TRIB3与糖尿病动脉粥样硬化关系的实验研究
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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,然而糖尿病加速动脉粥样硬化发生、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。寻找糖尿病加速动脉粥样硬化发生、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动脉粥样硬化研究的动物模型。目前,建立2型糖尿病动脉粥样硬化动物模型的方法主要有两种：一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；二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)由于人类的2型糖尿病是多基因遗传病,现有的基因敲除小鼠模型不能完全模拟类的2型糖尿病；(2)代谢相关基因敲除小鼠模型不能排除代谢相关的基因敲除之后对动脉粥样硬化的影响,且基因敲除为不可干预因素；(3)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,不能有效区分年龄和糖尿病相关的动脉粥样硬化；(4)单纯高脂饮食诱发的2型糖尿病动物模型,先有血脂代谢紊乱,而后逐渐出现血糖升高,这与人类糖尿病常有的糖脂代谢紊乱发病顺序不完全一致。因此,本研究针对2型糖尿病动脉粥样硬化动物模型的上述不足,采用ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素诱发糖尿病,通过监测体重、血压、血糖、糖耐量试验的动态变化,同时采用高分辨率显微超声技术监测动脉粥样硬化发生和发展过程,探讨建立2型糖尿病动脉粥样硬化动物模型的可行性。
目的
1.探讨高脂高糖饮食联合小剂量链脲佐菌素建立ApoE-/-/LDLR-/-小鼠2型糖尿病动物模型可行性；
2.探讨高脂高糖饮食联合小剂量链脲佐菌素建立2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化模型可行性；
方法
1.动物实验：3周龄雄性ApoE-/-/LDLR-/-小鼠30只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=15)和糖尿病组(n=15),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg/kg,2周后再次测IPGTT,随机血糖≥11.1mmol／L时,可作为2型糖尿病成模的动物入组；
2.IPGTT试验：小鼠空腹10-16h,按照1.5g/kg体重腹腔注射葡萄糖,然后于0min、15 min、30 min、60 min和120 min分别断尾取血检测血糖；
3.ApoE-/-/LDLR-/-小鼠体重的监测：定期监测小鼠体重的变化；
4.ApoE-/-/LDLR-/-小鼠血流动力学的评价：所有实验动物在20周龄、22周龄和24周龄时通过小鼠尾动脉血压测量仪监测血压和心率,每只动物测量3次,取其平均值；
5.ApoE-/-/LDLR-/-小鼠动脉粥样硬化斑块的显微超声监测：分别于相应观察时间点(14周龄、16周龄、18周龄、20周龄、22周龄和24周龄)进行动脉超声检查通过高分辨率显微超声技术监测主动脉、头臂干动脉和颈总动脉的动脉粥样硬化斑块的发生和发展,测量内膜中层厚度(IMT)；
6.ApoE-/-/LDLR-/-小鼠血清糖代谢指标的测定：实验末,处死动物,分离血清,测定血糖、血清胰岛素,计算胰岛素抵抗指数。
结果
1.ApoE-/-/LDLR-/-小鼠体重变化情况：3周龄时,两组雄性ApoE-/-/LDLR-/-小鼠体重差异没有统计学意义(P=0.213)；高脂饮食喂养6周后,糖尿病组ApoE-/-/LDLR-/-小鼠体重与普通饮食组ApoE-/-/LDLR-/-小鼠差异具有统计学意义(P=0.010)；注射链脲佐菌素后2周,糖尿病组ApoE-/-/LDLR-/-小鼠与普通饮食组ApoE-/-/LDLR-/-小鼠体重差异无统计学意义(P=0.081)；继续高脂饮食喂养至20周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重,差异具有统计学意义(P=0.0015)；随后小鼠体重基本稳定,至24周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重仍然明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重,差异具有统计学意义(P=0.0073)。
2.ApoE-/-/LDLR-/-小鼠血流动力学变化：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的心率未见明显改变(P=0.309),然而随着小鼠周龄的增加,小鼠的心率逐渐降低(P=0.014)。与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的收缩压显著升高(P=0.00004),小鼠周龄对收缩压的影响没有统计学意义(P=0.212),小鼠周龄与糖尿病在影响小鼠收缩压方面没有交互作用(P=0.390)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的舒张压显著升高(P=0.000003),小鼠周龄对舒张压的影响没有统计学意义(P=0.137),小鼠周龄与糖尿病在影响小鼠舒张压方面没有交互作用(P=0.652)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的平均动脉压显著升高(P=0.0001),小鼠周龄对平均动脉压的影响没有统计学意义(P=0.212),小鼠周龄与糖尿病在影响小鼠平均动脉压方面没有交互作用(P=0.263)。
3.ApoE-/-/LDLR-/-小鼠糖耐量试验结果：IPGTT的结果表明,3周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异没有统计学意义；9周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异具有统计学意义(P均<0.05)；随后注射小剂量链脲佐菌素,11周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异具有统计学意义(P均<0.05)。普通饮食组不同周龄的糖耐量试验结果差异没有统计学意义；糖尿病组不同周龄的糖耐量试验结果差异具有统计学意义。
4.ApoE-/-/LDLR-/-小鼠血糖代谢指标比较结果：在24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠相比,2型糖尿病ApoE-/-/LDLR-/-小鼠血糖、血清胰岛素以及胰岛素抵抗指数均明显升高(P=0.028,P=0.025,P=0.015),表明2型糖尿病ApoE-/-/LDLR-/-小鼠存在高血糖、高胰岛素血症和胰岛素抵抗。
5.ApoE-/-/LDLR-/-小鼠动脉IMT变化结果：ApoE-/-/LDLR-/-小鼠头臂干动脉IMT变化早于颈动脉。ApoE-/-/LDLR-/-小鼠头臂干动脉IMT在16周龄时,两组之间差异具有统计学意义(P=0.000)；差异维持至24周龄。ApoE-/-/LDLR-/-小鼠颈动脉IMT始终保持较慢的增长;至22周龄时,两组小鼠颈动脉IMT差异具有统计学意义(P=0.022)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT进一步增加(P=0.001)。
结论
(1)3周龄雄性ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素(75mg／kg)诱发糖尿病状态稳定；
(2)ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素诱发糖尿病后,体重和血压均增加,与人类的糖尿病状态基本类似；
(3)2型糖尿病ApoE-/-/LDLR-/-小鼠模型能够发生动脉粥样硬化斑块,并且发生时间提前；
(4)2型糖尿病ApoE-/-/LDLR-/-小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干预研究。
背景
糖尿病已经成为动脉粥样硬化和冠心病最重要的危险因素。但是糖尿病患者冠状动脉粥样硬化斑块病变严重、易损斑块发生率高的机制尚未完全阐明,因此,也缺乏以机制为基础的治疗措施。故而,探索糖尿病患者冠状动脉粥样硬化斑块病变严重、易损斑块发生率高的机制,建立以机制为导向的治疗措施成为近年来防治糖尿病心血管并发症的研究热点。
实现对糖尿病最重要的心血管并发症——急性冠脉综合征的有效遏制,关键在于寻找糖尿病和急性冠脉综合征发病的共同中间环节。目前对易损斑块的形成机制进行的基础和临床研究认为斑块是否破裂取决于斑块内在的组织特征和斑块所受的应力一应变关系的相互作用。而炎症机制、氧化应激、糖代谢异常、脂质代谢异常和血流动力学改变等均与易损斑块的形成有关。这些因素导致的动脉粥样斑块内细胞凋亡尤其巨噬细胞凋亡是导致易损斑块形成的重要机制。巨噬细胞凋亡的信号调控是一个复杂的网络体系。在巨噬细胞凋亡的调控机制中,Akt信号转导途径起关键作用。TRIB3通过抑制胰岛素激活的PI3K/Akt信号转导通路导致胰岛素抵抗,参与糖脂代谢,是2型糖尿发病的机制之一。流行病学研究发现,TRIB3基因Q84R错义突变患者发生严重胰岛素抵抗引起的相关病理改变如高胰岛素血症、高脂血症、代谢综合征及心血管疾病危险增加。TRIB3/Akt信号途径通过对巨噬细胞凋亡和糖代谢的影响,在胰岛素抵抗、糖尿病和易损斑块的形成过程中起重要作用。因此本课题拟通过体内转染腺病毒载体介导的TRIB3-shRNA特异性抑制2型糖尿病ApoE-/-/LDLR-/-小鼠体内TRIB3的表达,进而观察TRIB3基因沉默对2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样斑块稳定性影响,并阐述其作用机制。
目的
1.通过RNAi干扰技术设计合成针对小鼠TRIB3基因的shRNA,构建重组腺病毒载体pAdxsi-TRIB3-shRNA;
2.2型糖尿病ApoE-/-/LDLR-/-小鼠体内转染pAdxsi-TRIB3-shRNA,观察动脉粥样硬化斑块稳定性的变化;
3.在细胞分子水平、组织学水平和整体功能水平研究TRIB3基因沉默稳定2型糖尿病易损斑块的机制。
方法
1. pAdxsi-TRIB3-shRNA病毒载体的构建：根据RNAi干扰技术原则,设计合成4对针对小鼠TRIB3基因的siRNA,经过筛选之后,选择沉默效率最高的序列构建pGenesil-1.2-TRIB3-shRNA质粒,随后构建pShuttle-Basic-EGFP-TRIB3-shRNA重组穿梭载体质粒,最后构建pAdxsi-GFP-TRIB3-shRNA腺病毒载体。
2.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化动物模型：3周龄雄性ApoE-/-/LDLR-/-小鼠60只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=30)和糖尿病组(n=30),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg/kg,2周后再次测IPGTT,随机血糖≥11.1mmol/L时,可作为2型糖尿病成模的动物入组,超声监测至20周龄时开始体内转染腺病毒。
3. pAdxsi-TRIB3-shRNA病毒体内转染实验：在小鼠20周龄时,完成超声检查后,糖尿病+RNAi组(n=15)和普通饮食+RNAi组(n=15)经尾静脉注射pAdxsi-TRIB3-shRNA腺病毒,糖尿病组(n=15)和普通饮食组(n=15)经尾静脉注射pAdxsi空病毒载体。两周后,复查超声后再次追加腺病毒。24周龄时,完成超声检查,处死动物。
4.血液生化指标检测：检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平,测定血糖和血清胰岛素,计算胰岛素抵抗指数。
5.腹腔巨噬细胞功能的检测：分离小鼠腹腔巨噬细胞,经MOMA-2鉴定后,分别测定腹腔巨噬细胞的迁移功能、粘附功能和吞噬功能。
6.病理学检测：对头臂干动脉斑块分别进行HE染色、Masson染色、油红O染色、天狼猩红染色,并通过免疫组化方法检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-SM-actin)以及TRIB3的表达。测量斑块面积和纤维帽厚度,计算帽／核比值,观察斑块的形态结构,计算斑块内胶原、脂质、平滑肌、巨噬细胞的含量,并计算易损指数;
7.实时定量RT-PCR检测：取新鲜动脉斑块组织,提取RNA,实时定量荧光RT-PCR检测TRIB3的mRNA表达量;
8. Western bot检测：取新鲜动脉斑块组织,提取蛋白质,Western bot检测斑块内TRIB3/Akt/Caspase-3信号通路的蛋白表达量；
9.TUNEL检测：取冰冻切片,TUNEL法检测斑块内的细胞凋亡量以及巨噬细胞的凋亡量。
结果
1. pAdxsi-TRIB3-shRNA病毒载体的构建：将4对TRIB3 siRNA,转染巨噬细胞24h,采用实时定量RT-PCR检测GAPDH mRNA和TRIB3 mRNA的表达水平,以判断各对siRNA的抑制效率,筛选出抑制效率最佳的siRNA,其对TRIB3的最佳抑制效率为99.98%。选此序列构建pGenesil-1.2-TRIB3-shRNA质粒,经鉴定测序正确后,用于构建pShuttle-Basic-EGFP-TRIB3-shRNA重组穿梭载体；鉴定正确后,构建pAdxsi-GFP-TRIB3shRNA重组腺病毒质粒,再次鉴定正确后经Pad限制性内切酶线性化后,应用Lipofectamine2000脂质体转染HEK293细胞,行腺病毒包装,扩增,纯化得到滴度为2×1011 PFU/mL的腺病毒。
2. pAdxsi-TRIB3-shRNA病毒体内转染后2型糖尿病ApoE-/-/LDLR-/-小鼠—般状况的改变：
(1)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显升高(P=0.001)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的普通饮食组ApoE-/-/LDLR-/-小鼠,其体重略有降低(P=0.034)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠体重明显降低(P=0.003)；对于ApoE-/-/LDLR-/-小鼠体重而言,高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病能够增加体重(P=0.001),TRIB3的RNAi对降低ApoE-/-/LDLR-/-小鼠的体重无影响(P=0.112),高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病和TRIB3-shRNA之间没有交互作用(P=0.481),随着干预时间的增加ApoE-/-/LDLR-/-小鼠体重具有降低的趋势(P=0.0001)。
(2)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压明显升高(P均<0.001);与普通饮食组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的普通饮食组ApoE-/-/LDLR-/-小鼠,其收缩压、舒张压以及平均动脉压略有降低(P均<0.001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压明显降低(P均<0.001)；对于ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压而言,高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病能够增加收缩压、舒张压以及平均动脉压(P均<0.001),TRIB3的RNAi能够显著降低ApoE-/-/LDLR-/-小鼠的收缩压、舒张压以及平均动脉压(P均<0.05),高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病和TRIB3-shRNA之间没有交互作用(P均>0.05),ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压不随着干预时间的增加而发生变化(P均>0.05)。
3.血清学指标的改变：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血糖、血清胰岛素水平、胰岛素抵抗指数、胆固醇水平、LDL-C和HDL-C均明显升高(P均<0.05),甘油三酯水平略有降低,但是差异没有统计学意义(P=0.768)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血糖、胰岛素抵抗指数降低,而血清胰岛素、胆固醇、甘油三酯、LDL-C和HDL-C的差异没有统计学意义。
4.腹腔巨噬细胞功能的比较：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能、粘附功能、吞噬功能均明显增强；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能明显减低,吞噬功能明显增强,而粘附功能基本不变。
5.2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉的显微超声检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠IMT、动脉内径、血流速度的差异均无统计学意义(P均>0.05)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉IMT、动脉内径、血流速度的变化均无统计学意义(P均>0.05)。
6.病理学分析：
(1)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠主动脉动脉粥样硬化斑块负荷明显增加(17.00±3.12%vs 30.99±3.12%,P=0.000)：与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠主动脉动脉粥样硬化斑块负荷减轻(30.99±3.12%vs 22.54±2.13%,P=0.000)：
(2)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块面积、斑块内脂质含量、斑块内巨噬细胞含量明显升高(P均<0.05);与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块面积、斑块内脂质含量、斑块内巨噬细胞含量未见明显改变；
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块纤维帽厚度、帽核比值、斑块内胶原含量明显降低(P均<0.05)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块纤维帽厚度、帽／核比值、斑块内胶原含量明显增加(P均<0.05);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块内Ⅰ型和Ⅲ型胶原比值、斑块内平滑肌含量未见明显改变；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块内Ⅰ型和Ⅲ型胶原比值、斑块内平滑肌含量明显增加(P均<0.05);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块易损指数明显升高(1.75±0.45 vs 2.97±0.57,P=0.00001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块易损指数降低(2.97±0.57 vs 2.32±0.59,P=0.008)；
7.TUNEL检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的细胞凋亡明显升高(85.63±38.46 vs201.00±61.01,P=0.00003)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的细胞凋亡降低(201.00±61.01 vs 95.86±39.80,P=0.0001);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的巨噬细胞凋亡明显升高(48.31±28.39 vs 108.80±33.44,P=0.0001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的巨噬细胞凋亡降低(108.80±33.44 vs 46.20±15.21,P=0.00006);
8.实时定量RT-PCR检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3-mRNA表达明显增加(1.69±0.76 vs 4.97±1.30,P=1×10-7)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-mRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3-mRNA表达明显降低(4.97±1.30 vs 1.59±0.52,P=1×10-7)。
9.Western bot检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3蛋白质表达明显增加,Akt磷酸化程度降低,caspase-3表达增多;与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3蛋白质表达明显降低,Akt磷酸化程度增加,caspase-3表达降低。
结论
(1)2型糖尿病ApoE-/-/LDLR-/-小鼠主动脉的粥样硬化斑块负荷增加,TRIB3基因沉默能够减低主动脉的粥样硬化斑块负荷,TRIB3基因沉默能够减轻糖尿病所致的斑块负荷；
(2)2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块为易损斑块,主要表现为斑块的纤维帽厚度降低,纤维帽／脂质核心比例降低,斑块内脂质含量增加,胶原含量减少,巨噬细胞含量增加,斑块内巨噬细胞凋亡量增加,斑块的易损指数增加；
(3)TRIB3基因沉默主要通过增加斑块纤维帽的厚度,提升斑块纤维帽／脂质核心比例,增加斑块内胶原和平滑肌含量,减少凋亡细胞数量,稳定了巨噬细胞,降低了易损指数从而增加斑块的稳定性；
(4)TRIB3基因沉默能够有效的增加斑块内AKT的活性,减轻了胰岛素抵抗,减少了巨噬细胞的凋亡,避免脂质核心的进一步扩大,增强斑块的稳定性；
(5)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能增强;动脉粥样硬化晚期,TRIB3基因沉默能够降低腹腔巨噬细胞的迁移功能；TRIB3基因沉默能够部分阻断糖尿病所致的腹腔巨噬细胞迁移功能的增强；
(6)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞粘附功能增强,促进动脉粥样硬化的发生和发展；动脉粥样硬化晚期,TRIB3基因沉默能够增加腹腔巨噬细胞粘附功能,减少巨噬细胞的迁移,可能增加局部的吞噬清除能力；糖尿病和TRIB3基因沉默各自独立的增加腹腔巨噬细胞的粘附功能；
(7)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞吞噬脂质功能增强,造成斑块体积的迅速增加；动脉粥样硬化晚期,TRIB3基因沉默能够增加腹腔巨噬细胞吞噬功能,可以维持巨噬细胞的吞噬清除能力,避免巨噬细胞大量凋亡坏死造成脂质核心的扩大,引发斑块的不稳定；糖尿病和TRIB3基因沉默各自独立的增加腹腔巨噬细胞的吞噬功能；
(8)TRIB3基因沉默改善糖代谢和胰岛素抵抗,有效的减少了动脉粥样硬化发生的危险因素；
(9)高分辨率显微超声技术能够无创性监测2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉易损斑块的发生、发展以及干预后的转归。
背景
动脉粥样硬化的早期识别以及监测动脉粥样硬化斑块的进展对于2型糖尿病患者急性心血管事件的预防具有十分重要的意义。早期识别动脉粥样硬化,阻断并逆转动脉粥样硬化斑块的进展,是预防心脑血管事件的重要方法。对于易损斑块而言,早期识别和干预尤为重要。既往研究表明,ApoE-/-小鼠以头臂干动脉的斑块自发破裂率最高。这可能成为2型糖尿病动脉粥样硬化不稳定斑块的重要研究对象。然而,由于解剖位置的影响以及仪器功能的限制,以往对小动物模型动脉粥样硬化斑块的检测多集中于后期的组织学分析。尽管组织学分析能够准确的判断病变的程度,但是无法准确掌控活体病变的发展时相,也无法进行动态的观察研究,因而很难提供干预的最佳时机以及动态评价干预因素的有效性。此外,组织学分析相对较为局限,很难形成对动脉粥样硬化斑块的整体认识。因此,本研究采用显微超声仪UBM vevo770小鼠血管超声监测2型糖尿病ApoE-/-/LDLR-/-小鼠模型主动脉、头臂干和颈动脉动脉粥样硬化程度的变化,建立无创性评估糖尿病动脉粥样硬化斑块的方法。
目的
1.采用UBM vevo770小鼠血管超声监测糖尿病动物模型动脉粥样硬化程度的变化,建立无创性评估糖尿病动脉粥样硬化斑块的方法；
2.通过UBM vevo770小鼠血管超声比较2型糖尿病ApoE-/-/LDLR-/-小鼠全身动脉粥样硬化的发生发展的异同；
3.评价UBM vevo770小鼠血管超声在2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化研究中的应用价值；
方法
1.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉模型的建立：3周龄雄性ApoE-/-/LDLR-/-小鼠30只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=15)和糖尿病组(n=15),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg／kg,2周后再次测IPGTT,随机血糖≥11.1mmol／L时,可作为2型糖尿病成模的动物入组；
2.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化斑块的超声研究：应用UBMVevo770小鼠血管超声分别在14周龄、16周龄、18周龄、20周龄腺病毒转染之前、22周龄腺病毒追加转染之前以及实验结束时采用二维、脉冲多普勒技术,连续观察糖尿病组小鼠主动脉、头臂干和颈动脉血管动脉粥样硬化指标以及血流动力学指标的动态变化；
结果
1.动物的基本特征：高脂饮食喂养至20周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0015)；随后小鼠体重继续增加,至22周龄时达到最大,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0008)；至24周龄时,小鼠体重略有下降,但是糖尿病组ApoE-/-/LDLR-/-小鼠体重仍然明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0073)。
随着高脂饮食喂养时间的增加,小鼠的心率逐渐降低(P=0.014)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的收缩压、舒张压、平均动脉压显著升高(P均<0.001),小鼠周龄对血压的影响没有统计学意义(P=0.137),小鼠周龄与糖尿病在影响小鼠血压方面没有交互作用(P=0.652)。
2.ApoE-/-/LDLR-/-小鼠动脉IMT变化结果：在14周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,差异没有统计学意义；随后2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT迅速增厚,至20周龄时进展减缓,IMT保持基本稳定；而普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT始终保持较慢的增长,至24周龄时与2型糖尿病小鼠20周龄时斑块大小相当；16周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT明显增加(P=0.000)；18周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT仍然明显增加(P=0.0012)；20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT仅有明显增加的趋势(P=0.063)；24周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,差异没有统计学意义(P=0.656)。
在14周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,差异没有统计学意义(P=0.656)；随后两组小鼠的颈动脉IMT均缓慢的增厚；至20周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT增厚速度开始增加；而普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT始终保持较慢的增长；至22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT明显增加(P=0.022)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT进一步增加(P=0.001)。
3.ApoE-/-/LDLR-/-小鼠动脉血流动力学变化结果：20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度差异没有统计学意义(P=0.503)；头臂干动脉血流速度保持缓慢下降；至24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度明显降低(416.41±86.27 vs 310.05±175.99,P=0.045)。
20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度差异没有统计学意义(242.84±136.52vs 163.10±70.01,P=0.054);22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度明显降低(275.09±104.69 vs 188.45±80.92,P=0.017)；随后,颈动脉血流速度下降；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度差异没有统计学意义(P=0.968)。
4.ApoE-/-/LDLR-/-小鼠颈动脉弹性功能的改变：
(1)20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数差异均没有统计学意义(P均>0.05)；22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数明显升高(P均<0.05)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数差异没有统计学意义(P均>0.05)。
(2)20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)；22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)。
结论
(1)2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化负荷高于非糖尿病ApoE-/-/LDLR-/-小鼠；
(2)2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块进展迅速,至20周龄时进展减缓,而非糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块始终保持较慢的增长,至24周龄时与2型糖尿病小鼠20周龄时斑块大小相当；
(3)2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉粥样硬化斑块发生、发展迟于头臂干动脉；
(4)内膜中层厚度仍然是评价2型糖尿病ApoE-/-/LDLR-/-小鼠模型动脉粥样硬化发生和发展的有效指标；
(5)高分辨率显微超声能够无创性的动态监测2型糖尿病ApoE-/-/LDLR-/-小鼠模型动脉粥样硬化的发生和发展,有助于治疗研究中干预时机的选择。
Background
Diabetes mellitus has been recognized as an equivalence of coronary heart disease. However, the mechanism of diabetes-accelerated CHD remains to be clarified. Therefore, it is important to choose an appropriate diabetic animal model for each type of diabetes when doing diabetic atherosclerosis research. In the present, diabetic atherosclerosis animal models were established through gene knockout combined with high fat diet diabetes and long-term high fat diet. The limitations of these models include four major parts. The first one is that mouse models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to mice is not straightforward. The second one is that the effect of metabolism-associated gene on atherosclerosis cannot be excluded in knockout mice which could not be intervened. The third one is that long-term high fat diet induced diabetic models take a long time, which makes it difficult to distinguish the effect of aging from diabetes mellitus on the development of atherosclerosis. The last one is that long-term high fat diet induced diabetic models precede hyperglycemia with dyslipidemia. None of the currently used models is a perfect diabetic atherosclerosis mouse model. Thus, the present study focuses on the establishment of diabetic atherosclerosis ApoE-/-/LDLR-/-mouse models by high-fat and high-sugar diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose and glucose tolerance, detecting the initiation and development of atherosclerosis by ultrasound biomicroscopy(UBM) were performed to confirm the feasibility of establishing diabetic atherosclerosis model that closely resemble human diabetic atherosclerosis disease.
Objectives
1. To confirm the feasibility of establishing diabetic ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ;
2. To confirm the feasibility of establishing diabetic atherosclerosis ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ.
Methods
1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ (intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
2. IPGTT test:Mice were fasted for 10-16h before glucose tolerance tests. Intraperitoneal injection glucose load was administered at 1.5 g/kg of body weight. Glucose levels were measured from tail bleeds with a glucometer at specified time points after glucose administration.
3. Monitor body weight of ApoE-/-/LDLR-/- mice every week.
4. Assessment of hemodynamics of ApoE-/-/LDLR-/- mice:All ApoE-/-/LDLR-/-mice underwent hemodynamic examination at weeks 20,22 and 24, including blood pressure and heart rate.
5. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT.
6. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR.
Results
1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice show significant increased body weight (P=0.010). However, no significant difference was detected between the two groups at two weeks after administration of STZ (P=0.081). At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).
2. Hemodynamics alterations of ApoE-/-/LDLR-/- mice:Compared to the chow-diet mice, no significant difference was detected in the heart rate of diabetic mice (P=0.309). However, the heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased systolic blood pressure (P=0.00004), while the age of mice had no effect on systolic blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390). Compared to the chow-diet mice, diabetic mice show an increased diastolic blood pressure (P=0.000003), while the age of mice had no effect on diastolic blood pressure (P=0.137). No significant interactions were detected between the age and diabetes (P=0.652). Compared to the chow-diet mice, diabetic mice show an increased mean arterial blood pressure (P=0.0001), while the age of mice had no effect on mean arterial blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.263).
3. Results of IPGTT of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice showed significant increased blood glucose (P<0.05 for all). After administration of STZ, diabetic mice showed significant higher blood glucose than chow-diet mice (P<0.05 for all). No significant differences were detected among the chow-diet mice with different ages. Significant differences were detected among the diabetic mice with different ages.
4. Parameters of glucose metabolism of ApoE-/-/LDLR-/- mice:At the age of 24 weeks, compared to the chow-diet mice, diabetic mice showed significantly increased blood glucose, serum insulin, and HOMA-IR (P=0.028, P=0.025, P=0.015, respectively), indicating that diabetic mice have hyperglycemia, hyperinsulinemia and insulin resistance.
5. Monitoring IMT of ApoE-/-/LDLR-/- mice:Brachiocephalic IMT of ApoE-/-/LDLR-/- mice got thickened earlier than carotid IMT. At the age of 16 weeks, significant differences of brachiocephalic IMT were detected between the two groups (P=0.000), which will last till week 24. As for carotid IMT, it kept increased slowly. At week 22, significant differences of carotid IMT were detected between the two groups (P=0.022). At week 24, compared with the chow-diet mice, diabetic mice had significant thickened carotid IMT (P=0.001).
Conclusions
1. Male ApoE-/-/LDLR-/- mice induced by high fat and sugar diet combined with a small dose of STZ at the age of 3weeks would appear the stable state of diabetes mellitus.
2. Diabetes mellitus induced by high fat and sugar diet combined with a small dose of STZ in male ApoE-/-/LDLR-/- mice would resemble human diabetes mellitus.
3. Type 2 diabetic ApoE-/-/LDLR-/- mice would be attacked by atherosclerotic plaque. Furthermore, the atherosclerotic plaque formed ahead of time.
4. Type 2 diabetic ApoE-/-/LDLR-/- mouse atherosclerosis model was appropriate for intervention.
Background
Although diabetes mellitus is the most risk factors for atherosclerosis and CHD, the mechanism of more serious and vulnerable plaques in the diabetic patients remains to be understood. This has highlighted the importance and urgency of studying the mechanism of diabetic atherosclerosis and exploring therapeutic options.
It is critical to find the crossroad of diabetes mellitus and acute coronary diseases to effectively deal with acute coronary syndromes-the most important cardiovascular complication. Up to date, studies on the mechanism of vulnerable plaque have showed that innate characteristics and the stress that the plaque bears with determine the vulnerability of the atherosclerotic plaque. It has been demonstrated that inflammation, oxidative stress, impaired glucose metabolism, dyslipidemia and dyshemodynamics would make the atherosclerotic plaque vulnerable. All these risk factors would cause more macrophages apoptosis which would make plaque vulnerable. However, the signal transduction involved in the macrophages apoptosis is a network, among which Akt signal transduction might play an essential role. TRIB3 has been confirmed to inhibit PI3K/Akt stimulated by insulin, causing insulin resistance. Furthermore, TRIB3, as a scaffold protein, was involved in the lipid metabolism. Epidemiological studies showed that TRIB3 Q84R polymorphism was associated with insulin resistance and the increase of cardiovascular diseases, especially atherosclerosis. However, whether TRIB3/Akt signal transduction was involved in diabetes cardiovascular complications remained to be clarified. In the present study, TRIB3-shRNA was transfected into type 2 diabetic ApoE-/-/LDLR-/- mice, silencing TRIB3, to investigate whether TRIB3 silence could stabilize the plaque and the underlying mechanism.
Objectives
1. To design and synthesize siRNA against mouse TRIB3 according to RNAi principle, then to construct the pAdxsi-TRIB3-shRNA;
2. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice to investigate the vulnerability of atherosclerotic plaque;
3. To explore the mechanism by which silence of TRIB3 would stabilize atherosclerotic plaque.
Methods
1. To design and synthesize 4 pieces of siRNA against mouse TRIB3 according to RNAi principle, then construct pGenesil-1.2-TRIB3-shRNA plasmid using the most effective siRNA. Subsequently, pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.
2. Animal experiment:Sixty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow-diet (n=30) and diabetes (n=30) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
3. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice. At the age of 20 weeks, after echo examination, chow-diet with RNAi group (n=15) and diabetes with RNAi group (n=15) was injected by pAdxsi-TRIB3-shRNA through tail vein, while chow-diet group (n=15) and diabetes group (n=15) was injected by pAdxsi through tail vein. After 2 weeks, echo examination and transfection were performed again. At the age of 24 weeks, echo examination was performed before the animals were sacrificed.
4. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR. Furthermore, serum total cholesterol, triglycerides, HDL-C and LDL-C were also measured.
5. Assessment of peritoneal macrophages'function:Isolate peritoneal macrophage, identified by MOMA-2, then migration, adhesion and phagocytosis were assessed.
6. Pathology analyses:HE staining, Masson trichrome stain, oil red O, Sirius red, and Perl's staining were preformed on frozen sections from brachiocephalic plaque. Immunochemistry assay was performed to detect the macrophages, smooth muscle and TRIB3 in the brachiocephalic plaque. Plauqe area, fibrotic cap, collagen, lipid were measured to calculate cap/core ratio and vulnerability index.
7. Real-time RT-PCR:The mRNA of TRIB3 was quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green method.
8. Western bot:The proteins of TRIB3/Akt/Caspase-3 signal transduction were detected by western blotting.
9. TUNEL:Apoptotic cells and apoptotic macrophages were evaluated in frozen sections by TUNEL.
Results
1. Construction of pAdxsi-TRIB3-shRNA:After transfection of TRIB3 siRNA for 24h, the mRNA levels of GAPDH and TRIB3 were quantified by real-time RT-PCR using SYBR Green method. Based upon the inhibition rate of TRIB3, one piece was selected, which could silence 99.98% TRIB3. The chosen sequence was used to construct pGenesil-1.2-TRIB3-shRNA plasmid. Then pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. Subsequently, with the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.
2. Alterations of ApoE-/-/LDLR-/- mice after transfection of pAdxsi-TRIB3-shRNA
(1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased body weight(P=0.001). Compared to the chow-diet ApoE-/-/LDLR-/- mice, chow-diet with RNAi group showed a decreased body weight (P=0.034); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed a significantly decreased body weight (P=0.003). Therefore, diabetes could increase the body weight (P=0.001), while RNAi had no effect on body weight (P=0.112). No significant interactions were detected between the RNAi and diabetes (P=0.481).
(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased systolic blood pressure(SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MABP) (P<0.001 for all). Compared to the chow-diet ApoE-/-/LDLR-/-mice, chow-diet with RNAi group showed decreased SBP, DBP and MABP (P<0.001 for all); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed significantly decreased SBP, DBP and MABP (P<0.001 for all). Therefore, diabetes could increase SBP, DBP and MABP (P<0.001 for all), while RNAi could reduce SBP, DBP and MABP (P<0.05 for all). No significant interactions were detected between the RNAi and diabetes (P>0.05 for all).
3. Serum parameters of ApoE-/-/LDLR-/- mice:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased blood glucose, serum insulin, HOMA-IR, serum TC, serum LDL-C and serum HDL-C (P<0.05 for all), with serum TG unchanged (P=0.768). Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/-mice showed significantly decreased blood glucose and HOMA-IR (P<0.05 for both), but serum insulin, serum TC, serum TG, serum LDL-C and serum HDL-C remained unchanged.
4. Comparison of peritoneal macrophages' function:Compared to the chow-diet ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetic ApoE-/-/LDLR-/-mice showed significantly increased migration, adhesion and phagocytosis functions. Compared to diabetic ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased migration function, enchanced phagocytosis function but unaltered adhesion function.
5. UBM measurements of brachiocephalic artery:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic IMT, diameters and blood velocity. Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- showed no decrease in brachiocephalic IMT, diameters and blood velocity (P>0.05 for all).
6. Pathological analyses
(1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased aortic atherosclerotic burden (17.00±3.12% vs 30.99±3.12%, P=0.000); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased aortic atherosclerotic burden (30.99±3.12% vs 22.54±2.13%, P=0.000).
(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P>0.05 for all).
When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly decreased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all).
Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in I/III collagen ratio and SMC content of brachiocephalic plaque (P>0.05 for both); Compared to diabetic ApoE-/-/LDLR-/-mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increasedⅠ/Ⅲcollagen ratio and SMC content of brachiocephalic plaque (P<0.05 for both).
When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased vulnerability index of brachiocephalic plaque (1.75±0.45 vs 2.97±0.57, P=0.00001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased vulnerability index of brachiocephalic plaque (2.97±0.57 vs 2.32±0.59,P=0.008).
7. TUNEL analysis:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased apoptosis in brachiocephalic plaque (85.63±38.46 vs 201.00±61.01, P=0.00003); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased apoptosis in brachiocephalic plaque (201.00±61.01 vs 95.86±39.80, P=0.0001).
Furthermore, compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased macrophages'apoptosis in brachiocephalic plaque (48.31±28.39 vs 108.80±33.44, P=0.0001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased macrophages'apoptosis in brachiocephalic plaque (108.80±33.44 vs 46.20±15.21, P=0.00006).
8. Real-time RT-PCR:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 mRNA in atherosclerotic plaque (1.69±0.76 vs 4.97±1.30, P=1×10-7); Compared to diabetic ApoE-/-/LDLR-/ mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 mRNA in atherosclerotic plaque (4.97±1.30 vs 1.59±0.52, P=1×10-7).
9. Western bot:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 and caspase-3, but decreased phosphorylated Akt in atherosclerotic plaque; Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 and caspase-3, but increased phosphorylated Akt in atherosclerotic plaque.
Conclusions
1. Diabetic ApoE-/-/LDLR-/- mice showed significantly increased aortic atherosclerotic burden, which would be reversed by silencing TRIB3.
2. Brachiocephalic plaques of diabetic ApoE-/-/LDLR-/- mice were confirmed to be vulnerable, manifesting decreased fibrotic cap, reduced cap/core ratio, increased lipid in the plaque, decreased collagen content, incremental macrophages, increased apoptosis of macrophages, augmented vulnerability index and enhanced hemorrhage in the plaque.
3. Silencing of TRIB3 would stabilize plaque by augmenting the thickness of fibrotic cap, increasing cap/core ratio, increasing collagen and smooth muscle contents, reducing apoptotic cells and decreasing vulnerability index.
4. Silencing of TRIB3 would effectively increase the activity of Akt, improve insulin resistance, reduce apoptosis of macrophages, thereby protecting against the enchancement of lipid core, to stabilize plaques.
5. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased migration. In advanced atherosclerosis, silencing of TRIB3 would in part decrease migration of peritoneal macrophages.
6. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased adhesion. In advanced atherosclerosis, silencing of TRIB3 would enhance adhesion, reduce migration of peritoneal macrophages, thereby increasing local clearance. Diabetes mellitus and silencing of TRIB3 would enhance adhesion respectively.
7. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased phagocytosis of lipid, causing plaque size increased rapidly. In advanced atherosclerosis, silencing of TRIB3 would enhance phagocytosis of lipid, maintaining the ability of clearance, to avoid augmented lipid core due to apoptosis and necrosis. Diabetes mellitus and silencing of TRIB3 would enhance phagocytosis respectively.
8. Silencing of TRIB3 would effectively improve insulin resistance and glucose metabolism, elevate HDL and lower blood pressure, therefore reducing risk factors for atherosclerosis.
9. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of brachiocephalic plaque, highly identical with pathological analyses.
Background
Early identification of atherosclerosis and monitoring its development were essential for prevention of acute cardiovascular events in diabetic patients. Early identification to protect against atherosclerosis is an important method to prevent acute cardiovascular events, especially for vulnerable plaques. It has been demonstrated that spontaneous ruptures of atherosclerotic plaques mostly take place in brachiocephalic artery, which would be perfect for vulnerability of atherosclerotic plaques in type 2 diabetes mellitus. However, brachiocephalic artery was too deep to be detected. Therefore, the analyses nearly depend on the pathological analyses. Although pathological analyses could judge the development of atherosclerosis, it could not reflect the development of atherosclerosis in vivo. Furthermore, dynamic observations could not be realized, which make it difficult to choose the optimal opportunity and evaluate the effectiveness of interventions. Moreover, pathological analyses were restricted to the single sections without the integrity of atherosclerosis. Here, high resolution ultrasonic biomicroscopy UBM vevo770 was used to monitor the aortic, brachiocephalic and carotid atherosclerosis, to noninvasively evaluate diabetic atherosclerosis.
Objectives
1. To noninvasively evaluate diabetic atherosclerosis by monitoring atherosclerosis in diabetic animal model using UBM vevo770;
2. To compare the atherosclerosis in different arteries of diabetic ApoE-/-/LDLR-/- mice by UBM vevo770;
3. To evaluate the application of UBM vevo770 on studying atherosclerosis in diabetic ApoE-/-/LDLR-/- mice
Methods
1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
2. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT, diameters and blood velocity.
Results
1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).
The heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased blood pressure (P=0.00004), while the age of mice had no effect on blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390).
2. At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups. The brachiocephalic IMT rose rapidly in diabetic mice, but slowed down at week 20. The brachiocephalic IMT rose slowly in chow-diet mice. The brachiocephalic IMT at week 24 in chow-diet mice was similar to that at week 20 in diabetic mice. At weeks 16,18 and 20, diabetic mice show significantly increased brachiocephalic IMT when compared with chow-diet mice (P<0.005 for all). At the age of 24 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656).
At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656). At weeks 22 and 24, diabetic ApoE-/-/LDLR-/- mice show significantly increased brachiocephalic IMT when compared with chow-diet ApoE-/-/LDLR-/- mice (P<0.05 for both).
3. At the age of 20 weeks, no significant differences of brachiocephalic blood velocity were detected between the two groups (P=0.503). Then brachiocephalic blood velocity decreased slowly. At week 24, diabetic ApoE-/-/LDLR-/- mice show significantly decreased brachiocephalic blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (416.41±86.27 vs 310.05±175.99, P=0.045).
At the age of 20 weeks, no significant differences of carotid blood velocity were detected between the two groups(42.84±136.52 vs 163.10±70.01, P=0.054). At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly decreased carotid blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (275.09±104.69 vs 188.45±80.92, P=0.017). Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid blood velocity were detected between the two groups (P=0.968).
4. Alterations of carotid functions of diabetic ApoE-/-/LDLR-/- mice
(1) At the age of 20 weeks, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups. At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly increased carotid stiffness, elasticity, and standardized elasticity when compared with chow-diet ApoE-/-/LDLR-/- mice. Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups.
(2) At weeks 20,22 and 24, no significant differences of carotid expand coefficient and compliance were detected between the two groups.
Conclusions
1. Diabetic ApoE-/-/LDLR-/- mice had more atherosclerotic burden than chow-diet ApoE-/-/LDLR-/- mice.
2. Diabetic ApoE-/-/LDLR-/- mice experience a rapid development of brachiocephalic atherosclerosis, which would slow down at the age of 20 weeks. Chow-diet ApoE-/-/LDLR-/- mice keep a slow development of brachiocephalic atherosclerosis. The brachiocephalic IMT at week 24 in chow-diet mice was comparable to that at week 20 in diabetic mice.
3. Diabetic ApoE-/-/LDLR-/- mice's carotid atherosclerosis was preceded with brachiocephalic atherosclerosis.
4. IMT is still an effective index to evaluate the initiation and development of atherosclerosis in diabetic ApoE-/-/LDLR-/- mice.
5. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of atherosclerotic plaque, helping to select the optimal intervention opportunity.
糖尿病作为冠心病的等危症已经得到广泛共识,然而糖尿病加速动脉粥样硬化发生、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。寻找糖尿病加速动脉粥样硬化发生、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动脉粥样硬化研究的动物模型。目前,建立2型糖尿病动脉粥样硬化动物模型的方法主要有两种：一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；二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)由于人类的2型糖尿病是多基因遗传病,现有的基因敲除小鼠模型不能完全模拟类的2型糖尿病；(2)代谢相关基因敲除小鼠模型不能排除代谢相关的基因敲除之后对动脉粥样硬化的影响,且基因敲除为不可干预因素；(3)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,不能有效区分年龄和糖尿病相关的动脉粥样硬化；(4)单纯高脂饮食诱发的2型糖尿病动物模型,先有血脂代谢紊乱,而后逐渐出现血糖升高,这与人类糖尿病常有的糖脂代谢紊乱发病顺序不完全一致。因此,本研究针对2型糖尿病动脉粥样硬化动物模型的上述不足,采用ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素诱发糖尿病,通过监测体重、血压、血糖、糖耐量试验的动态变化,同时采用高分辨率显微超声技术监测动脉粥样硬化发生和发展过程,探讨建立2型糖尿病动脉粥样硬化动物模型的可行性。
目的
1.探讨高脂高糖饮食联合小剂量链脲佐菌素建立ApoE-/-/LDLR-/-小鼠2型糖尿病动物模型可行性；
2.探讨高脂高糖饮食联合小剂量链脲佐菌素建立2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化模型可行性；
方法
1.动物实验：3周龄雄性ApoE-/-/LDLR-/-小鼠30只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=15)和糖尿病组(n=15),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg/kg,2周后再次测IPGTT,随机血糖≥11.1mmol／L时,可作为2型糖尿病成模的动物入组；
2.IPGTT试验：小鼠空腹10-16h,按照1.5g/kg体重腹腔注射葡萄糖,然后于0min、15 min、30 min、60 min和120 min分别断尾取血检测血糖；
3.ApoE-/-/LDLR-/-小鼠体重的监测：定期监测小鼠体重的变化；
4.ApoE-/-/LDLR-/-小鼠血流动力学的评价：所有实验动物在20周龄、22周龄和24周龄时通过小鼠尾动脉血压测量仪监测血压和心率,每只动物测量3次,取其平均值；
5.ApoE-/-/LDLR-/-小鼠动脉粥样硬化斑块的显微超声监测：分别于相应观察时间点(14周龄、16周龄、18周龄、20周龄、22周龄和24周龄)进行动脉超声检查通过高分辨率显微超声技术监测主动脉、头臂干动脉和颈总动脉的动脉粥样硬化斑块的发生和发展,测量内膜中层厚度(IMT)；
6.ApoE-/-/LDLR-/-小鼠血清糖代谢指标的测定：实验末,处死动物,分离血清,测定血糖、血清胰岛素,计算胰岛素抵抗指数。
结果
1.ApoE-/-/LDLR-/-小鼠体重变化情况：3周龄时,两组雄性ApoE-/-/LDLR-/-小鼠体重差异没有统计学意义(P=0.213)；高脂饮食喂养6周后,糖尿病组ApoE-/-/LDLR-/-小鼠体重与普通饮食组ApoE-/-/LDLR-/-小鼠差异具有统计学意义(P=0.010)；注射链脲佐菌素后2周,糖尿病组ApoE-/-/LDLR-/-小鼠与普通饮食组ApoE-/-/LDLR-/-小鼠体重差异无统计学意义(P=0.081)；继续高脂饮食喂养至20周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重,差异具有统计学意义(P=0.0015)；随后小鼠体重基本稳定,至24周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重仍然明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重,差异具有统计学意义(P=0.0073)。
2.ApoE-/-/LDLR-/-小鼠血流动力学变化：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的心率未见明显改变(P=0.309),然而随着小鼠周龄的增加,小鼠的心率逐渐降低(P=0.014)。与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的收缩压显著升高(P=0.00004),小鼠周龄对收缩压的影响没有统计学意义(P=0.212),小鼠周龄与糖尿病在影响小鼠收缩压方面没有交互作用(P=0.390)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的舒张压显著升高(P=0.000003),小鼠周龄对舒张压的影响没有统计学意义(P=0.137),小鼠周龄与糖尿病在影响小鼠舒张压方面没有交互作用(P=0.652)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的平均动脉压显著升高(P=0.0001),小鼠周龄对平均动脉压的影响没有统计学意义(P=0.212),小鼠周龄与糖尿病在影响小鼠平均动脉压方面没有交互作用(P=0.263)。
3.ApoE-/-/LDLR-/-小鼠糖耐量试验结果：IPGTT的结果表明,3周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异没有统计学意义；9周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异具有统计学意义(P均<0.05)；随后注射小剂量链脲佐菌素,11周时普通饮食组ApoE-/-/LDLR-/-小鼠与糖尿病组ApoE-/-/LDLR-/-小鼠的糖耐量试验结果差异具有统计学意义(P均<0.05)。普通饮食组不同周龄的糖耐量试验结果差异没有统计学意义；糖尿病组不同周龄的糖耐量试验结果差异具有统计学意义。
4.ApoE-/-/LDLR-/-小鼠血糖代谢指标比较结果：在24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠相比,2型糖尿病ApoE-/-/LDLR-/-小鼠血糖、血清胰岛素以及胰岛素抵抗指数均明显升高(P=0.028,P=0.025,P=0.015),表明2型糖尿病ApoE-/-/LDLR-/-小鼠存在高血糖、高胰岛素血症和胰岛素抵抗。
5.ApoE-/-/LDLR-/-小鼠动脉IMT变化结果：ApoE-/-/LDLR-/-小鼠头臂干动脉IMT变化早于颈动脉。ApoE-/-/LDLR-/-小鼠头臂干动脉IMT在16周龄时,两组之间差异具有统计学意义(P=0.000)；差异维持至24周龄。ApoE-/-/LDLR-/-小鼠颈动脉IMT始终保持较慢的增长;至22周龄时,两组小鼠颈动脉IMT差异具有统计学意义(P=0.022)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT进一步增加(P=0.001)。
结论
(1)3周龄雄性ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素(75mg／kg)诱发糖尿病状态稳定；
(2)ApoE-/-/LDLR-/-小鼠经高脂高糖饮食联合小剂量链脲佐菌素诱发糖尿病后,体重和血压均增加,与人类的糖尿病状态基本类似；
(3)2型糖尿病ApoE-/-/LDLR-/-小鼠模型能够发生动脉粥样硬化斑块,并且发生时间提前；
(4)2型糖尿病ApoE-/-/LDLR-/-小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干预研究。
背景
糖尿病已经成为动脉粥样硬化和冠心病最重要的危险因素。但是糖尿病患者冠状动脉粥样硬化斑块病变严重、易损斑块发生率高的机制尚未完全阐明,因此,也缺乏以机制为基础的治疗措施。故而,探索糖尿病患者冠状动脉粥样硬化斑块病变严重、易损斑块发生率高的机制,建立以机制为导向的治疗措施成为近年来防治糖尿病心血管并发症的研究热点。
实现对糖尿病最重要的心血管并发症——急性冠脉综合征的有效遏制,关键在于寻找糖尿病和急性冠脉综合征发病的共同中间环节。目前对易损斑块的形成机制进行的基础和临床研究认为斑块是否破裂取决于斑块内在的组织特征和斑块所受的应力一应变关系的相互作用。而炎症机制、氧化应激、糖代谢异常、脂质代谢异常和血流动力学改变等均与易损斑块的形成有关。这些因素导致的动脉粥样斑块内细胞凋亡尤其巨噬细胞凋亡是导致易损斑块形成的重要机制。巨噬细胞凋亡的信号调控是一个复杂的网络体系。在巨噬细胞凋亡的调控机制中,Akt信号转导途径起关键作用。TRIB3通过抑制胰岛素激活的PI3K/Akt信号转导通路导致胰岛素抵抗,参与糖脂代谢,是2型糖尿发病的机制之一。流行病学研究发现,TRIB3基因Q84R错义突变患者发生严重胰岛素抵抗引起的相关病理改变如高胰岛素血症、高脂血症、代谢综合征及心血管疾病危险增加。TRIB3/Akt信号途径通过对巨噬细胞凋亡和糖代谢的影响,在胰岛素抵抗、糖尿病和易损斑块的形成过程中起重要作用。因此本课题拟通过体内转染腺病毒载体介导的TRIB3-shRNA特异性抑制2型糖尿病ApoE-/-/LDLR-/-小鼠体内TRIB3的表达,进而观察TRIB3基因沉默对2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样斑块稳定性影响,并阐述其作用机制。
目的
1.通过RNAi干扰技术设计合成针对小鼠TRIB3基因的shRNA,构建重组腺病毒载体pAdxsi-TRIB3-shRNA;
2.2型糖尿病ApoE-/-/LDLR-/-小鼠体内转染pAdxsi-TRIB3-shRNA,观察动脉粥样硬化斑块稳定性的变化;
3.在细胞分子水平、组织学水平和整体功能水平研究TRIB3基因沉默稳定2型糖尿病易损斑块的机制。
方法
1. pAdxsi-TRIB3-shRNA病毒载体的构建：根据RNAi干扰技术原则,设计合成4对针对小鼠TRIB3基因的siRNA,经过筛选之后,选择沉默效率最高的序列构建pGenesil-1.2-TRIB3-shRNA质粒,随后构建pShuttle-Basic-EGFP-TRIB3-shRNA重组穿梭载体质粒,最后构建pAdxsi-GFP-TRIB3-shRNA腺病毒载体。
2.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化动物模型：3周龄雄性ApoE-/-/LDLR-/-小鼠60只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=30)和糖尿病组(n=30),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg/kg,2周后再次测IPGTT,随机血糖≥11.1mmol/L时,可作为2型糖尿病成模的动物入组,超声监测至20周龄时开始体内转染腺病毒。
3. pAdxsi-TRIB3-shRNA病毒体内转染实验：在小鼠20周龄时,完成超声检查后,糖尿病+RNAi组(n=15)和普通饮食+RNAi组(n=15)经尾静脉注射pAdxsi-TRIB3-shRNA腺病毒,糖尿病组(n=15)和普通饮食组(n=15)经尾静脉注射pAdxsi空病毒载体。两周后,复查超声后再次追加腺病毒。24周龄时,完成超声检查,处死动物。
4.血液生化指标检测：检测血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的水平,测定血糖和血清胰岛素,计算胰岛素抵抗指数。
5.腹腔巨噬细胞功能的检测：分离小鼠腹腔巨噬细胞,经MOMA-2鉴定后,分别测定腹腔巨噬细胞的迁移功能、粘附功能和吞噬功能。
6.病理学检测：对头臂干动脉斑块分别进行HE染色、Masson染色、油红O染色、天狼猩红染色,并通过免疫组化方法检测斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-SM-actin)以及TRIB3的表达。测量斑块面积和纤维帽厚度,计算帽／核比值,观察斑块的形态结构,计算斑块内胶原、脂质、平滑肌、巨噬细胞的含量,并计算易损指数;
7.实时定量RT-PCR检测：取新鲜动脉斑块组织,提取RNA,实时定量荧光RT-PCR检测TRIB3的mRNA表达量;
8. Western bot检测：取新鲜动脉斑块组织,提取蛋白质,Western bot检测斑块内TRIB3/Akt/Caspase-3信号通路的蛋白表达量；
9.TUNEL检测：取冰冻切片,TUNEL法检测斑块内的细胞凋亡量以及巨噬细胞的凋亡量。
结果
1. pAdxsi-TRIB3-shRNA病毒载体的构建：将4对TRIB3 siRNA,转染巨噬细胞24h,采用实时定量RT-PCR检测GAPDH mRNA和TRIB3 mRNA的表达水平,以判断各对siRNA的抑制效率,筛选出抑制效率最佳的siRNA,其对TRIB3的最佳抑制效率为99.98%。选此序列构建pGenesil-1.2-TRIB3-shRNA质粒,经鉴定测序正确后,用于构建pShuttle-Basic-EGFP-TRIB3-shRNA重组穿梭载体；鉴定正确后,构建pAdxsi-GFP-TRIB3shRNA重组腺病毒质粒,再次鉴定正确后经Pad限制性内切酶线性化后,应用Lipofectamine2000脂质体转染HEK293细胞,行腺病毒包装,扩增,纯化得到滴度为2×1011 PFU/mL的腺病毒。
2. pAdxsi-TRIB3-shRNA病毒体内转染后2型糖尿病ApoE-/-/LDLR-/-小鼠—般状况的改变：
(1)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显升高(P=0.001)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的普通饮食组ApoE-/-/LDLR-/-小鼠,其体重略有降低(P=0.034)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠体重明显降低(P=0.003)；对于ApoE-/-/LDLR-/-小鼠体重而言,高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病能够增加体重(P=0.001),TRIB3的RNAi对降低ApoE-/-/LDLR-/-小鼠的体重无影响(P=0.112),高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病和TRIB3-shRNA之间没有交互作用(P=0.481),随着干预时间的增加ApoE-/-/LDLR-/-小鼠体重具有降低的趋势(P=0.0001)。
(2)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压明显升高(P均<0.001);与普通饮食组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的普通饮食组ApoE-/-/LDLR-/-小鼠,其收缩压、舒张压以及平均动脉压略有降低(P均<0.001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压明显降低(P均<0.001)；对于ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压而言,高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病能够增加收缩压、舒张压以及平均动脉压(P均<0.001),TRIB3的RNAi能够显著降低ApoE-/-/LDLR-/-小鼠的收缩压、舒张压以及平均动脉压(P均<0.05),高脂高糖饮食联合小剂量链脲佐菌素导致的糖尿病和TRIB3-shRNA之间没有交互作用(P均>0.05),ApoE-/-/LDLR-/-小鼠收缩压、舒张压以及平均动脉压不随着干预时间的增加而发生变化(P均>0.05)。
3.血清学指标的改变：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血糖、血清胰岛素水平、胰岛素抵抗指数、胆固醇水平、LDL-C和HDL-C均明显升高(P均<0.05),甘油三酯水平略有降低,但是差异没有统计学意义(P=0.768)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血糖、胰岛素抵抗指数降低,而血清胰岛素、胆固醇、甘油三酯、LDL-C和HDL-C的差异没有统计学意义。
4.腹腔巨噬细胞功能的比较：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能、粘附功能、吞噬功能均明显增强；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能明显减低,吞噬功能明显增强,而粘附功能基本不变。
5.2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉的显微超声检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠IMT、动脉内径、血流速度的差异均无统计学意义(P均>0.05)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉IMT、动脉内径、血流速度的变化均无统计学意义(P均>0.05)。
6.病理学分析：
(1)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠主动脉动脉粥样硬化斑块负荷明显增加(17.00±3.12%vs 30.99±3.12%,P=0.000)：与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠主动脉动脉粥样硬化斑块负荷减轻(30.99±3.12%vs 22.54±2.13%,P=0.000)：
(2)与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块面积、斑块内脂质含量、斑块内巨噬细胞含量明显升高(P均<0.05);与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块面积、斑块内脂质含量、斑块内巨噬细胞含量未见明显改变；
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块纤维帽厚度、帽核比值、斑块内胶原含量明显降低(P均<0.05)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块纤维帽厚度、帽／核比值、斑块内胶原含量明显增加(P均<0.05);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块内Ⅰ型和Ⅲ型胶原比值、斑块内平滑肌含量未见明显改变；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块内Ⅰ型和Ⅲ型胶原比值、斑块内平滑肌含量明显增加(P均<0.05);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块易损指数明显升高(1.75±0.45 vs 2.97±0.57,P=0.00001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块易损指数降低(2.97±0.57 vs 2.32±0.59,P=0.008)；
7.TUNEL检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的细胞凋亡明显升高(85.63±38.46 vs201.00±61.01,P=0.00003)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的细胞凋亡降低(201.00±61.01 vs 95.86±39.80,P=0.0001);
与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的巨噬细胞凋亡明显升高(48.31±28.39 vs 108.80±33.44,P=0.0001)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠头臂干动脉斑块的巨噬细胞凋亡降低(108.80±33.44 vs 46.20±15.21,P=0.00006);
8.实时定量RT-PCR检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3-mRNA表达明显增加(1.69±0.76 vs 4.97±1.30,P=1×10-7)；与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-mRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3-mRNA表达明显降低(4.97±1.30 vs 1.59±0.52,P=1×10-7)。
9.Western bot检测：与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3蛋白质表达明显增加,Akt磷酸化程度降低,caspase-3表达增多;与糖尿病组ApoE-/-/LDLR-/-小鼠相比,经过TRIB3-shRNA转染之后的糖尿病组ApoE-/-/LDLR-/-小鼠血管内TRIB3蛋白质表达明显降低,Akt磷酸化程度增加,caspase-3表达降低。
结论
(1)2型糖尿病ApoE-/-/LDLR-/-小鼠主动脉的粥样硬化斑块负荷增加,TRIB3基因沉默能够减低主动脉的粥样硬化斑块负荷,TRIB3基因沉默能够减轻糖尿病所致的斑块负荷；
(2)2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块为易损斑块,主要表现为斑块的纤维帽厚度降低,纤维帽／脂质核心比例降低,斑块内脂质含量增加,胶原含量减少,巨噬细胞含量增加,斑块内巨噬细胞凋亡量增加,斑块的易损指数增加；
(3)TRIB3基因沉默主要通过增加斑块纤维帽的厚度,提升斑块纤维帽／脂质核心比例,增加斑块内胶原和平滑肌含量,减少凋亡细胞数量,稳定了巨噬细胞,降低了易损指数从而增加斑块的稳定性；
(4)TRIB3基因沉默能够有效的增加斑块内AKT的活性,减轻了胰岛素抵抗,减少了巨噬细胞的凋亡,避免脂质核心的进一步扩大,增强斑块的稳定性；
(5)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞迁移功能增强;动脉粥样硬化晚期,TRIB3基因沉默能够降低腹腔巨噬细胞的迁移功能；TRIB3基因沉默能够部分阻断糖尿病所致的腹腔巨噬细胞迁移功能的增强；
(6)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞粘附功能增强,促进动脉粥样硬化的发生和发展；动脉粥样硬化晚期,TRIB3基因沉默能够增加腹腔巨噬细胞粘附功能,减少巨噬细胞的迁移,可能增加局部的吞噬清除能力；糖尿病和TRIB3基因沉默各自独立的增加腹腔巨噬细胞的粘附功能；
(7)2型糖尿病ApoE-/-/LDLR-/-小鼠腹腔巨噬细胞吞噬脂质功能增强,造成斑块体积的迅速增加；动脉粥样硬化晚期,TRIB3基因沉默能够增加腹腔巨噬细胞吞噬功能,可以维持巨噬细胞的吞噬清除能力,避免巨噬细胞大量凋亡坏死造成脂质核心的扩大,引发斑块的不稳定；糖尿病和TRIB3基因沉默各自独立的增加腹腔巨噬细胞的吞噬功能；
(8)TRIB3基因沉默改善糖代谢和胰岛素抵抗,有效的减少了动脉粥样硬化发生的危险因素；
(9)高分辨率显微超声技术能够无创性监测2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉易损斑块的发生、发展以及干预后的转归。
背景
动脉粥样硬化的早期识别以及监测动脉粥样硬化斑块的进展对于2型糖尿病患者急性心血管事件的预防具有十分重要的意义。早期识别动脉粥样硬化,阻断并逆转动脉粥样硬化斑块的进展,是预防心脑血管事件的重要方法。对于易损斑块而言,早期识别和干预尤为重要。既往研究表明,ApoE-/-小鼠以头臂干动脉的斑块自发破裂率最高。这可能成为2型糖尿病动脉粥样硬化不稳定斑块的重要研究对象。然而,由于解剖位置的影响以及仪器功能的限制,以往对小动物模型动脉粥样硬化斑块的检测多集中于后期的组织学分析。尽管组织学分析能够准确的判断病变的程度,但是无法准确掌控活体病变的发展时相,也无法进行动态的观察研究,因而很难提供干预的最佳时机以及动态评价干预因素的有效性。此外,组织学分析相对较为局限,很难形成对动脉粥样硬化斑块的整体认识。因此,本研究采用显微超声仪UBM vevo770小鼠血管超声监测2型糖尿病ApoE-/-/LDLR-/-小鼠模型主动脉、头臂干和颈动脉动脉粥样硬化程度的变化,建立无创性评估糖尿病动脉粥样硬化斑块的方法。
目的
1.采用UBM vevo770小鼠血管超声监测糖尿病动物模型动脉粥样硬化程度的变化,建立无创性评估糖尿病动脉粥样硬化斑块的方法；
2.通过UBM vevo770小鼠血管超声比较2型糖尿病ApoE-/-/LDLR-/-小鼠全身动脉粥样硬化的发生发展的异同；
3.评价UBM vevo770小鼠血管超声在2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化研究中的应用价值；
方法
1.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉模型的建立：3周龄雄性ApoE-/-/LDLR-/-小鼠30只,行糖耐量试验(IPGTT)后随机分为普通饮食组(n=15)和糖尿病组(n=15),糖尿病组饲以高脂饮食(20%脂肪,20%蔗糖,1.25%胆固醇)6周后,再次测IPGTT;出现胰岛素抵抗的小鼠腹腔注射链脲佐菌素(STZ)75mg／kg,2周后再次测IPGTT,随机血糖≥11.1mmol／L时,可作为2型糖尿病成模的动物入组；
2.2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化斑块的超声研究：应用UBMVevo770小鼠血管超声分别在14周龄、16周龄、18周龄、20周龄腺病毒转染之前、22周龄腺病毒追加转染之前以及实验结束时采用二维、脉冲多普勒技术,连续观察糖尿病组小鼠主动脉、头臂干和颈动脉血管动脉粥样硬化指标以及血流动力学指标的动态变化；
结果
1.动物的基本特征：高脂饮食喂养至20周龄时,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0015)；随后小鼠体重继续增加,至22周龄时达到最大,糖尿病组ApoE-/-/LDLR-/-小鼠体重明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0008)；至24周龄时,小鼠体重略有下降,但是糖尿病组ApoE-/-/LDLR-/-小鼠体重仍然明显大于普通饮食组ApoE-/-/LDLR-/-小鼠体重(P=0.0073)。
随着高脂饮食喂养时间的增加,小鼠的心率逐渐降低(P=0.014)；与普通饮食组ApoE-/-/LDLR-/-小鼠相比,糖尿病组ApoE-/-/LDLR-/-小鼠的收缩压、舒张压、平均动脉压显著升高(P均<0.001),小鼠周龄对血压的影响没有统计学意义(P=0.137),小鼠周龄与糖尿病在影响小鼠血压方面没有交互作用(P=0.652)。
2.ApoE-/-/LDLR-/-小鼠动脉IMT变化结果：在14周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,差异没有统计学意义；随后2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT迅速增厚,至20周龄时进展减缓,IMT保持基本稳定；而普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT始终保持较慢的增长,至24周龄时与2型糖尿病小鼠20周龄时斑块大小相当；16周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT明显增加(P=0.000)；18周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT仍然明显增加(P=0.0012)；20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT仅有明显增加的趋势(P=0.063)；24周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干IMT与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干IMT相比,差异没有统计学意义(P=0.656)。
在14周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,差异没有统计学意义(P=0.656)；随后两组小鼠的颈动脉IMT均缓慢的增厚；至20周龄时,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT增厚速度开始增加；而普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT始终保持较慢的增长；至22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT明显增加(P=0.022)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉IMT相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉IMT进一步增加(P=0.001)。
3.ApoE-/-/LDLR-/-小鼠动脉血流动力学变化结果：20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度差异没有统计学意义(P=0.503)；头臂干动脉血流速度保持缓慢下降；至24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉血流速度明显降低(416.41±86.27 vs 310.05±175.99,P=0.045)。
20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度差异没有统计学意义(242.84±136.52vs 163.10±70.01,P=0.054);22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度明显降低(275.09±104.69 vs 188.45±80.92,P=0.017)；随后,颈动脉血流速度下降；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉血流速度相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉血流速度差异没有统计学意义(P=0.968)。
4.ApoE-/-/LDLR-/-小鼠颈动脉弹性功能的改变：
(1)20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数差异均没有统计学意义(P均>0.05)；22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数明显升高(P均<0.05)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉僵硬度、弹性系数、标准化的弹性系数差异没有统计学意义(P均>0.05)。
(2)20周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)；22周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)；24周龄时,与普通饮食组ApoE-/-/LDLR-/-小鼠颈动脉相比,2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉扩张系数、顺应性差异没有统计学意义(P均>0.05)。
结论
(1)2型糖尿病ApoE-/-/LDLR-/-小鼠动脉粥样硬化负荷高于非糖尿病ApoE-/-/LDLR-/-小鼠；
(2)2型糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块进展迅速,至20周龄时进展减缓,而非糖尿病ApoE-/-/LDLR-/-小鼠头臂干动脉粥样硬化斑块始终保持较慢的增长,至24周龄时与2型糖尿病小鼠20周龄时斑块大小相当；
(3)2型糖尿病ApoE-/-/LDLR-/-小鼠颈动脉粥样硬化斑块发生、发展迟于头臂干动脉；
(4)内膜中层厚度仍然是评价2型糖尿病ApoE-/-/LDLR-/-小鼠模型动脉粥样硬化发生和发展的有效指标；
(5)高分辨率显微超声能够无创性的动态监测2型糖尿病ApoE-/-/LDLR-/-小鼠模型动脉粥样硬化的发生和发展,有助于治疗研究中干预时机的选择。
Background
Diabetes mellitus has been recognized as an equivalence of coronary heart disease. However, the mechanism of diabetes-accelerated CHD remains to be clarified. Therefore, it is important to choose an appropriate diabetic animal model for each type of diabetes when doing diabetic atherosclerosis research. In the present, diabetic atherosclerosis animal models were established through gene knockout combined with high fat diet diabetes and long-term high fat diet. The limitations of these models include four major parts. The first one is that mouse models cannot completely mimic human disease, so the extrapolation of relationships seen with human disease to mice is not straightforward. The second one is that the effect of metabolism-associated gene on atherosclerosis cannot be excluded in knockout mice which could not be intervened. The third one is that long-term high fat diet induced diabetic models take a long time, which makes it difficult to distinguish the effect of aging from diabetes mellitus on the development of atherosclerosis. The last one is that long-term high fat diet induced diabetic models precede hyperglycemia with dyslipidemia. None of the currently used models is a perfect diabetic atherosclerosis mouse model. Thus, the present study focuses on the establishment of diabetic atherosclerosis ApoE-/-/LDLR-/-mouse models by high-fat and high-sugar diet combined with a small dose of STZ. Then monitoring weight, blood pressure, blood glucose and glucose tolerance, detecting the initiation and development of atherosclerosis by ultrasound biomicroscopy(UBM) were performed to confirm the feasibility of establishing diabetic atherosclerosis model that closely resemble human diabetic atherosclerosis disease.
Objectives
1. To confirm the feasibility of establishing diabetic ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ;
2. To confirm the feasibility of establishing diabetic atherosclerosis ApoE-/-/LDLR-/- mouse model by high-fat and high-sugar diet combined with a small dose of STZ.
Methods
1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ (intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
2. IPGTT test:Mice were fasted for 10-16h before glucose tolerance tests. Intraperitoneal injection glucose load was administered at 1.5 g/kg of body weight. Glucose levels were measured from tail bleeds with a glucometer at specified time points after glucose administration.
3. Monitor body weight of ApoE-/-/LDLR-/- mice every week.
4. Assessment of hemodynamics of ApoE-/-/LDLR-/- mice:All ApoE-/-/LDLR-/-mice underwent hemodynamic examination at weeks 20,22 and 24, including blood pressure and heart rate.
5. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT.
6. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR.
Results
1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice show significant increased body weight (P=0.010). However, no significant difference was detected between the two groups at two weeks after administration of STZ (P=0.081). At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).
2. Hemodynamics alterations of ApoE-/-/LDLR-/- mice:Compared to the chow-diet mice, no significant difference was detected in the heart rate of diabetic mice (P=0.309). However, the heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased systolic blood pressure (P=0.00004), while the age of mice had no effect on systolic blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390). Compared to the chow-diet mice, diabetic mice show an increased diastolic blood pressure (P=0.000003), while the age of mice had no effect on diastolic blood pressure (P=0.137). No significant interactions were detected between the age and diabetes (P=0.652). Compared to the chow-diet mice, diabetic mice show an increased mean arterial blood pressure (P=0.0001), while the age of mice had no effect on mean arterial blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.263).
3. Results of IPGTT of ApoE-/-/LDLR-/- mice:At the age of 3 weeks, no significant difference was detected between the two groups. After high fat diet for 6 weeks, diabetic mice showed significant increased blood glucose (P<0.05 for all). After administration of STZ, diabetic mice showed significant higher blood glucose than chow-diet mice (P<0.05 for all). No significant differences were detected among the chow-diet mice with different ages. Significant differences were detected among the diabetic mice with different ages.
4. Parameters of glucose metabolism of ApoE-/-/LDLR-/- mice:At the age of 24 weeks, compared to the chow-diet mice, diabetic mice showed significantly increased blood glucose, serum insulin, and HOMA-IR (P=0.028, P=0.025, P=0.015, respectively), indicating that diabetic mice have hyperglycemia, hyperinsulinemia and insulin resistance.
5. Monitoring IMT of ApoE-/-/LDLR-/- mice:Brachiocephalic IMT of ApoE-/-/LDLR-/- mice got thickened earlier than carotid IMT. At the age of 16 weeks, significant differences of brachiocephalic IMT were detected between the two groups (P=0.000), which will last till week 24. As for carotid IMT, it kept increased slowly. At week 22, significant differences of carotid IMT were detected between the two groups (P=0.022). At week 24, compared with the chow-diet mice, diabetic mice had significant thickened carotid IMT (P=0.001).
Conclusions
1. Male ApoE-/-/LDLR-/- mice induced by high fat and sugar diet combined with a small dose of STZ at the age of 3weeks would appear the stable state of diabetes mellitus.
2. Diabetes mellitus induced by high fat and sugar diet combined with a small dose of STZ in male ApoE-/-/LDLR-/- mice would resemble human diabetes mellitus.
3. Type 2 diabetic ApoE-/-/LDLR-/- mice would be attacked by atherosclerotic plaque. Furthermore, the atherosclerotic plaque formed ahead of time.
4. Type 2 diabetic ApoE-/-/LDLR-/- mouse atherosclerosis model was appropriate for intervention.
Background
Although diabetes mellitus is the most risk factors for atherosclerosis and CHD, the mechanism of more serious and vulnerable plaques in the diabetic patients remains to be understood. This has highlighted the importance and urgency of studying the mechanism of diabetic atherosclerosis and exploring therapeutic options.
It is critical to find the crossroad of diabetes mellitus and acute coronary diseases to effectively deal with acute coronary syndromes-the most important cardiovascular complication. Up to date, studies on the mechanism of vulnerable plaque have showed that innate characteristics and the stress that the plaque bears with determine the vulnerability of the atherosclerotic plaque. It has been demonstrated that inflammation, oxidative stress, impaired glucose metabolism, dyslipidemia and dyshemodynamics would make the atherosclerotic plaque vulnerable. All these risk factors would cause more macrophages apoptosis which would make plaque vulnerable. However, the signal transduction involved in the macrophages apoptosis is a network, among which Akt signal transduction might play an essential role. TRIB3 has been confirmed to inhibit PI3K/Akt stimulated by insulin, causing insulin resistance. Furthermore, TRIB3, as a scaffold protein, was involved in the lipid metabolism. Epidemiological studies showed that TRIB3 Q84R polymorphism was associated with insulin resistance and the increase of cardiovascular diseases, especially atherosclerosis. However, whether TRIB3/Akt signal transduction was involved in diabetes cardiovascular complications remained to be clarified. In the present study, TRIB3-shRNA was transfected into type 2 diabetic ApoE-/-/LDLR-/- mice, silencing TRIB3, to investigate whether TRIB3 silence could stabilize the plaque and the underlying mechanism.
Objectives
1. To design and synthesize siRNA against mouse TRIB3 according to RNAi principle, then to construct the pAdxsi-TRIB3-shRNA;
2. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice to investigate the vulnerability of atherosclerotic plaque;
3. To explore the mechanism by which silence of TRIB3 would stabilize atherosclerotic plaque.
Methods
1. To design and synthesize 4 pieces of siRNA against mouse TRIB3 according to RNAi principle, then construct pGenesil-1.2-TRIB3-shRNA plasmid using the most effective siRNA. Subsequently, pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. With the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.
2. Animal experiment:Sixty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow-diet (n=30) and diabetes (n=30) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
3. Transfection of pAdxsi-TRIB3-shRNA into type 2 diabetic ApoE-/-/LDLR-/ mice. At the age of 20 weeks, after echo examination, chow-diet with RNAi group (n=15) and diabetes with RNAi group (n=15) was injected by pAdxsi-TRIB3-shRNA through tail vein, while chow-diet group (n=15) and diabetes group (n=15) was injected by pAdxsi through tail vein. After 2 weeks, echo examination and transfection were performed again. At the age of 24 weeks, echo examination was performed before the animals were sacrificed.
4. Serum parameters of ApoE-/-/LDLR-/- mice:At the end of experiment, blood glucose and serum insulin were measured to calculate the HOMA-IR. Furthermore, serum total cholesterol, triglycerides, HDL-C and LDL-C were also measured.
5. Assessment of peritoneal macrophages'function:Isolate peritoneal macrophage, identified by MOMA-2, then migration, adhesion and phagocytosis were assessed.
6. Pathology analyses:HE staining, Masson trichrome stain, oil red O, Sirius red, and Perl's staining were preformed on frozen sections from brachiocephalic plaque. Immunochemistry assay was performed to detect the macrophages, smooth muscle and TRIB3 in the brachiocephalic plaque. Plauqe area, fibrotic cap, collagen, lipid were measured to calculate cap/core ratio and vulnerability index.
7. Real-time RT-PCR:The mRNA of TRIB3 was quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green method.
8. Western bot:The proteins of TRIB3/Akt/Caspase-3 signal transduction were detected by western blotting.
9. TUNEL:Apoptotic cells and apoptotic macrophages were evaluated in frozen sections by TUNEL.
Results
1. Construction of pAdxsi-TRIB3-shRNA:After transfection of TRIB3 siRNA for 24h, the mRNA levels of GAPDH and TRIB3 were quantified by real-time RT-PCR using SYBR Green method. Based upon the inhibition rate of TRIB3, one piece was selected, which could silence 99.98% TRIB3. The chosen sequence was used to construct pGenesil-1.2-TRIB3-shRNA plasmid. Then pShuttle-Basic-EGFP-TRIB3-shRNA plasmid would be synthesized. Subsequently, with the shuttle plasmid, the pAdxsi-TRIB3-shRNA was constructed.
2. Alterations of ApoE-/-/LDLR-/- mice after transfection of pAdxsi-TRIB3-shRNA
(1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased body weight(P=0.001). Compared to the chow-diet ApoE-/-/LDLR-/- mice, chow-diet with RNAi group showed a decreased body weight (P=0.034); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed a significantly decreased body weight (P=0.003). Therefore, diabetes could increase the body weight (P=0.001), while RNAi had no effect on body weight (P=0.112). No significant interactions were detected between the RNAi and diabetes (P=0.481).
(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/ mice showed significant increased systolic blood pressure(SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MABP) (P<0.001 for all). Compared to the chow-diet ApoE-/-/LDLR-/-mice, chow-diet with RNAi group showed decreased SBP, DBP and MABP (P<0.001 for all); Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi group showed significantly decreased SBP, DBP and MABP (P<0.001 for all). Therefore, diabetes could increase SBP, DBP and MABP (P<0.001 for all), while RNAi could reduce SBP, DBP and MABP (P<0.05 for all). No significant interactions were detected between the RNAi and diabetes (P>0.05 for all).
3. Serum parameters of ApoE-/-/LDLR-/- mice:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased blood glucose, serum insulin, HOMA-IR, serum TC, serum LDL-C and serum HDL-C (P<0.05 for all), with serum TG unchanged (P=0.768). Compared to the diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/-mice showed significantly decreased blood glucose and HOMA-IR (P<0.05 for both), but serum insulin, serum TC, serum TG, serum LDL-C and serum HDL-C remained unchanged.
4. Comparison of peritoneal macrophages' function:Compared to the chow-diet ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetic ApoE-/-/LDLR-/-mice showed significantly increased migration, adhesion and phagocytosis functions. Compared to diabetic ApoE-/-/LDLR-/- mice, peritoneal macrophages from diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased migration function, enchanced phagocytosis function but unaltered adhesion function.
5. UBM measurements of brachiocephalic artery:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic IMT, diameters and blood velocity. Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- showed no decrease in brachiocephalic IMT, diameters and blood velocity (P>0.05 for all).
6. Pathological analyses
(1) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased aortic atherosclerotic burden (17.00±3.12% vs 30.99±3.12%, P=0.000); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased aortic atherosclerotic burden (30.99±3.12% vs 22.54±2.13%, P=0.000).
(2) Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly increased brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed no significant differences in brachiocephalic plaque area, lipid content in plaque, and macrophages in plaque (P>0.05 for all).
When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/-mice showed significantly decreased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increased thickness of fibrotic cap, cap/core ratio, and collagen content in brachiocephalic plaque (P<0.05 for all).
Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed no significant differences in I/III collagen ratio and SMC content of brachiocephalic plaque (P>0.05 for both); Compared to diabetic ApoE-/-/LDLR-/-mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly increasedⅠ/Ⅲcollagen ratio and SMC content of brachiocephalic plaque (P<0.05 for both).
When compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased vulnerability index of brachiocephalic plaque (1.75±0.45 vs 2.97±0.57, P=0.00001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased vulnerability index of brachiocephalic plaque (2.97±0.57 vs 2.32±0.59,P=0.008).
7. TUNEL analysis:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased apoptosis in brachiocephalic plaque (85.63±38.46 vs 201.00±61.01, P=0.00003); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased apoptosis in brachiocephalic plaque (201.00±61.01 vs 95.86±39.80, P=0.0001).
Furthermore, compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased macrophages'apoptosis in brachiocephalic plaque (48.31±28.39 vs 108.80±33.44, P=0.0001); Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased macrophages'apoptosis in brachiocephalic plaque (108.80±33.44 vs 46.20±15.21, P=0.00006).
8. Real-time RT-PCR:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 mRNA in atherosclerotic plaque (1.69±0.76 vs 4.97±1.30, P=1×10-7); Compared to diabetic ApoE-/-/LDLR-/ mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 mRNA in atherosclerotic plaque (4.97±1.30 vs 1.59±0.52, P=1×10-7).
9. Western bot:Compared to the chow-diet ApoE-/-/LDLR-/- mice, diabetic ApoE-/-/LDLR-/- mice showed significantly increased TRIB3 and caspase-3, but decreased phosphorylated Akt in atherosclerotic plaque; Compared to diabetic ApoE-/-/LDLR-/- mice, diabetes with RNAi ApoE-/-/LDLR-/- mice showed significantly decreased TRIB3 and caspase-3, but increased phosphorylated Akt in atherosclerotic plaque.
Conclusions
1. Diabetic ApoE-/-/LDLR-/- mice showed significantly increased aortic atherosclerotic burden, which would be reversed by silencing TRIB3.
2. Brachiocephalic plaques of diabetic ApoE-/-/LDLR-/- mice were confirmed to be vulnerable, manifesting decreased fibrotic cap, reduced cap/core ratio, increased lipid in the plaque, decreased collagen content, incremental macrophages, increased apoptosis of macrophages, augmented vulnerability index and enhanced hemorrhage in the plaque.
3. Silencing of TRIB3 would stabilize plaque by augmenting the thickness of fibrotic cap, increasing cap/core ratio, increasing collagen and smooth muscle contents, reducing apoptotic cells and decreasing vulnerability index.
4. Silencing of TRIB3 would effectively increase the activity of Akt, improve insulin resistance, reduce apoptosis of macrophages, thereby protecting against the enchancement of lipid core, to stabilize plaques.
5. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased migration. In advanced atherosclerosis, silencing of TRIB3 would in part decrease migration of peritoneal macrophages.
6. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased adhesion. In advanced atherosclerosis, silencing of TRIB3 would enhance adhesion, reduce migration of peritoneal macrophages, thereby increasing local clearance. Diabetes mellitus and silencing of TRIB3 would enhance adhesion respectively.
7. Peritoneal macrophages from diabetic ApoE-/-/LDLR-/- mice showed significantly increased phagocytosis of lipid, causing plaque size increased rapidly. In advanced atherosclerosis, silencing of TRIB3 would enhance phagocytosis of lipid, maintaining the ability of clearance, to avoid augmented lipid core due to apoptosis and necrosis. Diabetes mellitus and silencing of TRIB3 would enhance phagocytosis respectively.
8. Silencing of TRIB3 would effectively improve insulin resistance and glucose metabolism, elevate HDL and lower blood pressure, therefore reducing risk factors for atherosclerosis.
9. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of brachiocephalic plaque, highly identical with pathological analyses.
Background
Early identification of atherosclerosis and monitoring its development were essential for prevention of acute cardiovascular events in diabetic patients. Early identification to protect against atherosclerosis is an important method to prevent acute cardiovascular events, especially for vulnerable plaques. It has been demonstrated that spontaneous ruptures of atherosclerotic plaques mostly take place in brachiocephalic artery, which would be perfect for vulnerability of atherosclerotic plaques in type 2 diabetes mellitus. However, brachiocephalic artery was too deep to be detected. Therefore, the analyses nearly depend on the pathological analyses. Although pathological analyses could judge the development of atherosclerosis, it could not reflect the development of atherosclerosis in vivo. Furthermore, dynamic observations could not be realized, which make it difficult to choose the optimal opportunity and evaluate the effectiveness of interventions. Moreover, pathological analyses were restricted to the single sections without the integrity of atherosclerosis. Here, high resolution ultrasonic biomicroscopy UBM vevo770 was used to monitor the aortic, brachiocephalic and carotid atherosclerosis, to noninvasively evaluate diabetic atherosclerosis.
Objectives
1. To noninvasively evaluate diabetic atherosclerosis by monitoring atherosclerosis in diabetic animal model using UBM vevo770;
2. To compare the atherosclerosis in different arteries of diabetic ApoE-/-/LDLR-/- mice by UBM vevo770;
3. To evaluate the application of UBM vevo770 on studying atherosclerosis in diabetic ApoE-/-/LDLR-/- mice
Methods
1. Animal experiment:Thirty 3-week-old male ApoE-/-/LDLR-/- mice were randomized into 2 groups after intraperitoneal glucose tolerance test (IPGTT): chow(n=15) and diabetes(n=15) groups. Diabetes group were fed on high fat and sugar diet (20% fat,20% sugar and 1.25%cholesterol) for 6 weeks. IPGTT was performed to confirm the appearance of insulin resistance. Those insulin resistant mice were injected once with low dose of STZ(intraperitoneal at 75mg/kg). After 2 weeks, IPGTT was performed. Those with random blood glucose more than 11.1 mmol/l were regarded as diabetic mouse model.
2. Evaluation of atherosclerosis of ApoE-/-/LDLR-/- mice by UBM:All ApoE-/-/LDLR-/- mice underwent echocardiography at weeks 14,16,18,20,22 and 24 to monitor the initiation and development of atherosclerotic plaques in aortic, brachiocephalic and carotid arteries by measuring IMT, diameters and blood velocity.
Results
1. Comparison of body weight of ApoE-/-/LDLR-/- mice:At week 20, diabetic mice show significant increased body weight (P=0.0015). Then the body weight of mice kept stable. At week 24, diabetic mice still show significant increased body weight (P=0.0073).
The heart rate went down with the age of mice increased (P=0.014). Compared to the chow-diet mice, diabetic mice show an increased blood pressure (P=0.00004), while the age of mice had no effect on blood pressure (P=0.212). No significant interactions were detected between the age and diabetes (P=0.390).
2. At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups. The brachiocephalic IMT rose rapidly in diabetic mice, but slowed down at week 20. The brachiocephalic IMT rose slowly in chow-diet mice. The brachiocephalic IMT at week 24 in chow-diet mice was similar to that at week 20 in diabetic mice. At weeks 16,18 and 20, diabetic mice show significantly increased brachiocephalic IMT when compared with chow-diet mice (P<0.005 for all). At the age of 24 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656).
At the age of 14 weeks, no significant differences of brachiocephalic IMT were detected between the two groups (P=0.656). At weeks 22 and 24, diabetic ApoE-/-/LDLR-/- mice show significantly increased brachiocephalic IMT when compared with chow-diet ApoE-/-/LDLR-/- mice (P<0.05 for both).
3. At the age of 20 weeks, no significant differences of brachiocephalic blood velocity were detected between the two groups (P=0.503). Then brachiocephalic blood velocity decreased slowly. At week 24, diabetic ApoE-/-/LDLR-/- mice show significantly decreased brachiocephalic blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (416.41±86.27 vs 310.05±175.99, P=0.045).
At the age of 20 weeks, no significant differences of carotid blood velocity were detected between the two groups(42.84±136.52 vs 163.10±70.01, P=0.054). At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly decreased carotid blood velocity when compared with chow-diet ApoE-/-/LDLR-/- mice (275.09±104.69 vs 188.45±80.92, P=0.017). Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid blood velocity were detected between the two groups (P=0.968).
4. Alterations of carotid functions of diabetic ApoE-/-/LDLR-/- mice
(1) At the age of 20 weeks, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups. At the age of 22 weeks, diabetic ApoE-/-/LDLR-/- mice show significantly increased carotid stiffness, elasticity, and standardized elasticity when compared with chow-diet ApoE-/-/LDLR-/- mice. Then carotid blood velocity decreased slowly. At week 24, no significant differences of carotid stiffness, elasticity, and standardized elasticity were detected between the two groups.
(2) At weeks 20,22 and 24, no significant differences of carotid expand coefficient and compliance were detected between the two groups.
Conclusions
1. Diabetic ApoE-/-/LDLR-/- mice had more atherosclerotic burden than chow-diet ApoE-/-/LDLR-/- mice.
2. Diabetic ApoE-/-/LDLR-/- mice experience a rapid development of brachiocephalic atherosclerosis, which would slow down at the age of 20 weeks. Chow-diet ApoE-/-/LDLR-/- mice keep a slow development of brachiocephalic atherosclerosis. The brachiocephalic IMT at week 24 in chow-diet mice was comparable to that at week 20 in diabetic mice.
3. Diabetic ApoE-/-/LDLR-/- mice's carotid atherosclerosis was preceded with brachiocephalic atherosclerosis.
4. IMT is still an effective index to evaluate the initiation and development of atherosclerosis in diabetic ApoE-/-/LDLR-/- mice.
5. High resolution ultrasonic biomicroscopy could noninvasively monitor the initiation, development and prognosis of atherosclerotic plaque, helping to select the optimal intervention opportunity.
引文
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