表达有所降低；糖尿病siRNA组较糖尿病空载体组collagen Ⅰ蛋白表达显著降低,collagen Ⅲ蛋白表达下降不显著。定量分析Western blot结果,以各自空载体组collagen Ⅰ/collagen Ⅲ比值为100%,高脂siRNA组collagen Ⅰ/collagen Ⅲ比值为94%,糖尿病siRNA组collagen Ⅰ/collagen Ⅲ比值为83%,糖尿病siRNA组较其空载体组有统计学差异。
(8)TRB3基因沉默后对心肌组织炎症的改善：
RT-PCR结果显示,与高脂空载体组相比,高脂siRNA组TNF-α及IL-6mRNA含量均明显降低(P均<0.01)；与糖尿病空载体组相比,糖尿病siRNA组TNF-α及IL-6mRNA含量均显著降低(P均<0.001)。免疫组化结果显示：与高脂空载体组相比,高脂siRNA组TNF-α及IL-6阳性表达有所减少；糖尿病siRNA组较糖尿病空载体组TNF-α及IL-6表达显著减少。Western blot结果显示：定量分析Western blot结果,与高脂空载体组相比,高脂siRNA组TNF-α及IL-6蛋白表达有所降低(P均<0.001)；糖尿病siRNA组较糖尿病空载体组TNF-α及IL-6蛋白表达显著降低(P均<0.001)。
(9)TRB3基因沉默后Akt、MAPK信号通路的改变：
高脂siRNA组与高脂空载体组相比Akt的磷酸化水平升高；糖尿病siRNA组较糖尿病空载体组Akt的磷酸化水平明显升高,其蛋白表达为空载体组的1.45倍(P<0.001)。
与高脂空载体组相比,高
Background
Diabetes mellitus, as CHD risk equivalents, has got the extensive consensus. The risks of cardiovascular complications are markedly increased. Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. However, the underlying mechanism of DCM has not been clarified, so there was no a targeted treatment strategy.
Therefore, it is important to choose an appropriate diabetic animal model for each of diabetes when diabetic cardiomyopathy research. In the present, experimental models of diabetes can be obtained by high-fat diet and/or chemical induction, or the use of spontaneous or genetically derived animal strains. The main limitations of these methodologies include four major parts. The first one is that genetic models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to rat is not straightforward. The second one is that the effect of metabolism-associated gene knockout on DCM cannot be excluded which could not be intervened. The third one is that an excess of chemical results in type1diabetes. The last one is that long-term high fat diet induced diabetic models take a long time, and can not be sustained over the long term.
Thus, the present study focuses on the establishment of diabetic rat model induced by high-fat diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose, blood lipids, glucose tolerance, insulin tolerance and ultrasound biomicroscopy (UBM) were performed to detect the initiation and development of DCM, investigating the feasibility of establishing the type2diabetic rat model.
Objectives
1. To confirm the feasibility of establishing diabetic rat model induced by high-fat diet combined with a small dose of STZ;
2. To confirm the feasibility of establishing diabetic cardiomyopathy rat model induced by high-fat diet combined with a small dose of STZ.
Methods
1. Generation of Diabetes.
Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IP ITT) were performed. Then the rats were randomized into4groups:control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IP ITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBG×FINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg·kg-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.
2. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).
Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose. To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.
3. Blood analyses.
After rats fasted overnight, we collected jugular blood. Total cholesterol (TC), triglycercide levels (TG) and fasting blood glucose (FBG) were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E0877Or, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.
4. Measurements of blood pressures and heart rate.
Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan).
5. Echocardiography.
Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.
For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), standardized integrated backscatter (IB%) and cyclic variation of integrated backscatter (CVIB).
6. Hemodynamic measurement.
Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) was measured.
Results
1. General characteristics of diabetic rats
The rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight and HW/BW were significantly higher in the DM group than control group (P<0.01). No significant differences were seen in body weight in DM group when compared to the control.
2. Glucose and insulin tolerance after a4-week HF diet
The HF and DM groups showed impaired glucose tolerance on IPGTT as compared with the control and chow+STZ groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the HF and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. The DM and HF groups showed the higher mean AUC on IPITT. Thus, the DM group showed insulin resistance after a4-week HF diet.
3. Glucose and insulin tolerance at12weeks after the onset of diabetes
The DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.
4. Glucose and insulin tolerance at the end of the experiment
The DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).
5. TC, TG, FFA, and FBG concentrations
After4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.
6. Blood pressures and heart rate:there were no differences in SBP, DBP and MAP among4groups during the experimental process. HR was higher in HF group compared with the control and chow+STZ groups at the end of the experiment.
7. Left ventricle (LV) dysfunction assessed by echocardiography
We evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group. Additionally, LVEDD, IVRT, IVCT, and Tei index were significantly increased in DM group compared with the control(P<0.01-P<0.001) at the end of the experiment.
The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control group (P<0.05) from6weeks after the onset of diabetes (at week11). And this deteriorated at the end of the experiment.
8. Electrocardiogram
There was obvious arrhythmia in DM groups at the end of experiment.
9. Catheterization
To further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.
Conclusions
1. The combination of high-fat diet and low-dose streptozotocin would successfully establish type2diabetic rat model, resembling human diabetes mellitus;
2. Both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function;
3. The type2diabetic rat model was appropriate for gene intervention.
Background
Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricle (LV) systolic and diastolic dysfunction. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM. Insulin resistance is associated with increased LV mass and deterioration of LV diastolic function. However, the underlying relevance of insulin resistance leading to altered myocardial structure remains incompletely understood.
Tribbles3(TRB3) is a pseudokinase with increased activity in response to fasting that binds to and inhibits the activation of the serine-threonine kinase Akt in the liver. Indeed, humans with a gain-of-function mutation in TRB3show increased insulin resistance and diabetes-associated complications. These observations have led to the suggestion that TRB3elevation contributes to insulin resistance. TRB3also serves as a molecular switch and regulates the activation of the three classes of mitogen-activated protein kinases (MAPKs). TRB3binds to and regulates MAPK kinase, thus controlling the activation of MAPK by incoming signals. However, the TRB3/MAPK signal-transduction pathway has not been investigated in vivo on cardiac tissues directly.
Akt and MAPK are the most important pathways involved in "selective" insulin resistance, and activated MAPK contributes to the development of cardiac fibrosis. So we hypothesized that upregulated TRB3induced by insulin resistance might participate in the pathophysiological process of DCM.
Objectives
1. We established the type2DCM model and determined the relationships among TRB3expression, cardiac remodeling, and cardiac function in the model.
2. To further elucidate the role of TRB3in DCM, we used TRB3gene silencing in vivo to explore the mechanisms of TRB3in DCM as a potential target for treatment.
Methods
1. To design and synthesize4pieces of siRNA against mouse TRB3according to RNAi principle, then construct pGenesil-1.2-TRB3-shRNA plasmid using the most effective siRNA. Next, pShuttle-Basic-EGFP-TRB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRB3-shRNA was constructed.
2. Induction of Diabetes.
Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). The animals were housed at220C with12-h light-dark cycles. After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. Then the rats were randomized into4groups: control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IPITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBGXFINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg-k9-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.
3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).
Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose.
To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.
4. Blood analyses.
After rats fasted overnight, we collected jugular blood. Total cholesterol, triglycercide levels and fasting blood glucose were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E08770r, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.
5. Echocardiography and measurement of BP.
Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.
For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), cyclic variation of integrated backscatter (CVIB) and standardized integrated backscatter (IB%).
Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan) as previously described.
6. Hemodynamic measurement.
Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the LVED pressure (LVEDP) was measured.
7. Histology and morphometric analysis.
Paraformaldehyde (4%)-fixed hearts were bisected transversely at the mid-ventricular level, embedded in paraffin, and cut into4-μm sections. A single myocyte was measured with images captured from hematoxylin and eosin (H&E)-stained sections. The myocyte cross-sectional area was assessed under×400magnification within the left ventricle, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0; Media Cybernatics, Houston, TX, USA).
Dark green-stained collagen fibers were quantified as a measure of fibrosis in Masson's trichrome-stained sections. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were analyzed by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0). CVF was calculated as previously reported. Perivascular collagen was excluded from the CVF measurement. To normalize the area of perivascular collagen around vessels with different sizes, the perivascular collagen content was represented as the PVCA/LA ratio.
Interstitial and perivascular fibrosis were evaluated by Picrosirius red staining. Sections were stained with0.5%sirius red (Sigma, St. Louis, MO) in saturated picric acid for25min. Collagen stained an intense red color.
Myocardial frozen sections (5-μm) were stained with Oil Red O (Sigma, St. Louis, MO) for10min, washed, and then counterstained with hematoxylin for30s. A Nikon microscope (Nikon, Melville, NY) was used to capture the Oil Red O-stained tissue sections.
8. Hydroxyproline analysis. The collagen content of myocardial tissue was determined by hydroxyproline assay. Tissue hydrolysate was detected by use of an ELISA kit (F15649, WESTANG BIO-TECH, SHANGHAI, CHINA). Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%hydroxyproline.
9. Immunohistochemical staining.
Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method. The sections were incubated with primary rabbit polyclonal anti-collagen Ⅰ, anti-collagen Ⅲ, anti-TNF-α, and anti-IL-6antibodies (Abcam, Cambridge, MA, USA) overnight, then a matching biotinylated secondary antibody for30min at37℃. Negative controls were omission of the primary antibody. The stained sections were developed with diaminobenzidine and counterstained with hematoxylin. The results were viewed under a confocal FV1000SPD Laser Scanning microscope (Olympus, Japan).
10. Quantitative real-time RT-PCR.
Total RNA was prepared with the TRIzol reagent (Gibco/Invitrogen, Carlsbad, CA). RT-PCR was performed using the following primers:β-actin forward5'AGA CCT TCA ACA CCC CAG3', reverse5'CAC GAT TTC CCT CTC AGC3'; brain natriuretic protein (BNP) forward5'GGG CTG TGA CGG GCT GAG GTT3', reverse5'AGT TTG TGC TGG AAG ATA AGA3'; TRB3forward5'TGA TGC TGT CTG GAT GAC AA3'; reverse5'GTG AAT GGG GAC TTT GGT CT3'; TNF-α forward5'CAC GCT CTT CTG TCT ACT GA3'; reverse5'GGA CTC CGT GAT GTC TAA GT3'; IL-6forward5'ACC ACT TCA CAA GTC GGA GG3', reverse5'ACA GTG CAT CAT CGC TGT TC3'. Reactions were carried out on a real-time PCR thermocycler (IQ5Real-Time PCR cycler, Bio-Rad), using SYBR green as fluorescence dye. Relative expression analysis involved the2method.
11. Western blot analysis.
Western blot analysis was as previously described. We used antibodies against TRB3(Calbiochem, La Jolla, CA), collagen Ⅰ, collagen Ⅲ, TNF-α, IL-6(Abcam, Cambridge, MA, USA), phospho-ERK/ERK, phospho-p38/p38MAPK, phospho-JNK/JNK, and phospho-Akt/Akt (Cell Signaling Technology, Beverly, MA), followed by anti-IgG horseradish peroxidase-conjugated secondary antibody. TRB3, collagen Ⅰ, collagen Ⅲ, TNF-α, and IL-6protein levels were normalized to that of β-actin as an internal control and phospho-specific proteins to total protein.
12. Gene silencing of TRB3.
Sixty rats were randomized to receive TRB3-siRNA or vehicle treatment. Gene silencing occurred immediately after the appearance of LV diastolic dysfunction. After12weeks of diabetes, both E/A and E'/A' were<1.0as assessed by echocardiography in DM rats. Then animals were injected via the jugular vein with2.5×1010plaque-forming units of an adenovirus harboring TRB3gene (TRB3-siRNA) or a control empty virus (vehicle). Adenovirus transfer was repeated in2weeks. According to our present and previous studies, TRB3level was increased in HF-diet-fed and high fructose-fed rats. Previous studies have shown that insulin resistance was a hallmark of obesityand metabolic syndrome. In light of the interaction between TRB3and insulin resistance, we also investigated the effect of TRB3-siRNA on HF-diet-induced cardiac injury. Four weeks after first adenovirus injection, rats were sacrificed. The heart was excised and weighed.
Results
1. Generation of type2DCM model
(1) General characteristics of diabetic rats
As expected, the rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight was significantly higher in the DM group than control and chow+STZ groups (P<0.01).
(2) Glucose and insulin tolerance
①After a4-week HF diet, insulin resistance was confirmed by IPGTT and IPITT. By IPGTT, the levels of blood glucose in the DM group were significantly higher at week4than at baseline at all time points tested except15min. The AUC across the time for glucose level was higher at week4than at baseline (29.08±1.27vs.25.09±0.73, respectively, P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. Thus, the DM group showed insulin resistance after a4-week HF diet.
②At12weeks after the onset of diabetes (at week17), The DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.
③At the end of the experiment, the DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).
(3) TC, TG, FFA, and FBG concentrations
After4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.
(4) Left ventricle (LV) dysfunction assessed by echocardiography and catheterization
We evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group.
To further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.
(5) Pathology characteristics of diabetic rats
The HW/BW ratio was25%higher in the DM than control group (P<0.01), and LV myocyte size was40%and31%higher, respectively, than the control and HF groups (P<0.001). Furthermore, the relative mRNA expression of BNP, a marker of LV hypertrophy, was higher in DM than control and HF rats (P<0.05).
The DM group showed cardiac fibrosis, with a diffuse, small, patchy and nonuniform pattern, as well as destroyed and disorganized collagen network structure in the interstitial and perivascular areas. CVF and collagen content were higher in the DM than other groups (P<0.05～P<0.001), as was PVCA/LA (P<0.001). These histological changes were confirmed by echocardiographic results. The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control and HF groups (P<0.05). Similarly, CVF, collagen content and PVCA/LA were significantly increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
Coincident with cardiac dysfunction and hypertrophy, myocardial lipid analysis revealed striking myocardial accumulation of triglycerides in diabetic rats. DM rats had higher oil red O staining areas than other groups. The mRNA expression levels of TNF-α and IL-6were significantly higher in DM group as compared with the control (P<0.05, P<0.01). The protein expression levels of TNF-α and IL-6were significantly increased in DM group as compared with the other three groups. Likewise, the protein expression of TNF-a and IL-6content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
(6)Immunohistochemistry and western blot analysis revealed the protein expression of collagen Ⅰ and Ⅲ content increased in the DM group, and the ratio of collagen Ⅰ/Ⅲ significantly elevated as compared with the control group (216±16%vs.100±6%, respectively,P<0.001). Likewise, the protein expression of collagen Ⅰ and Ⅲ content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
These results show the established type2DCM model, with insulin resistance, severe LV dysfunction and myocardial remodeling.
(7) Activated TRB3/MAPK signaling pathway in DCM
Cardiac TRB3mRNA and protein expression was significantly increased in DM rats. We detected Akt expression in the myocardium. The phosphorylation of Akt was significantly lower in the DM group than the control and HF groups.
Accompanied by TRB3overexpression, the phosphorylation of ERK1/2and JNK was markedly increased, whereas that of p38MAPK was decreased, which suggested that the TRB3/MAPK signaling pathway participates in the pathogenesis of DCM.
2. TRB3gene silencing reverses DCM
(1) Detection of cardiac and hepatic TRB3expression by western blot analysis after gene silencing
Compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated mRNA and protein expression of cardiac TRB3. The mRNA and protein expression of hepatic TRB3was also downregulated as compared to the vehicles.
(2) TRB3gene silencing ameliorated metabolism
Water intake and urine volume were decreased in the DM group with TRB3-siRNA treatment. The elevated serum levels of TC, TG, FFA and FBG were greatly reduced after4-week transfection.
(3) TRB3gene silencing improved glucose tolerance and insulin sensitivity
With TRB3gene silencing, ISI was higher in the DM TRB3-siRNA group than vehicle (-5.17±0.14vs.-5.67±0.14, respectively, P<0.05). With TRB3-siRNA treatment, IPGTT and IPITT results showed blood glucose level significantly lower for TRB3-siRNA treated rats than vehicle group (P<0.05). The AUC for glucose and insulin was lower for the DM than vehicle group (P<0.05). The TRB3-siRNA HF group showed lower mean AUC on both IPGTT and IPITT than vehicle group (P<0.05).
(4) Recovery of
糖尿病作为冠心病的等危症已经得到广泛共识,糖尿病患者的心血管风险显著增加。糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的以左室舒张功能受损和心功能不全为主要表现的心肌病变,是糖尿病患者心力衰竭发生率高和死亡率高的主要原因。然而糖尿病性心肌病发生、发展的具体机制目前仍然不详,因而缺少以机制为基础的靶向性治疗。寻找糖尿病性心肌病发生、发展的具体机制并探索可能的有效治疗方法成为目前国内外研究的热点。
开展相应研究的首要问题是建立适宜开展糖尿病性心肌病研究的动物模型,而2型糖尿病动物模型的建立是前提。目前,建立2型糖尿病动物模型的方法主要有五种：一是遗传相关模型,即自发性糖尿病模型；二是通过敲除与代谢有关的基因联合高脂饮食喂养诱发2型糖尿病；三是化学药物损伤联合高脂饮食所诱发的2型糖尿病；四是胰腺部分切除；五是通过长期高脂饮食喂养诱发2型糖尿病。然而,这些建立动物模型的方法仍然存在部分不足：(1)遗传相关模型,由于遗传因素在其发病过程中起主导作用,不完全与临床相符,使其应用受到限制；(2)由于人类的2型糖尿病是多基因遗传病,现有的基因敲除模型不能完全模拟人类2型糖尿病,而且此模型不能排除代谢相关的基因敲除之后对后续基因干预的影响；(3)化学药物剂量过大或胰腺切除过多造成胰腺过度损伤会使血糖过高,而使动物模型倾向于1型糖尿病；(4)长期高脂饮食诱发的2型糖尿病动物模型的造模周期较长,且停用高脂饮食之后,血糖会逐渐恢复正常。
本课题针对2型糖尿病动物模型的上述不足,采用了高脂饮食联合小剂量链脲佐菌素(streptozocin, STZ)诱导的2型糖尿病模型,通过监测体重、血压、血糖、血脂、糖耐量试验及胰岛素耐量试验的动态变化,并采用高分辨率显微超声技术监测糖尿病性心肌病发生和发展过程,探讨建立2型糖尿病性心肌病动物模型的可行性。
目的
1、探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病大鼠动物模型的可行性；
2、探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病性心肌病大鼠动物模型的可行性。
方法
1.选择5周龄健康雄性SD (Sprague Dawley)大鼠60只,体重120g±20g左右,购自山东中医药大学实验动物中心。行腹腔葡萄糖耐量试验(intraperitoneal glucose tolerance test, IPGTT)及胰岛素耐量试验(intraperitoneal insulin tolerance test, IPITT)后随机分为4组：对照组(control组)、普通饮食STZ组(chow+streptozocin组)、高脂饮食组(high fat, HF组)和糖尿病组(diabetes mellitus, DM组)。Control组和chow+STZ组大鼠喂以基础饲料,主要组成为：20%粗蛋白,3%粗脂肪,3%粗纤维,其他74%(包括碳水化合物、微量元素等)。HF组和DM组大鼠喂以高糖高脂高热量饲料(北京华阜康生物有限公司提供),主要成分为：34.5%脂肪、17.5%蛋白质、48%碳水化合物。4周后再次行IPGTT及IPITT, DM组大鼠出现胰岛素抵抗者给予一次性腹腔注射链脲佐菌素(Streptozotocin, STZ)27.5mg/kg,同时chow+STZ组大鼠给予一次性腹腔注射STZ27.5mg/kgo Control组和HF组大鼠给予同等剂量枸橼酸钠缓冲液腹腔注射。各组以原饲料继续喂养1周后,测定空腹血糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),计算胰岛素敏感指数[ISI, ISI=In (FINS×FBG)-1]。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感性减低且有多尿、多饮、多食现象的大鼠纳入实验。糖尿病成模后16周实施动物安乐死。
2.腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT):大鼠禁食12h后行IPGTT,葡萄糖按1g/kg腹腔注射,于0、15、30、60及120分钟采集尾静脉血,使用强生One-Touch血糖仪测量血糖,并计算血糖曲线下面积(area under curve, AUC)。大鼠禁食4h后行IPITT,胰岛素按1unit/kg腹腔注射,余同IPGTT。
3.血液生化指标的测定：大鼠禁食12h后颈静脉取血,检测血清总胆固醇(Total cholesterol, TC)、甘油三酯(triglycercide, TG)、游离脂肪酸(free fatty acid, FFA),血糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),并计算胰岛素敏感指数(insulin sensitivity index, ISI)。
4.血压及心率监测：应用BP-98A智能鼠尾无创血压计测定收缩压、舒张压、平均动脉压及心率；
5.超声心动图和血流动力学监测：采用二维、M超、脉冲多普勒和组织多普勒超声心动图技术以及超声背向散射技术,连续观察DCM发生和发展过程中左心室收缩和舒张功能变化；
6.应用心导管技术测定左室舒张末压(left ventricular end-diastolic pressure, LVEDP):大鼠深麻醉后,经右侧颈动脉插管至左心室,测量左室收缩压及舒张末期压力,监测2型DCM大鼠心室功能的改变。
结果
1、实验末各组大鼠的一般状况比较：
与对照组相比,普通饮食STZ组大鼠心脏重量/体重、饮水量、摄食量及尿量均明显增加(P<0.05)；与对照组相比,高脂组大鼠体重明显增加,心脏重量、饮水量均有所增加(P<0.05)；与对照组相比,糖尿病组大鼠心脏重量、心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均显著增加(P<0.01)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心脏重量、饮水量、摄食量、尿量均明显增加(P<0.001)；与高脂组大鼠相比,糖尿病组大鼠饮水量、摄食量、尿量均显著增加(P<0.001)。
2、高脂4周各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组大鼠在IPGTT试验60min、120min的血糖明显升高(P<0.05)；与对照组相比,高脂组大鼠在IPGTT试验15min、30min、60min、120min的血糖值明显升高(P<0.05)；与普通饮食STZ组大鼠相比,高脂组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(area under curve, AUC)均明显增大(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组血糖曲线下面积(area under curve, AUC)均明显增大。
(2) IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验0min及15min的血糖值均显著升高(P<0.05)；与普通饮食STZ组大鼠相比,糖尿病组大鼠在IPITT试验15min及120min的血糖明显升高(P<0.05)；与对照组相比,高脂组大鼠在IPITT试验0mmin及15min的血糖值明显升高(P<0.05)；与普通饮食STZ组大鼠相比,高脂组大鼠在IPITT试验15min及120mmin的血糖明显升高(P<0.05)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)；与普通饮食STZ组大鼠相比,高脂组及糖尿病组血糖曲线下面积(AUC)均明显增大。
以上结果说明,高脂饮食4周后,高脂组及糖尿病组大鼠出现了明显的胰岛素抵抗。
3、糖尿病成模后12周各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPGTT试验30min及60min的血糖值明显升高(P<0.05)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPGTT试验60mmin的血糖明显升高(P<0.05)。与对照组相比,普通饮食STZ组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(2) IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min、30min、60min及120min的血糖值均显著升高(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05～P<0.01)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPITT试验60min及120mmin的血糖明显升高(P<0.05～P<0.01)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组血糖曲线下面积(AUC)明显增大(P<0.05)。
4、实验末各组大鼠腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
(1) IPGTT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验Omin、15min、30min、60min、120mmin的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在OGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在OGTT试验60min、120min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(2) IPITT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验15min、60min的血糖值均明显升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验15min的血糖值明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验Omin、15min、30min、60min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
5、生化指标的测定：高脂饮食4周即STZ注射前,与对照组相比,高脂组及糖尿病组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),空腹血糖无显著变化,提示注射STZ前糖尿病组大鼠已存在胰岛素抵抗；STZ注射后一周,与对照组相比,普通饮食STZ组及糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),高脂组及糖尿病组血清TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后6周,与对照组相比,糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),糖尿病组血清TC、TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后12周,与对照组相比,糖尿病组血清TC、TG、FFA明显升高(P<0.05),ISI显著降低(P<0.05),FINS无显著差异；实验末,与对照组相比,糖尿病组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。
以上结果提示,本研究以高脂饮食诱导联合小剂量链脲佐菌素注射建立的2型糖尿病动物模型,具有中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点。
6、血压及心率监测：实验过程中各组大鼠收缩压、舒张压及平均动脉压均无差异；实验末,与对照组及普通饮食STZ组相比,糖尿病组大鼠心率明显增快(P<0.05)。
7、超声心动图监测：
在糖尿病成模后6周,与对照组相比,糖尿病组E’/A’开始下降(P<0.05)；在糖尿病成模后12周,与对照组相比,糖尿病组LVEF和FS也开始受损(P<0.05),且E’/A’下降更为明显。实验末与对照组大鼠相比,普通饮食STZ组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均明显增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均降低(P<0.05)；与对照组大鼠比较,高脂组大鼠舒张末期左室后壁厚度、左室质量有所增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.01),E’/A’降低(P<0.05)；与对照组大鼠相比,糖尿病组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均显著增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均显著降低(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠左室质量明显增加(P<0.05),等容舒张时间、等容收缩时间明显延长(P<0.01),FS有所降低(P<0.05)。超声背向散射积分结果显示,与对照组大鼠比较,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05~P<0.001),CVIB显著降低(P<0.01～P<0.001)；与对照组大鼠相比,普通饮食STZ组大鼠室间隔及左室侧壁IB%明显增加(P<0.01～P<0.001),室间隔、左室后壁及左室侧壁CVIB显著降低(P<0.05～P<0.001)；与高脂组相比,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001), CVIB明显降低(P<0.001)。
8、心电图检查：与对照组相比,糖尿病组大鼠出现了多种心电图异常。
9、血流动力学检测：与对照组相比,普通饮食STZ组大鼠左室舒张末期压力明显增加(P<0.001)；与对照组相比,高脂组大鼠左室舒张末期压力有所增加(P<0.05)；与对照组相比,糖尿病组左室舒张末期压力显著增加(P<0.001)。
结论
(1)高脂高热量饮食诱导联合小剂量链脲佐菌素注射建立的2型糖尿病动物模型,具有糖尿病状态稳定,中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点；
(2)高分辨显微超声技术可以无创性地监测2型糖尿病性心肌病的发生发展,2型糖尿病性心肌病大鼠以左室舒张功能障碍为主要表现,伴随有轻微的收缩功能异常；
(3)2型糖尿病大鼠模型的建模因素中没有不可干预因素,适于开展后续的糖尿病基因干预研究。
背景
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的以左室舒张功能受损和心功能不全为主要表现的心肌病变,是糖尿病患者心力衰竭发生率高和死亡率高的主要原因。自从1972年Ruber等首先提出DCM这一概念后,国内外学者在此领域中进行了大量的基础和临床研究。虽然临床研究显示,严格控制血糖可明显降低糖尿病微血管病变,但并未显著降低心血管事件的发生,糖尿病患者心力衰竭的发生率仍居高不下。因此,有必要对DCM的发生机制进行深入研究。
DCM发生的始动因素和关键环节是胰岛素抵抗(insulin resistance)。胰岛素抵抗通过抑制胰岛素信号传导和葡萄糖转运,降低心肌对葡萄糖的利用,使机体抑制脂肪分解的能力减弱,使血浆游离脂肪酸(FFA)和甘油三酯升高,而FFA可呈剂量依赖性地抑制细胞葡萄糖的转运和磷酸化作用,造成心肌能量代谢的异常,进一步加重胰岛素抵抗,造成恶性循环。胰岛素缺乏,能量代谢异常,酶系活性受抑导致心肌细胞核皱缩,线粒体肿胀及糖原沉积,形成DCM的病理基础。临床实验同时证实胰岛素抵抗与左室病理性重构和左室舒张功能障碍密切相关。但胰岛素抵抗与糖尿病性心肌病之间的深层链接机制尚不清楚,因此缺乏有针对性的治疗靶点。
TRBs基因是Groβhans等于2000年在果蝇体内新发现的一类丝氨酸/苏氨酸蛋白激酶样蛋白基因家族,是抑制有丝分裂,诱导细胞凋亡的核基因,也被称为神经细胞死亡诱导蛋白激酶。人类TRBs家族包括TRB1、TRB2、TRB3,它们含有一个的相同的中心区,以及70-100氨基酸残基的N-末端和25个残基的C-末端区。果蝇TRB3是哺乳动物的同系物,因此,目前有关TRBs基因的研究主要集中在TRB3。TRB3具有广泛的生物活性,与糖脂代谢及胰岛素抵抗密切相关。研究发现,TRB3基因可以通过抑制Akt的功能影响胰岛素信号传导通路；TRB3是MAPK活性重要的调节蛋白,在MAPKK水平调控MAPK的磷酸化作用和活性,少量的TRB3增加即可显著增强ERK1/2和JNK活性,轻度抑制P38活性。但关于TRB3/MAPK信号通路的研究未见到后续的报道。
Akt和MAPK是选择性“胰岛素抵抗”两个关键通路,而且MAPK是调控心肌间质纤维化的关键信号分子。因此,我们推测在2型糖尿病状态下,胰岛素抵抗刺激TRB3过表达,并可能通过调控Akt及MAPK信号途径而影响DCM的发生和进展,但目前尚未见类似报道。据此,本课题拟通过2型DCM大鼠模型,研究TRB3在DCM发生、发展中的作用,并应用TRB3-siRNA特异性抑制2型糖尿病大鼠体内TRB3的表达,进而探讨TRB3基因沉默是否可以通过调控“胰岛素抵抗”相关信号通路从而起到改善DCM的作用。
目的
1.建立2型糖尿病性心肌病大鼠动物模型,探讨TRB3在2型糖尿病性心肌病发生、发展中的作用；
2.2型糖尿病大鼠体内转染TRB3腺病毒,观察2型糖尿病性心肌病的心脏结构和功能的变化；
3.在分子生物学、组织学和整体功能水平研究TRB3基因沉默改善2型糖尿病性心肌病的机制。
方法
1.pAdxsi-TRB3-shRNA病毒载体的构建：根据RNAi干扰技术原则,设计4对针对大鼠的TRB3基因的siRNA,经筛选后,选择沉默效率最高的序列构建pGenesil-1.2-TRB3-shRNA质粒,随后构建pShuttle-Basic-EGFP-TRB3-shRNA重组穿梭载体质粒,最后构建pAdxsi-GFP-TRB3-shRNA腺病毒载体。
2.选择健康雄性SD大鼠60只,体重120g±20g左右,购自山东中医药大学实验动物中心。行腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)后随机分为4组：对照组(control组)、普通饮食+链脲佐菌素组(chow+STZ组)、高脂组(HF组)、糖尿病组(DM组)。Control组和chow+STZ组大鼠喂以基础饲料,主要组成为：20%粗蛋白,3%粗脂肪,3%粗纤维,其他74%(包括碳水化合物、微量元素等)。HF组和DM组大鼠喂以高糖高脂高热量饲料(北京华阜康生物有限公司提供),主要成分为：34.5%脂肪、17.5%蛋白质、48%碳水化合物。4周后再次行IPGTT及IPITT,DM组大鼠出现胰岛素抵抗者给予一次性腹腔注射STZ27.5mg/kg,同时chow+STZ组大鼠给予一次性腹腔注射STZ27.5mg/kg。Control组和HF组大鼠给予同等剂量枸橼酸钠缓冲液腹腔注射。各组以原饲料继续喂养1周后,测定空腹血糖(FBG)和胰岛素(FINS),计算胰岛素敏感指数[ISI, ISI=In (FINS×FBG)-1]。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感性减低且有多尿、多饮、多食现象的大鼠纳入实验。糖尿病成模后16周实施动物安乐死,称取心脏重量,计算心脏重量/体重比值。
3.腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IP ITT):大鼠禁食12h后行IPGTT,葡萄糖按1g/kg腹腔注射,于0、15、30、60及120分钟采集尾静脉血,使用强生One-Touch血糖仪测量血糖,并计算血糖曲线下面积。大鼠禁食4h后行IPITT,胰岛素按1unit/kg腹腔注射,余同IPGTT。
4.血液生化指标的测定：大鼠禁食12h后颈静脉取血,检测血清总胆固醇(Total cholesterol, TC)、甘油三酯(triglycercide, TG)、游离脂肪酸(free fatty acid, FFA),皿糖(fasting blood glucose, FBG)和胰岛素(fasting insulin, FINS),并计算胰岛素敏感指数(insulin sensitivity index, ISI)。
5.超声心动图和血流动力学监测：采用二维、M超、脉冲多普勒和组织多普勒超声心动图技术以及超声背向散射技术,连续观察DCM发生和发展过程中左心室收缩和舒张功能变化；
6.应用心导管技术测定左室舒张末压：大鼠深麻醉后,经右侧颈动脉插管至左心室,测量左室收缩压及舒张末期压力,监测2型DCM大鼠心室功能的改变。
7.病理学检测：对左室心肌组织分别进行H&E染色、Masson染色、天狼猩红染色以及油红O染色,应用图象分析仪测定心肌细胞大小、胶原体积分数、管周胶原面积/管腔面积及心肌脂质含量。
8.心肌羟脯氨酸含量测定：将干燥至恒重的左室心肌组织称重后置于6NHCL中110℃水解16h,应用ELISA试剂盒测定水解物中羟脯氨酸的含量。因羟脯氨酸在胶原组织中占13.5%,所以最终结果显示为胶原含量(ug/mg左室干重)。
9.免疫组织化学染色：应用免疫组织化学染色法,光镜下观察心肌间质的Ⅰ型胶原、Ⅲ型胶原、肿瘤坏死因子-α (TNF-α)及白介素6(IL-6)的分布情况。
10.实时定量RT-PCR检测：取新鲜左室心肌组织,Trizol法提取RNA,实时定量荧光RT-PCR技术检测TRB3、脑钠肽(brain natriuretic peptide, BNP)、TNF-α及IL-6的mRNA相对表达量。
11. Western blot检测：取新鲜左室心肌组织,提取蛋白,Western blot检测心肌组织内TRB3、胶原Ⅰ、胶原Ⅲ、TNF-α、IL-6及磷酸化及非磷酸化的ERK1/2、p38、JNK、 Akt的蛋白表达量。
12. TRB3-siRNA腺病毒体内转染实验：选择健康雄性SD大鼠60只(体重120g±20g左右),随机分为HF+空载体组、HF+TRB3siRNA组、DM+空载体组、DM+TRB3siRNA组。糖尿病组造模过程同上。糖尿病成模后12周,HF+TRB3siRNA组、DM+TRB3siRNA组大鼠经颈静脉注射TRB3腺病毒,HF+空载体组及DM+空载体组注射pAdxsi空病毒载体,2周后补充注射一次,TRB3腺病毒注射4周后处死动物留取标本。
结果
1.2型糖尿病性心肌病大鼠模型的建立：
(1)实验末各组大鼠的一般状况比较：
与对照组相比,普通饮食STZ组大鼠心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均明显增加(P<0.05)；与对照组相比,高脂组大鼠体重明显增加,心脏重量、饮水量及左室舒张末期压力均有所增加(P<0.05)；与对照组相比,糖尿病组大鼠心脏重量、心脏重量/体重、饮水量、摄食量、尿量及左室舒张末期压力均显著增加(P<0.01)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心脏重量、饮水量、摄食量、尿量均明显增加(P<0.001)；与高脂组大鼠相比,糖尿病组大鼠饮水量、摄食量、尿量均显著增加(P<0.001)。
(2)腹腔葡萄糖耐量试验(IPGTT)及胰岛素耐量试验(IPITT)结果：
①DM组大鼠高脂4周后与实验前IPGTT比较：
IPGTT结果显示：糖尿病组大鼠高脂4周后与基线状态相比,在IPGTT试验0min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积(AUC)明显增大(P<0.05)。
②DM组大鼠高脂4周后与实验前IPITT比较：
IPITT结果显示：糖尿病组大鼠高脂4周后与基线状态相比,在IPGTT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积(AUC)明显增大(P<0.05)。
③糖尿病成模后12周各组大鼠IPGTT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPGTT试验30min及60min的血糖值明显升高(P<0.05)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPGTT试验60min的血糖明显升高(P<0.05)。与对照组相比,普通饮食STZ组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
④糖尿病成模后12周各组大鼠IPITT结果分析：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min、30min、60min及120min的血糖值均显著升高(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验Omin、15min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05-P<0.01)；与高脂组大鼠相比,普通饮食STZ组大鼠在IPITT试验60min及120min的血糖明显升高(P<0.05-P<0.01)。与对照组相比,高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05～P<0.01)；与高脂组大鼠相比,糖尿病组血糖曲线下面积(AUC)明显增大(P<0.05)。
⑤实验末IPGTT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPGTT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在OGTT试验120min的血糖明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在OGTT试验60min、120min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
⑥实验末IPITT试验结果：
与对照组大鼠相比,糖尿病组大鼠在IPITT试验15min、60min的血糖值均明显升高(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠在IPITT试验15min的血糖值明显升高(P<0.05)；与对照组相比,普通饮食STZ组大鼠在IPITT试验0min、15min、30min、60min的血糖值明显升高(P<0.05)。与对照组相比,普通饮食STZ组、高脂组及糖尿病组的血糖曲线下面积(AUC)均明显增大(P<0.05)。
(3)生化指标的测定：
STZ注射前,与对照组相比,高脂组及糖尿病组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),空腹血糖无显著变化,提示注射STZ前糖尿病组大鼠已存在胰岛素抵抗；STZ注射后一周,与对照组相比,普通饮食STZ组及糖尿病组FBG明显升高(P<0.05),ISI显著降低(P<0.05),高脂组及糖尿病组血清TG、FFA明显升高(P<0.05),FINS无显著差异；实验末,与对照组相比,糖尿病组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。
以上结果提示,本研究以高脂饮食诱导联合小剂量链脲佐菌素注射建立的动物模型,具有中度胰岛素抵抗,中度高血糖、高血脂等表现,基本符合2型糖尿病的临床特点。在糖尿病成模后12周这些异常已出现,至实验末稳定存在。
(4)超声心动图及心导管结果比较：
在糖尿病成模后6周,与对照组相比,糖尿病组E/A和E’/A’略有下降(P<0.05)；在糖尿病成模后12周,与对照组相比,糖尿病组LVEF和FS也开始受损(P<0.05)。实验末与对照组大鼠相比,普通饮食STZ组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均明显增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均降低(P<0.05)；与对照组大鼠比较,高脂组大鼠舒张末期左室后壁厚度、左室质量有所增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.01),E’/A’降低(P<0.05)；与对照组大鼠相比,糖尿病组大鼠舒张末期左室内径、室间隔厚度、左室后壁厚度、左室质量及左室容积均显著增加(P<0.05),等容舒张时间、等容收缩时间及Tei指数均明显延长(P<0.001),EF、FS及E’/A’均显著降低(P<0.05)；与高脂组大鼠相比,糖尿病组大鼠左室质量明显增加(P<0.05),等容舒张时间、等容收缩时间明显延长(P<0.01),FS有所降低(P<0.05)。超声背向散射积分结果显示,与对照组大鼠比较,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001),CVIB显著降低(P<0.01～P<0.001)；与对照组大鼠相比,普通饮食STZ组大鼠室间隔及左室侧壁IB%明显增加(P<0.01～P<0.001),室间隔、左室后壁及左室侧壁CVIB显著降低(P<0.05～P<0.001)；与高脂组相比,糖尿病组大鼠室间隔、左室后壁及左室侧壁IB%均显著增加(P<0.05～P<0.001),CVIB明显降低(P<0.001)。
与对照组相比,普通饮食STZ组大鼠左室舒张末期压力明显增加(P<0.001)；与对照组相比,高脂组大鼠左室舒张末期压力有所增加(P<0.05)；与对照组相比,糖尿病组左室舒张末期压力显著增加(P<0.001)。
(5)左室心肌病理学分析：
①心脏大体形态及HE染色结果如下：
心脏大体标本显示糖尿病组心脏大小较其他三组明显增加,定量分析心脏重量／体重比值(HW/BW),结果显示：与正常对照组大鼠相比,糖尿病组HW/BW明显增大(P<0.01)；与正常对照组大鼠相比,普通饮食STZ组大鼠HW/BW增加(P<0.05)；
左室乳头肌部位横截面HE染色显示,与正常对照组比较,糖尿病组大鼠心腔明显扩张,室壁厚度明显增加；
左室局部心肌纵切面及横切面HE染色可见正常对照组大鼠心肌细胞排列整齐、致密,结构清晰,细胞核大小均一,胞浆染色均匀；普通饮食STZ组可见心肌细胞间隙增大、排列紊乱,可见少量肌纤维断裂；高脂组大鼠心肌细胞排列较为整齐,间隙略有增大；糖尿病组大鼠心肌细胞肥大、扭曲、排列紊乱,细胞核大小不甚规则,可见大量心肌纤维断裂,心肌细胞间隙明显增大。
选取×400左室心肌横切面图片,采用Image-pro Plus对各组大鼠心肌细胞横截面积进行分析(每组选6只大鼠),结果显示：与正常对照组大鼠相比,普通饮食STZ组大鼠左室心肌细胞横截面积明显增加(P<0.01)；与正常对照组大鼠相比,高脂组大鼠左室心肌细胞横截面积略有增加(P<0.05)；与正常对照组大鼠相比,糖尿病组大鼠左室心肌细胞横截面积显著增加(P<0.001)；与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠左室心肌细胞横截面积显著增加(P<0.05～P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠左室心肌细胞横截面积明显增加(P<0.05)
BNP是反映心肌肥厚的主要指标之一。与对照组相比,普通饮食STZ组大鼠BNPmRNA表达明显升高,较对照组增加了183%(P<0.001)；高脂组BNP mRNA表达有所增加,较对照组增加了39%；糖尿病组大鼠BNP mRNA表达水平显著升高,较对照组增加了323%(P<0.05)。与高脂组相比,普通饮食STZ组和糖尿病组大鼠BNP mRNA表达均有统计学意义(P<0.01,P<0.05)。
②Masson染色及天狼猩红染色结果如下：
Masson染色显示,心肌细胞染色呈红色,间质胶原呈蓝绿色。正常对照组大鼠心肌胶原纤维分布均匀、纤细,相邻细胞的胶原纤维网完好,血管周围纤维化程度很轻；普通饮食STZ组大鼠胶原纤维增多,排列紊乱,管周纤维化较为明显；高脂组大鼠心肌间质及管周纤维化较对照组有所增多,但胶原纤维仍排列较均匀；糖尿病组心肌内胶原组织明显增多,粗大胶原纤维相互连接成网状或团块状,排列紊乱,分布不匀,紧密围绕于心肌细胞周围及小血管周围。
天狼猩红染色：普通显微镜下胶原组织呈红色,心肌组织呈黄色；偏振光显微镜观察显示：Ⅰ型胶原纤维,紧密排列,显示很强的双折光性,呈黄色或红色的纤维；Ⅱ型胶原纤维,显示弱的双折光,呈多种色彩的疏松网状分布；Ⅲ型胶原纤维,显示弱的双折光,呈绿色的细纤维；Ⅳ型胶原纤维,显示弱的双折光的基膜,呈淡黄色。对照组大鼠胶原纤维均匀纤细,血管周围有极少量胶原；普通饮食STZ组大鼠胶原纤维明显增多,排列紊乱,以黄红色的Ⅰ型胶原为主；高脂组胶原纤维较对照组有所增加,排列较为整齐；糖尿病组大鼠心肌间质胶原纤维以红色及黄红色的Ⅰ型胶原为主,胶原纤维断裂明显,大量红色胶原组织呈不规则网状或团块状围绕在小血管周围。
采用Image-pro Plus对各组大鼠Masson染色图片进行分析,半定量分析结果显示,与正常对照组大鼠相比,普通饮食STZ组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有明显增加(P均<0.001)；与正常对照组大鼠相比,高脂组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有增加(P<0.01；P<0.001)；与正常对照组大鼠相比,糖尿病组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有显著增加(P均<0.001)；与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠胶原容积分数(CVF%)及血管周围胶原面积/管腔面积(PVCA/LA)均有显著增加(P<0.05～P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠胶原容积分数(CVF%)明显增加(P<0.05)
采用羟脯氨酸法测定心肌胶原含量,结果显示：与正常对照组相比,普通饮食STZ组大鼠心肌胶原含量明显增加(P<0.001)；与正常对照组相比,高脂组大鼠心肌组织胶原含量略有升高(P<0.01)；与正常对照组相比,糖尿病组大鼠心肌组织胶原含量显著增加(P<0.001);与高脂组大鼠相比,普通饮食STZ组和糖尿病组大鼠心肌胶原含量明显增加(P<0.001)；与普通饮食STZ组大鼠相比,糖尿病组大鼠心肌组织胶原含量显著增加(P<0.01)。
(6)油红O染色结果分析：
脂滴呈橙红色,胞核显示蓝色。正常对照组大鼠心肌细胞内几无红色脂滴沉积；普通饮食组及高脂组大鼠心肌细胞内可见散在的脂滴沉着；糖尿病组大鼠心肌细胞内可见大量红色脂滴沉着。采用Image-pro Plus对各组大鼠油红O染色图片进行分析,半定量分析结果显示,与正常对照组相比,普通饮食STZ组、高脂组及糖尿病组脂质沉积均有增加(P<0.01～P<0.001),其中糖尿病组增加最为显著(P<0.001)且糖尿病组较普通饮食STZ组及高脂组均有明显差异(P<0.01,P<0.001)。
(7)心肌炎症浸润结果分析：
与对照组相比,普通饮食STZ组大鼠TNF-a mRNA表达明显升高,较对照组增加了139%(P<0.05)；高脂组TNF-a mRNA表达有所增加,较对照组增加了46%(P<0.05)；糖尿病组大鼠TNF-a mRNA表达水平显著升高,较对照组增加了213%(P<0.05)
与对照组相比,普通饮食STZ组大鼠IL-6mRNA表达明显升高,较对照组增加了119%(P<0.05)；高脂组IL-6mRNA表达有所增加,较对照组增加了106%；糖尿病组大鼠IL-6mRNA表达水平显著升高,较对照组增加了206%(P<0.05)
免疫组化结果显示：与对照组相比,普通饮食STZ组大鼠TNF-α、IL-6阳性表达明显增多；高脂组TNF-α、IL-6表达有所增加；糖尿病组大鼠TNF-α、IL-6阳性表达显著增多。
TNF-α及IL-6的Western blot结果显示：与对照组相比,普通饮食STZ组大鼠TNF-α及IL-6蛋白表达明显增加(P<0.01),高脂组TNF-α及IL-6蛋白含量略有增加(P<0.01～P<0.001),糖尿病组大鼠TNF-α及IL-6蛋白表达显著增加(P<0.001)
(8)心肌胶原Ⅰ及胶原Ⅲ的表达：
免疫组化结果显示：与对照组相比,普通饮食STZ组大鼠collagen Ⅰ及collagen Ⅲ阳性表达明显增多；高脂组collagen Ⅰ及collagen Ⅲ表达有所增加；糖尿病组大鼠collagen Ⅰ及collagen Ⅲ阳性表达显著增多。
Collagen Ⅰ及collagen Ⅲ的Western blot结果显示：与对照组相比,普通饮食STZ组大鼠collagen Ⅰ及collagen Ⅲ蛋白表达明显增加(P<0.001),高脂组collagen Ⅰ及collagen Ⅲ蛋白含量略有增加(P<0.01～P<0.001),糖尿病组大鼠collagen Ⅰ及collagenⅢ蛋白表达显著增加(P<0.001),且collagen Ⅰ蛋白含量增加更为显著。定量分析Western blot结果,以对照组collagen Ⅰ/collagen Ⅲ比值为100%,普通饮食STZ组、高脂组及糖尿病组的比值分别为124%、116%和216%,普通饮食STZ组及糖尿病组较对照组有统计学差异(P<0.05,P<0.001),说明其Collagen Ⅰ蛋白含量增加更为显著。
(9)左室心肌TRB3的表达：
与对照组相比,普通饮食STZ组大鼠TRB3mRNA表达明显升高,较对照组增加了47%(P<0.01)；高脂组TRB3mRNA表达有所增加,较对照组增加了24%(P<0.05)；糖尿病组大鼠TRB3mRNA表达水平显著升高,较对照组增加了285%(P<0.01)；且糖尿病组与高脂组、普通饮食STZ组相比均有统计学意义(P均<0.01)
与对照组相比,普通饮食STZ组大鼠TRB3蛋白表达明显升高,较对照组增加了2.26倍；高脂组TRB3蛋白含量有所增加,较对照组增加了92%；糖尿病组大鼠TRB3蛋白表达水平显著升高,较对照组增加了3.13倍；且糖尿病组与高脂组、普通饮食STZ组相比均有统计学意义(P<0.001,P<0.05)
(10) Western blot检测Akt及MAPK通路各关键分子的表达：
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化Akt与总Akt的比值分别为：0.89,0.53,0.71,0.49,普通饮食STZ组、高脂组及糖尿病组Akt的磷酸化水平与对照组相比均明显降低(P<0.01),其中糖尿病组Akt的磷酸化水平最低,为对照组的55%；与高脂组相比,普通饮食STZ组及糖尿病组Akt的磷酸化水平均有下降,糖尿病组降低更为显著,为高脂组的69%；与普通饮食STZ组相比,糖尿病组Akt的磷酸化水平有所下降,但无统计学意义。
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化ERK1/2与总ERK1/2比值分别为：0.11,0.28,0.21,0.54,普通饮食STZ组、高脂组及糖尿病组ERK1/2的磷酸化水平与对照组相比均明显升高(P<0.001)
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化P38与总P38比值分别为：0.77,0.34,0.43,0.22,普通饮食STZ组、高脂组及糖尿病组P38的磷酸化水平与对照组相比均明显降低(P<0.001)
对照组、普通饮食STZ组、高脂组及糖尿病组磷酸化JNK与总JNK比值分别为：0.36,0.63,0.44,0.69,普通饮食STZ组、高脂组及糖尿病组JNK的磷酸化水平与对照组相比均明显升高(P<0.001)。
2.TRB3基因沉默改善2型糖尿病性心肌病的机制：
(1)TRB3基因沉默后心肌及肝脏TRB3mRNA及蛋白的表达
①心肌TRB3mRNA及蛋白的表达：RT-PCR结果显示：与高脂空载体组相比,高脂siRNA组心肌TRB3mRNA表达明显降低(P<0.001),糖尿病siRNA组较糖尿病空载体组心肌TRB3mRNA表达显著降低,大约降低60%(P<0.001)。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组心肌TRB3蛋白水平下降至空载体组的56%(P<0.001)；糖尿病siRNA组较糖尿病空载体组TRB3表达降低,其蛋白水平下降了约52%(P<0.001)。
②肝脏TRB3mRNA及蛋白的表达：与高脂空载体组相比,高脂siRNA组肝脏TRB3mRNA表达明显降低(P<0.001),糖尿病siRNA组较糖尿病空载体组肝脏TRB3mRNA表达显著降低(P<0.001)。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组肝脏TRB3蛋白水平下降至空载体组的59%(P<0.001)；糖尿病siRNA组较糖尿病空载体组TRB3表达降低,其蛋白水平下降至空载体组的45%(P<0.001)。
(2)TRB3基因沉默后2型糖尿病大鼠一般状况的改变：
与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠心脏重量／体重、饮水量及尿量均显著降低(P<0.05),其他指标如体重、饮食量、收缩压、舒张压、平均动脉压及心率等未见统计学差异。
(3)TRB3基因沉默后IPGTT及IPITT的改变：
与高脂空载体组大鼠相比,高脂siRNA组大鼠在OGTT试验60min、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=0.043)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠在OGTT试验30min、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)减少(P<0.05)。
与高脂空载体组大鼠相比,高脂siRNA组大鼠在IPITT试验30min的血糖值明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=0.028)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠在IPITT试验Omin、120min的血糖值均明显降低(P<0.05),血糖曲线下面积(AUC)明显减少(P=-0.029)。
(4)TRB3基因沉默后生化指标的变化：
经TRB3腺病毒干预2周后,与糖尿病空载体组比较,糖尿病siRNA组大鼠血清TG明显降低(P<0.05)；糖尿病siRNA组大鼠TC、FFA及FBG有所降低但未达到统计学差异；糖尿病siRNA组大鼠ISI有所升高但未达到统计学差异。高脂空载体组较高脂siRNA组各指标变化无统计学差异。
经TRB3腺病毒干预4周后,与糖尿病空载体组比较,糖尿病siRNA组大鼠血清TC、TG、FFA及FBG均明显降低(P<0.05),ISI显著升高(P<0.05)。高脂空载体组较高脂siRNA组各指标变化无统计学差异。
(5)TRB3基因沉默后超声心动图及心导管结果的变化：
高脂siRNA组较高脂空载体组,舒张末期左室后壁厚度及左室质量均减少(P<0.05),等容舒张时间及Tei指数均缩短(P<0.01),室间隔及左室后壁IB%有所降低(P<0.05)；糖尿病siRNA组较糖尿病空载体组,舒张末期左室后壁厚度及左室质量均明显减少(P<0.05),等容舒张时间及Tei指数均缩短(P<0.05),FS及E’/A’均有所升高(P<0.05),室间隔及左室侧壁IB%明显降低,左室后壁及侧壁CVIB明显升高。
与高脂空载体组大鼠相比,高脂siRNA组大鼠左室舒张末期压力明显降低(P<0.05)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠左室舒张末期压力均显著降低(P<0.01)。
(6)TRB3基因沉默后组织病理学的改变：
HE染色显示高脂siRNA组较高脂空载体组心肌细胞间隙减小,心肌细胞排列更为整齐、致密；糖尿病siRNA组较糖尿病空载体组心肌细胞体积减小,心肌细胞扭曲、断裂减少,排列较为整齐,细胞间隙减小。RT-PCR检测心肌组织BNP的mRNA表达,与高脂空载体组大鼠相比,高脂siRNA组大鼠心肌组织BNP的mRNA表达明显降低(P<0.001)；与糖尿病空载体组大鼠相比,糖尿病siRNA组大鼠心肌组织BNP的mRNA表达显著降低(P<0.001)。
油红O染色显示高脂siRNA组较高脂空载体组心肌细胞内脂质沉积有所减少；糖尿病siRNA组较糖尿病空载体组心肌细胞内脂质沉积明显减少。采用Image-pro Plus对各组大鼠油红O染色图片进行分析,半定量分析结果显示,高脂siRNA组较高脂空载体组油红O着色面积减少(P<0.01)；糖尿病siRNA组较糖尿病空载体组脂质沉积明显减少(P<0.001)。
Masson染色显示高脂siRNA组较高脂空载体组间质纤维化及管周纤维化均有所减轻,纤维较为纤细、均匀；糖尿病siRNA组较糖尿病空载体组胶原沉积明显减少,排列较为规整,管周纤维化减轻。天狼猩红染色显示高脂siRNA组较高脂空载体组红黄色胶原沉积有所减轻,纤维组织较为纤细均匀；糖尿病siRNA组较糖尿病空载体组胶原沉积明显减少,排列较为规整,管周可见较少量的纤维组织围绕。采用Image-pro Plus对各组大鼠Masson染色图片进行分析,半定量分析结果显示,高脂siRNA组较高脂空载体组CVF%及PVCA/LA均有所降低(P<0.05,P<0.01)；糖尿病siRNA组较糖尿病空载体组CVF%及PVCA/LA均明显降低(P<0.001,P<0.01)。
羟脯氨酸法测定心肌胶原含量(collagen content)结果显示高脂siRNA组较高脂空载体组胶原含量有所降低(P<0.01)；糖尿病siRNA组较糖尿病空载体组胶原含量明显降低(P<0.001)。
(7)TRB3基因沉默后胶原Ⅰ及胶原Ⅲ表达的变化：
免疫组化结果显示：与高脂空载体组相比,高脂siRNA组Collagen Ⅰ、Ⅲ阳性表达有所减少；糖尿病siRNA组较糖尿病空载体组collagen Ⅰ表达显著减少,collagen Ⅲ阳性表达稍有下降。
Western blot结果显示：与高脂空载体组相比,高脂siRNA组collagen Ⅰ、Ⅲ蛋白表达有所降低；糖尿病siRNA组较糖尿病空载体组collagen Ⅰ蛋白表达显著降低,collagen Ⅲ蛋白表达下降不显著。定量分析Western blot结果,以各自空载体组collagen Ⅰ/collagen Ⅲ比值为100%,高脂siRNA组collagen Ⅰ/collagen Ⅲ比值为94%,糖尿病siRNA组collagen Ⅰ/collagen Ⅲ比值为83%,糖尿病siRNA组较其空载体组有统计学差异。
(8)TRB3基因沉默后对心肌组织炎症的改善：
RT-PCR结果显示,与高脂空载体组相比,高脂siRNA组TNF-α及IL-6mRNA含量均明显降低(P均<0.01)；与糖尿病空载体组相比,糖尿病siRNA组TNF-α及IL-6mRNA含量均显著降低(P均<0.001)。免疫组化结果显示：与高脂空载体组相比,高脂siRNA组TNF-α及IL-6阳性表达有所减少；糖尿病siRNA组较糖尿病空载体组TNF-α及IL-6表达显著减少。Western blot结果显示：定量分析Western blot结果,与高脂空载体组相比,高脂siRNA组TNF-α及IL-6蛋白表达有所降低(P均<0.001)；糖尿病siRNA组较糖尿病空载体组TNF-α及IL-6蛋白表达显著降低(P均<0.001)。
(9)TRB3基因沉默后Akt、MAPK信号通路的改变：
高脂siRNA组与高脂空载体组相比Akt的磷酸化水平升高；糖尿病siRNA组较糖尿病空载体组Akt的磷酸化水平明显升高,其蛋白表达为空载体组的1.45倍(P<0.001)。
与高脂空载体组相比,高
Background
Diabetes mellitus, as CHD risk equivalents, has got the extensive consensus. The risks of cardiovascular complications are markedly increased. Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. However, the underlying mechanism of DCM has not been clarified, so there was no a targeted treatment strategy.
Therefore, it is important to choose an appropriate diabetic animal model for each of diabetes when diabetic cardiomyopathy research. In the present, experimental models of diabetes can be obtained by high-fat diet and/or chemical induction, or the use of spontaneous or genetically derived animal strains. The main limitations of these methodologies include four major parts. The first one is that genetic models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to rat is not straightforward. The second one is that the effect of metabolism-associated gene knockout on DCM cannot be excluded which could not be intervened. The third one is that an excess of chemical results in type1diabetes. The last one is that long-term high fat diet induced diabetic models take a long time, and can not be sustained over the long term.
Thus, the present study focuses on the establishment of diabetic rat model induced by high-fat diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose, blood lipids, glucose tolerance, insulin tolerance and ultrasound biomicroscopy (UBM) were performed to detect the initiation and development of DCM, investigating the feasibility of establishing the type2diabetic rat model.
Objectives
1. To confirm the feasibility of establishing diabetic rat model induced by high-fat diet combined with a small dose of STZ;
2. To confirm the feasibility of establishing diabetic cardiomyopathy rat model induced by high-fat diet combined with a small dose of STZ.
Methods
1. Generation of Diabetes.
Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IP ITT) were performed. Then the rats were randomized into4groups:control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IP ITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBG×FINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg·kg-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.
2. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).
Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose. To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.
3. Blood analyses.
After rats fasted overnight, we collected jugular blood. Total cholesterol (TC), triglycercide levels (TG) and fasting blood glucose (FBG) were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E0877Or, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.
4. Measurements of blood pressures and heart rate.
Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan).
5. Echocardiography.
Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.
For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), standardized integrated backscatter (IB%) and cyclic variation of integrated backscatter (CVIB).
6. Hemodynamic measurement.
Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) was measured.
Results
1. General characteristics of diabetic rats
The rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight and HW/BW were significantly higher in the DM group than control group (P<0.01). No significant differences were seen in body weight in DM group when compared to the control.
2. Glucose and insulin tolerance after a4-week HF diet
The HF and DM groups showed impaired glucose tolerance on IPGTT as compared with the control and chow+STZ groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the HF and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. The DM and HF groups showed the higher mean AUC on IPITT. Thus, the DM group showed insulin resistance after a4-week HF diet.
3. Glucose and insulin tolerance at12weeks after the onset of diabetes
The DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.
4. Glucose and insulin tolerance at the end of the experiment
The DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).
5. TC, TG, FFA, and FBG concentrations
After4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.
6. Blood pressures and heart rate:there were no differences in SBP, DBP and MAP among4groups during the experimental process. HR was higher in HF group compared with the control and chow+STZ groups at the end of the experiment.
7. Left ventricle (LV) dysfunction assessed by echocardiography
We evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group. Additionally, LVEDD, IVRT, IVCT, and Tei index were significantly increased in DM group compared with the control(P<0.01-P<0.001) at the end of the experiment.
The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control group (P<0.05) from6weeks after the onset of diabetes (at week11). And this deteriorated at the end of the experiment.
8. Electrocardiogram
There was obvious arrhythmia in DM groups at the end of experiment.
9. Catheterization
To further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.
Conclusions
1. The combination of high-fat diet and low-dose streptozotocin would successfully establish type2diabetic rat model, resembling human diabetes mellitus;
2. Both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function;
3. The type2diabetic rat model was appropriate for gene intervention.
Background
Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality. Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricle (LV) systolic and diastolic dysfunction. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM. Insulin resistance is associated with increased LV mass and deterioration of LV diastolic function. However, the underlying relevance of insulin resistance leading to altered myocardial structure remains incompletely understood.
Tribbles3(TRB3) is a pseudokinase with increased activity in response to fasting that binds to and inhibits the activation of the serine-threonine kinase Akt in the liver. Indeed, humans with a gain-of-function mutation in TRB3show increased insulin resistance and diabetes-associated complications. These observations have led to the suggestion that TRB3elevation contributes to insulin resistance. TRB3also serves as a molecular switch and regulates the activation of the three classes of mitogen-activated protein kinases (MAPKs). TRB3binds to and regulates MAPK kinase, thus controlling the activation of MAPK by incoming signals. However, the TRB3/MAPK signal-transduction pathway has not been investigated in vivo on cardiac tissues directly.
Akt and MAPK are the most important pathways involved in "selective" insulin resistance, and activated MAPK contributes to the development of cardiac fibrosis. So we hypothesized that upregulated TRB3induced by insulin resistance might participate in the pathophysiological process of DCM.
Objectives
1. We established the type2DCM model and determined the relationships among TRB3expression, cardiac remodeling, and cardiac function in the model.
2. To further elucidate the role of TRB3in DCM, we used TRB3gene silencing in vivo to explore the mechanisms of TRB3in DCM as a potential target for treatment.
Methods
1. To design and synthesize4pieces of siRNA against mouse TRB3according to RNAi principle, then construct pGenesil-1.2-TRB3-shRNA plasmid using the most effective siRNA. Next, pShuttle-Basic-EGFP-TRB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRB3-shRNA was constructed.
2. Induction of Diabetes.
Sixty male Sprague-Dawley (SD) rats (120-140g) were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine (Jinan, China). The animals were housed at220C with12-h light-dark cycles. After1week of acclimatization, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. Then the rats were randomized into4groups: control, chow+streptozotocin (STZ), high-fat diet (HF) and diabetes mellitus (DM). HF and DM groups were fed a high-fat diet (34.5%fat,17.5%protein,48%carbohydrate, Beijing HFK Bio-Technology Co. Ltd, China), and the other2groups received normal chow. Four weeks later, IPGTT and IPITT were performed again, and blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index [ISI=ln(FBGXFINS)-1] was calculated. Diabetes mellitus was induced by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO)(27.5mg-k9-1intraperitoneally [ip] in0.1mol/1citrate buffer, pH4.5) to rats with insulin resistance. Rats in the chow+STZ group received the same dose of STZ. The control and HF groups received citrate buffer (ip) alone. One week after STZ administration, rats with FBG>11.1mmol/1in2consecutive analyses were considered the diabetic rat model. After16weeks of diabetes, rats were sacrificed. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.
3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).
Glucose tolerance was assessed by IPGTT after rats fasted for12h. A bolus of glucose (1g/kg ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60, and120min and tested for glucose. Plasma glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The mean area under the receiver operating characteristic curve (AUC) was calculated for glucose.
To evaluate insulin tolerance, IPITT was performed after rats fasted for4h. A bolus of insulin (1unit/kg ip) was administered, and blood samples were taken for glucose measurement as described above.
4. Blood analyses.
After rats fasted overnight, we collected jugular blood. Total cholesterol, triglycercide levels and fasting blood glucose were analyzed with use of the Bayer1650blood chemistry analyzer (Bayer, Tarrytown, NY, USA). Free fatty acids (FFA) concentrations were measured using an enzymatic test kit (CSB-E08770r, HuaMei BIO-TECH, Wuhan, CHINA). Fasting insulin level was measured by ELISA. ISI was calculated.
5. Echocardiography and measurement of BP.
Echocardiography involved use of the Vevo770imaging system (VisualSonics, Inc, Toronto, Canada). Images were obtained from2-D, M-mode, pulsed-wave (PW) Doppler and tissue Doppler imaging (TDI). All measurements were performed by the same observer and were the average of6consecutive cardiac cycles. Wall thickness and left ventricle dimensions were obtained from a long-axis view at the level of chordae tendineae. Diameters of left ventricular end-diastole (LVEDd), end-diastolic posterior wall (LVPWd) and septum thickness (IVSd), as well as ejection fraction (LVEF) and fractional shortening (FS) were measured according to the American Society of Echocardiography guidelines. The mitral-valve pulsed Doppler recordings were obtained from the apical four-chamber view. After pulsed Doppler, we evaluated transmitral flow velocity variables, including peak E, peak A, and the E/A ratio. Isovolumetric contraction time (IVCT), isovolumetric relaxation time (IVRT) and ejection time (ET) were measured and were used to calculate the Tei index (Tei index=IVCT+IVRT/ET). TDI of the mitral annulus was obtained from the apical four-chamber view. We analyzed early (E') and late diastolic velocity (A') and calculated E'/A' and E/E'.
For quantitative analysis of integrated backscatter (IBS) of the left ventricle, we used a commercially available software package (acoustic densitometry; Phillips Medical Systems, Netherlands).2-D echocardiographic images, including left ventricle long-axis and apical four-chamber views were obtained. The following variables were measured:time-averaged integrated backscatter (IBS), cyclic variation of integrated backscatter (CVIB) and standardized integrated backscatter (IB%).
Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan) as previously described.
6. Hemodynamic measurement.
Rats under deep anesthesia underwent hemodynamic measurement. A fluid-filled catheter was advanced from the right carotid artery into the left ventricle, and the LVED pressure (LVEDP) was measured.
7. Histology and morphometric analysis.
Paraformaldehyde (4%)-fixed hearts were bisected transversely at the mid-ventricular level, embedded in paraffin, and cut into4-μm sections. A single myocyte was measured with images captured from hematoxylin and eosin (H&E)-stained sections. The myocyte cross-sectional area was assessed under×400magnification within the left ventricle, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0; Media Cybernatics, Houston, TX, USA).
Dark green-stained collagen fibers were quantified as a measure of fibrosis in Masson's trichrome-stained sections. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were analyzed by quantitative morphometry with automated image analysis (Image-Pro Plus, Version5.0). CVF was calculated as previously reported. Perivascular collagen was excluded from the CVF measurement. To normalize the area of perivascular collagen around vessels with different sizes, the perivascular collagen content was represented as the PVCA/LA ratio.
Interstitial and perivascular fibrosis were evaluated by Picrosirius red staining. Sections were stained with0.5%sirius red (Sigma, St. Louis, MO) in saturated picric acid for25min. Collagen stained an intense red color.
Myocardial frozen sections (5-μm) were stained with Oil Red O (Sigma, St. Louis, MO) for10min, washed, and then counterstained with hematoxylin for30s. A Nikon microscope (Nikon, Melville, NY) was used to capture the Oil Red O-stained tissue sections.
8. Hydroxyproline analysis. The collagen content of myocardial tissue was determined by hydroxyproline assay. Tissue hydrolysate was detected by use of an ELISA kit (F15649, WESTANG BIO-TECH, SHANGHAI, CHINA). Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%hydroxyproline.
9. Immunohistochemical staining.
Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method. The sections were incubated with primary rabbit polyclonal anti-collagen Ⅰ, anti-collagen Ⅲ, anti-TNF-α, and anti-IL-6antibodies (Abcam, Cambridge, MA, USA) overnight, then a matching biotinylated secondary antibody for30min at37℃. Negative controls were omission of the primary antibody. The stained sections were developed with diaminobenzidine and counterstained with hematoxylin. The results were viewed under a confocal FV1000SPD Laser Scanning microscope (Olympus, Japan).
10. Quantitative real-time RT-PCR.
Total RNA was prepared with the TRIzol reagent (Gibco/Invitrogen, Carlsbad, CA). RT-PCR was performed using the following primers:β-actin forward5'AGA CCT TCA ACA CCC CAG3', reverse5'CAC GAT TTC CCT CTC AGC3'; brain natriuretic protein (BNP) forward5'GGG CTG TGA CGG GCT GAG GTT3', reverse5'AGT TTG TGC TGG AAG ATA AGA3'; TRB3forward5'TGA TGC TGT CTG GAT GAC AA3'; reverse5'GTG AAT GGG GAC TTT GGT CT3'; TNF-α forward5'CAC GCT CTT CTG TCT ACT GA3'; reverse5'GGA CTC CGT GAT GTC TAA GT3'; IL-6forward5'ACC ACT TCA CAA GTC GGA GG3', reverse5'ACA GTG CAT CAT CGC TGT TC3'. Reactions were carried out on a real-time PCR thermocycler (IQ5Real-Time PCR cycler, Bio-Rad), using SYBR green as fluorescence dye. Relative expression analysis involved the2method.
11. Western blot analysis.
Western blot analysis was as previously described. We used antibodies against TRB3(Calbiochem, La Jolla, CA), collagen Ⅰ, collagen Ⅲ, TNF-α, IL-6(Abcam, Cambridge, MA, USA), phospho-ERK/ERK, phospho-p38/p38MAPK, phospho-JNK/JNK, and phospho-Akt/Akt (Cell Signaling Technology, Beverly, MA), followed by anti-IgG horseradish peroxidase-conjugated secondary antibody. TRB3, collagen Ⅰ, collagen Ⅲ, TNF-α, and IL-6protein levels were normalized to that of β-actin as an internal control and phospho-specific proteins to total protein.
12. Gene silencing of TRB3.
Sixty rats were randomized to receive TRB3-siRNA or vehicle treatment. Gene silencing occurred immediately after the appearance of LV diastolic dysfunction. After12weeks of diabetes, both E/A and E'/A' were<1.0as assessed by echocardiography in DM rats. Then animals were injected via the jugular vein with2.5×1010plaque-forming units of an adenovirus harboring TRB3gene (TRB3-siRNA) or a control empty virus (vehicle). Adenovirus transfer was repeated in2weeks. According to our present and previous studies, TRB3level was increased in HF-diet-fed and high fructose-fed rats. Previous studies have shown that insulin resistance was a hallmark of obesityand metabolic syndrome. In light of the interaction between TRB3and insulin resistance, we also investigated the effect of TRB3-siRNA on HF-diet-induced cardiac injury. Four weeks after first adenovirus injection, rats were sacrificed. The heart was excised and weighed.
Results
1. Generation of type2DCM model
(1) General characteristics of diabetic rats
As expected, the rats in DM group had the highest values of water intake, food intake and urine volume as compared with the other3groups (P<0.01～P<0.001). Heart weight was significantly higher in the DM group than control and chow+STZ groups (P<0.01).
(2) Glucose and insulin tolerance
①After a4-week HF diet, insulin resistance was confirmed by IPGTT and IPITT. By IPGTT, the levels of blood glucose in the DM group were significantly higher at week4than at baseline at all time points tested except15min. The AUC across the time for glucose level was higher at week4than at baseline (29.08±1.27vs.25.09±0.73, respectively, P<0.05). Similarly, IPITT revealed impaired insulin sensitivity. Thus, the DM group showed insulin resistance after a4-week HF diet.
②At12weeks after the onset of diabetes (at week17), The DM group showed impaired glucose tolerance on IPGTT as compared with the control and HF groups; especially blood glucose levels were significantly elevated at all time points as compared with the control except0min (P<0.05). The AUC across the time for glucose level was higher at week4in the chow+STZ and DM groups than the control (P<0.05). Similarly, IPITT revealed impaired insulin sensitivity; especially blood glucose levels in DM group were significantly elevated at all time points as compared with the control (P<0.05～P<0.01).The DM group showed the highest mean AUC on IPITT when compared to the other3groups.
③At the end of the experiment, the DM group showed impaired glucose tolerance on IPGTT as compared with the other3groups; especially blood glucose levels were significantly elevated at all time points as compared with the control (P<0.05). The DM group showed the highest mean AUC on both IPGTT and IPITT. Similarly, the chow+STZ and HF groups showed higher mean AUC on both IPGTT and IPITT as compared with the control (P<0.05).
(3) TC, TG, FFA, and FBG concentrations
After4weeks of a high-fat diet, serum TG and FFA levels were significantly higher in the HF and DM groups than control and chow+STZ groups (P<0.05). The ISI was markedly decreased in the HF and DM groups (P<0.05). Insulin resistance appeared at week4in rats fed a HF diet. One week after STZ injection, FBG was remarkably elevated in the DM group and remained elevated until the end of the experiment. ISI consistently decreased in the DM group after the onset of diabetes. Simultaneously, in the DM group, serum TC, TG, and FFA levels were maintained at higher levels than the control (P<0.05) during diabetes. Thus, the diabetic model induced by a HF diet and low-dose STZ was characterized by insulin resistance, moderate hyperglycemia and hyperlipidemia resembling the state of chemical diabetes in humans.
(4) Left ventricle (LV) dysfunction assessed by echocardiography and catheterization
We evaluated EF, fractional shortening (FS), E/A, and E'/A' to investigate changes in systolic and diastolic function. Similar to the pattern in humans, in rats, diastolic dysfunction precedes systolic dysfunction, beginning from2to3months after the induction of DM. In our study, at6weeks after the onset of diabetes (at week11), the DM rats showed a moderate decrease in E/A and E'/A' as compared with the control (P<0.05); LVEF and FS were impaired from week17. At the end of the experiment, LVEF, FS, E/A, and E'/A' were further decreased in the DM group, with the reduction in E/A and E'/A' more pronounced. LVEF, FS, E/A, and E'/A' were also reduced in the chow+STZ group compared with the control (P<0.05) at the end of the experiment. LVEDd was the highest in the DM group.
To further confirm the LV diastolic dysfunction, LVEDP was measured by cardiac catheterization. DM rats had the highest LV pressure. The chow+STZ and HF groups showed higher LV pressure as compared with the control (P<0.01～P<0.001). In summary, both systolic and diastolic dysfunction developed and progressed during DCM, with predominant deterioration of diastolic function.
(5) Pathology characteristics of diabetic rats
The HW/BW ratio was25%higher in the DM than control group (P<0.01), and LV myocyte size was40%and31%higher, respectively, than the control and HF groups (P<0.001). Furthermore, the relative mRNA expression of BNP, a marker of LV hypertrophy, was higher in DM than control and HF rats (P<0.05).
The DM group showed cardiac fibrosis, with a diffuse, small, patchy and nonuniform pattern, as well as destroyed and disorganized collagen network structure in the interstitial and perivascular areas. CVF and collagen content were higher in the DM than other groups (P<0.05～P<0.001), as was PVCA/LA (P<0.001). These histological changes were confirmed by echocardiographic results. The DM group showed increased IB%and decreased cyclic variation of integrated backscatter (CVIB) in the IVS, LV posterior and lateral walls as compared with the control and HF groups (P<0.05). Similarly, CVF, collagen content and PVCA/LA were significantly increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
Coincident with cardiac dysfunction and hypertrophy, myocardial lipid analysis revealed striking myocardial accumulation of triglycerides in diabetic rats. DM rats had higher oil red O staining areas than other groups. The mRNA expression levels of TNF-α and IL-6were significantly higher in DM group as compared with the control (P<0.05, P<0.01). The protein expression levels of TNF-α and IL-6were significantly increased in DM group as compared with the other three groups. Likewise, the protein expression of TNF-a and IL-6content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
(6)Immunohistochemistry and western blot analysis revealed the protein expression of collagen Ⅰ and Ⅲ content increased in the DM group, and the ratio of collagen Ⅰ/Ⅲ significantly elevated as compared with the control group (216±16%vs.100±6%, respectively,P<0.001). Likewise, the protein expression of collagen Ⅰ and Ⅲ content was increased in chow+STZ and HF groups as compared with the control (P<0.01～P<0.001).
These results show the established type2DCM model, with insulin resistance, severe LV dysfunction and myocardial remodeling.
(7) Activated TRB3/MAPK signaling pathway in DCM
Cardiac TRB3mRNA and protein expression was significantly increased in DM rats. We detected Akt expression in the myocardium. The phosphorylation of Akt was significantly lower in the DM group than the control and HF groups.
Accompanied by TRB3overexpression, the phosphorylation of ERK1/2and JNK was markedly increased, whereas that of p38MAPK was decreased, which suggested that the TRB3/MAPK signaling pathway participates in the pathogenesis of DCM.
2. TRB3gene silencing reverses DCM
(1) Detection of cardiac and hepatic TRB3expression by western blot analysis after gene silencing
Compared with vehicle treatment, TRB3-siRNA treatment conferred downregulated mRNA and protein expression of cardiac TRB3. The mRNA and protein expression of hepatic TRB3was also downregulated as compared to the vehicles.
(2) TRB3gene silencing ameliorated metabolism
Water intake and urine volume were decreased in the DM group with TRB3-siRNA treatment. The elevated serum levels of TC, TG, FFA and FBG were greatly reduced after4-week transfection.
(3) TRB3gene silencing improved glucose tolerance and insulin sensitivity
With TRB3gene silencing, ISI was higher in the DM TRB3-siRNA group than vehicle (-5.17±0.14vs.-5.67±0.14, respectively, P<0.05). With TRB3-siRNA treatment, IPGTT and IPITT results showed blood glucose level significantly lower for TRB3-siRNA treated rats than vehicle group (P<0.05). The AUC for glucose and insulin was lower for the DM than vehicle group (P<0.05). The TRB3-siRNA HF group showed lower mean AUC on both IPGTT and IPITT than vehicle group (P<0.05).
(4) Recovery of
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