TRIB3基因沉默改善2型糖尿病大血管病变的实验研究
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摘要
研究背景
糖尿病的发病率逐年增高,在发达国家已被列为继心血管疾病及肿瘤之后的第三大疾病。研究结果显示,糖尿病患者的医疗服务使用是非糖尿病患者的3至4倍(包括门诊和住院次数)；糖尿病患者的医疗支出是同年龄、同性别无糖尿病患者的9倍。另一项研究显示,我国糖尿病患者医疗花费中,只有不到20%是用于控制血糖,超过80%的费用是治疗糖尿病引起的并发症。目前糖尿病对人类健康危害最大的是在动脉硬化及微血管病变基础上产生的多种慢性并发症。糖尿病患者的血管病变发病率是非糖尿病患的2至4倍。现有研究证实,糖尿病血管病变的基本病理基础为动脉粥样硬化及血管平滑肌细胞和成纤维细胞大量增殖所致的动脉中膜、外膜不规则增厚、纤维化和钙化。UKPDS研究证实,严格控制血糖可以明显降低糖尿病患者微血管病变的发生率。由此,奠定了糖尿病血管并发症的防治基础。然而,2008年相继揭晓的三项重要强化降糖试验(ACCORD.ADVANCE及VADT)显示,积极控制血糖可以降低糖尿病患者微血管病变的发生率,但却未能证实强化降糖治疗可降低2型糖尿病患者大血管事件发生率。因此,亟需深入探索糖尿病患者心血管并发症风险增高的深层机制,奠定糖尿病患者心血管事件源头性防治的理论基础。
胰岛素抵抗贯穿于糖尿病的整个病程。UKPDS资料表明,在血糖未明显升高以前,单胰岛素抵抗就可引起大血管病变。胰岛素抵抗是糖尿病代谢性异常和心血管疾病的原始动因和致病基础。胰岛素抵抗的选择性使血管平滑肌和成纤维细胞增殖增强。“选择性胰岛素抵抗”的研究指出,在胰岛素抵抗状态下,MAPK增殖通路活性明显增强,而Akt代谢通路显著受损。TRIB3作为Akt和MAPK共同的调节蛋白,与“选择性胰岛素抵抗”密切相关。因此,我们推测TRIB3不仅通过Akt信号途径引起胰岛素抵抗和参与糖尿病发病,并可能通过调控Akt及MAPK信号途径而影响糖尿病大血管病变的发生和进展。因此,本课题将建立2型糖尿病大血管病变大鼠模型,采用RNAi技术,构建能够在哺乳动物细胞中表达siRNA的腺病毒表达载体,诱导TRIB3基因沉默,研究TRIB3在糖尿病大血管病变发生发展中的作用,综合运用各种病理学组织学染色技术研究TRIB3基因沉默后大血管形态结构的变化。通过分析上述实验结果,探讨TRIB3在糖尿病大血管病变中的作用及其可能机制。这些结果将为糖尿病心血管并发症患者的早期防治提供新靶点,具有重要的学术理论意义和潜在的临床应用价值。
研究目的
1.探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病大血管病变大鼠动物模型的可行性；
2.2型糖尿病大血管病变大鼠体内转染TRIB3-shRNA腺病毒,观察糖尿病大血管病变改善情况；
3.在细胞分子水平、组织学水平和整体功能水平研究TRIB3基因沉默改善2型糖尿病大血管病变的机制；研究方法
1.5周龄健康雄性SD (Sprague-Dawley)大鼠60只,行腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT)后随机分为4组：对照组(Control组)、普通饮食STZ组(Chow+STZ组)、高脂高糖饮食组(HF组)、DM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,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,留取静脉血,测定空腹血糖(FBG)和空腹胰岛素(FINS),计算胰岛素敏感指数(ISI, ISI=Ln(FINS×FBG)-1)。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感指数降低且有多尿、多饮、多食现象的大鼠作为成模大鼠纳入实验；
2.5周龄健康雄性SD (Sprague-Dawley)大鼠30只,糖尿病造模过程同上,造模成功后随机分为DM-Vector组和DM-siRNA组。糖尿病成模12周后,DM-siRNA组大鼠经颈静脉注射pAdxsi-TRIB3-shRNA腺病毒2.5×1010PFU/只,DM-Vector组注射pAdxsi空病毒载体2.5×1010PFU/只,2周后补充注射一次,剂量同第一次,腺病毒注射4周后处死动物留取标本。
3.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(禁食不禁水),断尾取血测定空腹血糖,继而给予腹腔注射20%葡萄糖(1g葡萄糖/kg体重),于注射葡萄糖后15min、30min、60min、120min按时从尾静脉取血测定血糖水平。采用强生稳步血糖仪和试纸条测定血糖。通过IPGTT结果计算血糖曲线下面积(AUC of glucose, AUCg).大鼠禁食4h后(禁食不禁水)行IPITT,给予腹腔注射胰岛素(1U胰岛素/kg体重),余同IPGTT；
4.血液生化指标的测定：检测血清总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)的水平,测定血糖和血清胰岛素,计算胰岛素敏感指数；
5.血流动力学监测：所有实验动物通过大鼠尾动脉血压测量仪监测血压和心率；
6.腹主动脉超声：采用二维、脉冲多普勒和背向散射积分等超声技术,连续观察2型糖尿病大血管病变发生和发展过程中腹主动脉结构和功能的变化；
7.病理学检测：对大鼠主动脉组织分别进行HE染色、Masson染色、天狼猩红染色以及Verhoeff染色,应用图象分析仪测定主动脉中层厚度,主动脉血管周围胶原面积/管腔面积,主动脉中膜胶原比例,主动脉中膜弹力纤维比例,主动脉胶原与弹力纤维的比值,计算平均主动脉壁结构积分；
8.免疫组织化学染色：应用免疫组织化学染色法,光镜下观察大鼠主动脉间质的Ⅰ型胶原、Ⅱ工型胶原分布情况；
9.大鼠主动脉羟脯氨酸含量测定：将干燥至恒重的大鼠主动脉组织称重后置于6mol/L HCl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主动脉干重)；
10.实时定量RT-PCR检测：取新鲜大鼠主动脉组织,Trizol法提取RNA,实时荧光定量RT-PCR技术检测TRIB3mRNA相对表达量；
11Western blot检测：取新鲜大鼠主动脉组织,提取蛋白,Western blot检测主动脉组织内TRIB3、胶原Ⅰ、胶原Ⅲ、磷酸化及非磷酸化的MKK4、JNK、PI3K、Akt的蛋白表达量。
结果
1、实验末各组大鼠的一般状况比较：与Control组大鼠相比,Chow+STZ组大鼠日饮水量、日摄食量及24h尿量均明显增加(P<0.05)；与Control组大鼠相比,HF组大鼠体重明显增加,日饮水量、日摄食量及24h尿量均有所增加(P<0.05)；与Control组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均显著增加(P<0.01)。与HF组大鼠相比,Chow+STZ组大鼠日饮水量、24h尿量明显增多(P<0.05～0.001)；与HF组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均显著增加(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均明显增加(P<0.001)。
2、实验期间各组大鼠腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT)结果分析：
(1)IPGTT结果分析：
DM组大鼠高脂高糖饮食4周后与高脂高糖饮食前IPGTT结果显示：与高脂高糖饮食前相比,DM组大鼠高脂高糖饮食4周后在IPGTT试验Omin、30min、60min、120min的血糖值均明显升高,差异具有统计学意义(P均<0.05)；血糖曲线下面积(AUCg)明显增大(P 2型糖尿病成模后12周各组大鼠IPGTT结果显示：与Control组大鼠相比,Chow+STZ组大鼠在IPGTT试验30min及60min的血糖值明显升高(P<0.05)；与Control组大鼠相比,HF组大鼠在IPGTT试验各时间点血糖值差异无统计学意义：与Control组大鼠相比,DM组大鼠在IPGTT试验15min、60min的血糖值均显著升高(P 实验末IPGTT试验结果显示：与Control组大鼠相比,Chow+STZ组大鼠在IPGTT试验60min、120min的血糖值均显著升高(P<0.05)；与Control组大鼠相比,HF组大鼠在IPGTT试验各时间点血糖值差异无统计学意义；与Control组大鼠相比,DM组大鼠在IPGTT试验15min、60min、120min的血糖值均显著升高(P<0.05)。与Chow+STZ组相比,DM组大鼠在IPGTT试验15min、30min的血糖值明显升高(P<0.05)。与HF组相比,DM组大鼠在IPGTT试验60min、120min的血糖明显升高(P<0.05)。与Control组相比,Chow+STZ组、HF组及DM组的血糖曲线下面积(AUCg)均明显增大(P<0.05)。与HF组大鼠相比,DM组血糖曲线下面积明显增大(P<0.05)。
(2)IPITT结果分析：
DM组大鼠高脂高糖饮食4周后与高脂高糖饮食前IPITT结果显示：与高脂高糖饮食前相比,DM组大鼠高脂高糖饮食4周后在IPITT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积明显增大(P<0.01)。
2型糖尿病糖尿病成模后12周各组大鼠IPITT结果显示：与Control组大鼠相比,Chow+STZ组大鼠IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05～P<0.01)；与Control组大鼠相比,HF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Control组大鼠相比,DM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Chow+STZ组相比,HF组大鼠在IPITT试验各时间点血糖值差异无统计学意义；与Chow+STZ组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与HF组相比,DM组大鼠在IPITT试验Omin、15min、30min、60min的血糖值均显著升高(P<0.05)。与Control组相比,Chow+STZ组、HF组及DM组的血糖曲线下面积均明显增大(P<0.05～P<0.01)；与HF组大鼠相比,DM组血糖曲线下面积明显增大(P<0.05)。
实验末IPITT试验结果显示：与Contro1组大鼠相比,Chow+STZ组大鼠IPITT试验15min的血糖值明显升高(P<0.05)；与Control组大鼠相比,HF组大鼠在IPITT试验各时间点血糖值差异无统计学意义；与Control组大鼠相比,DM组大鼠在IPITT试验15min、60min、120min的血糖值均明显升高(P<0.05)。与Chow+STZ组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与HF组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与Control组相比,Chow+STZ组、HF组及DM组的血糖曲线下面积均明显增大(P<0.05)。与HF组大鼠相比,DM组血糖曲线下面积明显增大(P<0.05)。
3、生化指标的测定：实验前各组大鼠生化指标无明显差异；高脂饮食4周后,与Control相比,HF组及DM组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),FBG无显著变化,提示注射STZ前DM组大鼠已存在胰岛素抵抗；STZ注射后一周,与Control相比,Chow+STZ组及DM组FBG明显升高(P<0.05),ISI显著降低(P<0.05),HF组及DM组血清TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后12周,与Control相比,DM组FBG明显升高(P<0.05),ISI显著降低(P<0.05),DM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14周,与Control相比,DM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Control相比,DM组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。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。
4、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Control组大鼠及Chow+STZ组大鼠相比,DM组大鼠心率略有增加,但是差异没有统计学意义。
5、腹主动脉超声监测
实验结束时,与Control组大鼠比较,HF组大鼠各项指标变化均无统计学意义；与Control组大鼠相比,Chow+STZ组大鼠腹主动脉Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～0.001),舒张末期流速、横断面扩张系数均降低,差异具有统计学意义(P<0.05～0.001,)；与Control组大鼠相比,DM组大鼠腹主动脉收缩期内径、舒张期内径、Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～P<0.001),收缩期峰值流速、舒张末期流速、横断面扩张系数均降低,差异具有统计学意义(P<0.05～0.001)。与HF组大鼠相比,Chow+STZ组大鼠腹主动脉僵硬度指数明显增加,差异具有统计学意义(P<0.01)；与HF组大鼠相比,DM组大鼠腹主动脉舒张期内径、Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～P<0.001),收缩期峰值流速、舒张末期流速均降低,差异具有统计学意义(P<0.05～P<0.001)。与Chow+STZ组大鼠相比,DM组大鼠腹主动脉收缩期内径和舒张期内径明显增加,差异具有统计学意义(P<0.05～0.001)。余各组大鼠指标比较,差异均无统计学意义。
实验结束时,超声背向散射积分结果显示,与Control组大鼠比较,Chow+STZ组和DM组大鼠腹主动脉校正平均背向散射积分(IBS%)均明显增加(P<0.01)；与Control组大鼠比较,Chow+STZ组、HF组和DM组大鼠腹主动脉背向散射积分周期变化幅度(CVIB)均明显降低(P<0.001)；与HF组比较,Chow+STZ组和DM组大鼠腹主动脉IBS%明显增加(P<0.05～0.01),DM组大鼠腹主动脉CVIB明显降低(P<0.01)。
6、主动脉病理学分析
(1)主动脉HE染色,与Control组大鼠相比,DM组大鼠内皮细胞排列紊乱、致密,仍紧贴于内弹力板上。血管平滑肌细胞肥大、扭曲、排列紊乱,层数增多,细胞核大小不一,细胞膜及核膜欠清晰、完整,胞浆染色不均,可见大量肌纤维断裂。定量分析结果显示：与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉中膜厚度明显增加,差异具有统计学意义(72.73.09±2.12vs.58.09±0.97μ m,P<0.01；62.69±0.67vs.58.09±0.97μ m,P<0.05；72.38±2.30vs.58.09±0.97μ m,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉中膜厚度略有增加,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜厚度略有增加,差异无统计学意义。
(2)Masson染色显示,大鼠主动脉血管平滑肌细胞及弹力纤维呈红色,间质胶原纤维呈蓝绿色。Control组大鼠主动脉胶原纤维分布均匀、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；与Control组相比,DM组大鼠主动脉胶原组织明显增多,排列紊乱,分布不匀,血管周围纤维化明显。
天狼猩红染色：在普通显微镜下,Control组大鼠主动脉胶原纤维均匀纤细,血管周围有极少量胶原；偏振光显微镜下,Control组大鼠主动脉中膜未见明显显色,外膜以Ⅰ型及Ⅲ型胶原纤维为主。在普通显微镜下,DM组大鼠主动脉间质胶原纤维明显增多,迂曲,排列紊乱,断裂明显增多；偏振光显微镜下,DM组大鼠主动脉中膜和外膜Ⅰ型及Ⅲ型胶原明显增加,胶原纤维断裂明显。半定量分析结果显示,与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉管周胶原面积/管腔面积(PVCA/LA)均有明显增加,差异具有统计学意义(0.06±0.0029vs.0.03±0.0015,P<0.001；0.04±0.0016vs.0.03±0.0015,P<0.05；0.10±0.007vs.0.03±0.0015,P<0.01),中膜胶原比例均有明显增加,差异具有统计学意义(4.42±0.48vs.1.12±0.14,P<0.001；2.45±0.22vs.1.12±0.14,P<0.01；5.70±0.31vs.1.12±0.14,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉中膜胶原比例及管周胶原面积/管腔面积(PVCA/LA)均有明显增加,差异具有统计学意义(P<0.01～0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉管周胶原面积/管腔面积(PVCA/LA)有所增加,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜胶原比例有所增加,但差异没有统计学意义。
(3)Verhoeff弹力纤维染色显示：Control组大鼠主动脉弹力纤维分布均匀、整齐,完整无断裂；DM组大鼠主动脉弹力纤维迂曲,排列紊乱、疏松,分布欠均匀,可见明显断裂。半定量分析结果显示,与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉中膜弹力纤维比例均明显降低,差异具有统计学意义(5.19±0.76vs.11.96±0.75,P<0.001；8.83±0.98vs.11.96±0.75,P<0.05；4.70±0.36vs.11.96±0.75,P<0.001)；与HF组大鼠相比,DM组大鼠主动脉中膜弹力纤维比例明显降低,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜弹力纤维比例略有降低,差异没有统计学意义。与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉胶原/弹力纤维比值均明显增加,差异具有统计学意义(0.57±0.09vs.0.05±0.007,P<0.01；0.17±0.0024vs.0.05±0.007,P<0.01；0.71±0.075vs.0.05±0.007,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉胶原/弹力纤维比值均明显增加,差异具有统计学意义(P<0.05～0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉胶原/弹力纤维比值略有增加,差异没有统计学意义。与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉平均主动脉壁结构积分均明显增加,差异具有统计学意义(P均<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉平均主动脉壁结构积分均明显增加,差异具有统计学意义(P<0.01、0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉平均主动脉壁结构积分略有增加,差异具有统计学意义(P<0.05)。
(4)采用羟脯氨酸法测定大鼠主动脉胶原含量。结果显示：与Control组相比,Chow+STZ组、HF组、DM组大鼠主动脉胶原含量均明显增加,差异具有统计学意义(13.34±0.37vs.7.01±0.21ug/mg血管干重,P<0.001；8.47±0.21vs.7.01±0.21ug/mg血管干重,P<0.01；15.34±0.47vs7.01±0.21ug/mg血管干重,P<0.001)；与HF组大鼠相比,Chow+STZ组和DM组大鼠主动脉胶原含量明显增加,差异具有统计学意义(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉组织胶原含量明显增加,差异具有统计学意义(P<0.001)。
(5)免疫组化结果显示：与Control组相比,Chow+STZ组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达明显增多,HF组大鼠主动脉内collagen Ⅰ、collagen Ⅲ表达有所增加,DM组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达显著增多；与HF组相比,Chow+STZ组、DM组大鼠主动脉内collagen Ⅰ、collagen Ⅲ阳性表达均明显增多；与Chow+STZ组相比,DM组大鼠主动脉collagenⅠ、collagen Ⅲ阳性表达均明显增多。
(6)Collagen Ⅰ、collagen Ⅲ的Western blot结果显示：与Control组相比,Chow+STZ组大鼠主动脉collagen Ⅰ、collagen Ⅲ蛋白表达明显增加,差异具有统计学意义(collagen Ⅰ:0.82±0.05vs.0.51±0.05,P<0.001：collagen Ⅲ:0.62±0.03vs.0.31±0.02,P<0.001),HF组主动脉collagen Ⅰ、collagen Ⅲ蛋白含量略有增加,差异具有统计学意义(collagen Ⅰ:0.60±0.03vs.0.51±0.05,P<0.01；collagen Ⅲ：0.47±0.03vs.0.31±0.02,P (7)与Control组相比,Chow+STZ组大鼠主动脉TRIB3蛋白表达量增加了1.33倍,差异具有统计学意义(P<0.001),HF组大鼠主动脉TRIB3蛋白表达量增加了72%,差异具有统计学意义(P<0.001),DM组大鼠主动脉TRIB3蛋白表达量增加了1.78倍,差异具有统计学意义(P<0.001)；与HF组大鼠相比,Chow+STZ组大鼠主动脉TRIB3蛋白表达量增加了35%,差异具有统计学意义(P<0.001),DM组大鼠主动脉TRIB3蛋白表达量增加了61%,差异具有统计学意义(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉TRIB3蛋白表达量增加了19%,差异具有统计学意义(P<0.001)
(8)Western blot检测Akt和MAPK通路各关键信号分子
①Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化P13K与总P13K比值分别为：0.97,0.67,0.75,0.45。与Control组大鼠相比,Chow+STZ组、HF组及DM组大鼠主动脉P13K磷酸化水平均明显降低,差异具有统计学意义(P均<0.001)；与HF组大鼠相比较,Chow+STZ组及DM组大鼠主动脉P13K磷酸化水平均明显降低(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠P13K磷酸化水平明显降低(P<0.01)
②Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化Akt与总Akt的比值分别为：0.90,0.48,0.61,0.29。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉Akt的磷酸化水平均明显降低,差异具有统计学意义(P<0.01～P<0.001),其中DM组大鼠主动脉Akt的磷酸化水平最低,为Control组的32%(P<0.001)；与HF组相比,Chow+STZ组及DM组大鼠主动脉Akt的磷酸化水平均有下降,以DM组降低最为显著,为HF组的47%(P<0.001)；与Chow+STZ组相比,DM组大鼠主动脉Akt的磷酸化水平有所下降,但差异无统计学意义。
③Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化MKK4与总MKK4比值分别为：0.59,0.85,0.64,1.03。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉MKK4磷酸化水平均明显升高,差异具有统计学意义(P<0.05～0.001),其中DM组大鼠主动脉MKK4的磷酸化水平最高,为Control组的1.86倍(P<0.001)；与HF组比较,Chow+STZ组、DM组大鼠主动脉MKK4磷酸化水平明显升高,差异具有统计学意义(P均<0.001)；与Chow+STZ组相比,DM组大鼠主动脉MKK4磷酸化水平明显升高,差异具有统计学意义(P<0.01)
④Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化JNK与总JNK比值分别为：0.45,0.78,0.60,0.97。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉JNK磷酸化水平均明显升高,差异具有统计学意义(P<0.01、0.001),其中DM组大鼠主动脉JNK的磷酸化水平最高,为Control组的2.15倍(P<0.001)；与HF组比较,DM组大鼠主动脉JNK磷酸化水平明显升高,差异具有统计学意义(P<0.001),Chow+STZ组大鼠主动脉JNK磷酸化水平略有升高,差异没有统计学意义；与Chow+STZ组相比,DM组大鼠主动脉JNK磷酸化水平略有升高,差异没有统计学意义。
7、TRIB3基因沉默后主动脉TRIB3mRNA及蛋白的表达
主动脉TRIB3mRNA及蛋白的表达：RT-PCR结果显示：与糖尿病空载体(DM-Vector)组相比,糖尿病siRNA (DM-siRNA)组大鼠主动脉TRIB3mRNA表达降低了66%,差异具有统计学意义(P 8、TRIB3基因沉默对2型糖尿病大鼠一般情况的改善
经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠日饮水量显著减少,差异具有统计学意义(P<0.05),同时DM-siRNA组大鼠24h尿量也显著减少,差异具有统计学意义(P<0.05)。DM-siRNA组大鼠日摄食量较DM-Vector组均有所降低,但未达到统计学意义。DM-siRNA组大鼠体重、收缩压、舒张压、平均动脉压及心率较DM-Vector组无变化。
9、TRIB3基因沉默对IPGTT及IPITT的改变
(1)IPGTT结果显示：经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠在IPGTT试验30min、120min的血糖值均明显降低(Ｐ均<0.05),血糖曲线下面积明显减少(P=0.041),差异具有统计学意义。
(2)IPITT结果显示：经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠在IPITT试验0min、120min的血糖值均明显降低(P均<0.05),血糖曲线下面积明显减少(P=0.030),差异具有统计学意义。
10、TRIB3基因沉默对血清生化指标的影响
经TRIB3腺病毒干预4周后,与DM-Vector组大鼠相比,DM-siRNA组大鼠血清总胆固醇明显降低,差异具有统计学意义(4.98±1.62vs2.57±0.62mmol/L,P<0.05)；DM-siRNA组大鼠血清甘油三酯明显降低,差异具有统计学意义(3.96±1.01vs.7.74±2.15mmol/L;P<0.05)；DM-siRNA组大鼠血清游离脂肪酸明显降低,差异具有统计学意义(355.85±26.24vs.464.69±31.23μmol/L,P<0.05)；DM-siRNA组大鼠血清空腹血糖明显降低,差异具有统计学意义(15.63±2.04vs.24.58±3.04mmol/L,P<0.05)；DM-siRNA组大鼠胰岛素敏感指数明显升高,差异具有统计学意义(-5.17±0.14vs.-5.67±0.14,P<0.05)；DM-siRNA组大鼠空腹胰岛素水平较空载体组略有降低,但差别没有统计学意义。
11、TRIB3基因沉默对腹主动脉超声指标的影响
经TRIB3腺病毒干预4周后,与DM-Vector组大鼠相比,DM-siRNA组大鼠腹主动脉舒张期内径明显减小,差异具有统计学意义(P<0.05)；DM-siRNA组大鼠收缩期峰值流速和舒张末期流速均明显增加,差异具有统计学意义(P 经TRIB3腺病毒干预4周后,超声背向散射积分结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠腹主动脉IBS%明显减小,差异具有统计学意义(1.76±0.14vs1.42±0.15,P<0.001),CVIB明显增加,差异具有统计学意义(4.17±0.81vs5.21±0.57,P<0.01)。
12、TRIB3基因沉默改善糖尿病大鼠主动脉重构
(1)主动脉HE染色可见,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉仅有部分内皮细胞部分突起、断裂,结构不完整,排列趋于整齐。血管平滑肌细胞呈长梭形,平行排列,层数减少,细胞核大小基本均一,细胞膜及核膜清晰、完整,胞浆染色均匀,肌纤维断裂明显减少。分析结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜厚度明显减少(74.88±1.21μ m vs.69.38±1.11μ m,P<0.01),差异具有统计学意义；
(2)Masson染色显示,与DMVector组大鼠相比,DM-siRNA组大鼠主动脉胶原纤维明显减少,胶原纤维变细,排列区域规整,分布基本均匀。天狼猩红染色,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉间质胶原纤维明显减少,排列较为整齐,断裂明显减少；偏振光显微镜下,DM-siRNA组大鼠主动脉中膜和外膜Ⅰ型及Ⅲ型胶原纤维均明显减少,胶原纤维断裂减少。半定量分析结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉血管周围胶原面积/管腔面积(PVCA/LA)明显减少,差异具有统计学意义(0.10±0.005vs.0.06±0.006,P<0.001)；与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜胶原比例明显减少,差异具有统计学意义(3.77±0.36vs.2.48±0.22,P<0.01)
(3)Verhoeff弹力纤维染色染色显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉弹力纤维趋于均匀,整齐,断裂明显减少。半定量分析结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜弹力纤维比例均明显增加,差异具有统计学意义(5.29±0.36vs.7.79±0.72,P<0.01)；DM-siRNA组大鼠主动脉胶原/弹力纤维比值明显降低,差异具有统计学意义(0.75±0.09vs.0.36±0.07,P<0.01)；DM-siRNA组大鼠主动脉平均主动脉壁结构积分有所降低,差异具有统计学意义(P<0.05)
(4)采用羟脯氨酸法测定大鼠主动脉胶原含量,结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉胶原含量明显减少,差异具有统计学意义(14.16±0.30vs.10.70±0.19μ g/mg血管干重,P<0.001)
(5)TRIB3基因沉默降低了主动脉Ⅰ、Ⅲ型胶原的表达
免疫组化结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达均有所减少。Western blot结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagenⅠ、collagen Ⅲ蛋白表达有所降低。定量分析Western blot结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagen Ⅰ、 collagen Ⅲ蛋白表达明显降低,差异具有统计学意义(P<0.001),其中collagen Ⅰ蛋白表达降至DM-Vector组大鼠的61%(P<0.01),collagen Ⅲ蛋白表达下降至DM-Vector组大鼠的59%(P<0.01)。
(6)TRIB3基因沉默后主动脉PI3K/Akt及MKK4/JNK通路各关键分子的表达
①与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉PI3K的磷酸化水平明显升高,其蛋白表达为DM-Vector组大鼠的1.79倍(P<0.001)。
②与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉Akt的磷酸化水平明显升高,其蛋白表达为DM-Vector组大鼠的1.94倍(P<0.001)。
③与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉MKK4的磷酸化水平明显降低,其蛋白表达降为DM-Vector组大鼠的37%(P<0.001)
④与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉JNK的磷酸化水平明显降低,其蛋白表达下降至DM-Vector组大鼠的66%(P<0.001)
结论
(1)高糖高脂高热量饮食喂养联合小剂量链脲佐菌素诱发的2型糖尿病状态稳定,符合2型糖尿病的临床特点,能够出现糖尿病大血管病变,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；
(2)2型糖尿病大鼠主动脉重构以血管硬化为主,主要表现为血管壁内胶原含量明显增加,主动脉中膜厚度增加,主动脉内径增加,主动脉僵硬度指数明显增加；
(3)2型糖尿病状态下,TRIB3表达上调,进而激活MMK4及JNK信号传导途径引起Ⅰ、Ⅲ型胶原合成明显增加,最终导致血管间质纤维化,是2型糖尿病大血管病变发生的重要分子机制；
(4)2型糖尿病大鼠发生主动脉重构的同时,主动脉组织TRIB3表达明显升高,PI3K/Akt信号通路活性减弱,而MAPK信号通路明显增强,提示选择性胰岛素抵抗的信号通路参与糖尿病大血管病变的发病；
(5)TRIB3基因沉默能够通过减少主动脉内胶原含量,降低主动脉中膜厚度,调整主动脉内胶原/弹性纤维比值,从而减轻主动脉硬化,延缓了主动脉重构的进展；
(6)TRIB3基因沉默延缓2型糖尿病大鼠主动脉重构的同时,主动脉组织TRIB3表达明显降低,PI3K/Akt信号通路活性随之增加,而MAPK信号通路明显降低,进一步证实以TRIB3为核心的选择性胰岛素抵抗参与了糖尿病大血管病变的发生和发展；
(7)TRIB3基因沉默能够改善2型糖尿病大鼠胰岛素抵抗和糖脂代谢异常,有效地减少了糖尿病大血管病变发生的危险因素；
(8)TRIB3基因沉默主要通过增加主动脉组织内Akt的磷酸化水平,阻断主动脉组织内MMK4及JNK的活化,尤其是通过对MMK4的抑制,从分子水平有效改善选择性胰岛素抵抗,纠正代谢性信号通路与增殖性信号通路的失衡,延缓了糖尿病主动脉重构的进展,改善了大血管的功能。
Background
Diabetes mellitus, the incidence of which increasing year by year, has been listed as the third mighty disease following cardiovascular disease and tumor in the Developed Countries. A research result shows that the times of clinical service and hospitalization in diabetic patients increased obviously, and the use of medical services of diabetic patients was two to four times that of non-diabetic patients, the medical costs of diabetic populations were nine times that of non-diabetic populations with the same age and sex. Another research indicates that for diabetic patients in china less than20%of the diabetic medical expenses were spent in blood-glucose control, and more than80%of that spent in the treatment of diabetic complications. The serious harm of diabetes mellitus to human health is the chronic diabetic complications developed from arteriosclerosis and microangiopathy. The incidence of angiopathy in diabetic patients is three to four times that of non-diabetics patients. It has been suggested that the fundamental pathological characteristics of diabetic angiopathy were atherosclerosis and the anomaly thickening, fibrosis and calcification of tunica media and tunica adventitia. The UKPDS shows conclusively that the incidence of diabetic microangiopathy could be obviously reduced by good glycemia control. However, the three important trials in intensive glucose lowering (ACCORD, ADVANCE and VADT) published in2008show that intensive blood-glucose control could reduce the rate of diabetic microangiopathy, but did not reduce the rate of major CVD events in patients with type2diabetes. So it is desiderated to further explore the mechanisms of the high rate of cardiovascular diseases in patients with type2diabetes, in order to find the theoretical basis of upstream treatment strategies for cardiovascular complications in patients with type2diabetes.
Insulin resistance runs through the whole course of type2diabetes. The UKPDS shows that insulin resistance could cause macroangiopathy before the onset of hyperglycemia. Insulin resistance is the primitive cause and pathogenic basis of metabolic abnormality and cardiovascular complications in type2diabetes. Selective insulin resistance could enhance the proliferation of vascular smooth muscle cell and fibroblasts. Studies on selective insulin resistance indicate that in insulin-resistant states, the metabolic Akt pathway of insulin effect is impaired obviously, while the proliferative MAPK pathway is enhanced. TRIB3, served as the common regulatory protein of the Akt and MAPK pathway, is closely related to selective insulin resistance. So we hypothesize that TRIB3not only participates in pathogenesis of type2diabetes by inducing insulin resistance through the metabolic Akt pathway, but also participates in the initiation and progression of diabetic macroangiopathy through the proliferative MAPK pathway. So in this study, we aim to establish the type2diabetic model, determine the effects of TRIB3on the initiation and progression of diabetic macroangiopathy through inducing in vivo TRIB3gene silencing by RNAi technique, and investigate the morph metric and structural changes of great vessels after TRIB3gene silencing by various pathological and histological staining techniques. We aim to explore the effects of TRIB3on diabetic macroangiopathy and its possible mechanisms. The results will provide potential new targets for the early treatment of cardiovascular complications in type2diabetes, and have important academic theoretical significance and potential clinical application perspective.
Objectives
1.To confirm the feasibility of establishing diabetic macroangiopathy rat model induced by a high fat and sugar diet combined with a small dose of STZ;
2.To investigate the improvement of diabetic macroangiopathy by transfection of pAdxsi-TRIB3-shRNA into type2diabetic rat;
3. To explore the mechanism by which TRIB3silencing would ameliorate diabetic macroangiopathy in molecular, histological and functional levels.
Methods
1. Sixty5-week-old male SD (Sprague-Dawley) rats were randomized into Four groups after intraperitoneal glucose tolerance test(IPGTT) and intraperitoneal insulin tolerance test (IPITT):Control, chow+streptozotocin (Chow+STZ), high-fat and sugar diet (HF) and diabetes mellitus (DM).Control and Chow+STZ groups rats were fed on normal chow (20%crude protein,3%crude fat,3%crude fiber, others74%,including carbohydrates, microelements, etc). HF and DM groups rats received high-fat and sugar diet (34.5%fat,17.5%protein and48%carbohydrates). Four weeks later, IPGTT and IPITT were performed again, rats with insulin resistant in DM group were injected once with low dose of STZ (intraperitoneal at27.5mg/kg), and meanwhile Chow+STZ group rats received same dose of STZ. Control and HF groups rats received same dose of citrate buffer. One week after STZ administration, blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index(ISI, ISI=Ln(FINS×FBG)-1)was calculated. Rats with FBG>11.1mmol/1in two consecutive analyses, lower insulin sensitivity and characterized by polyuria, polydipsia and polyphagia were considered the diabetic rat model.
2.Thirty5-week-old male SD (Sprague-Dawley) rats were made into diabetic rat model according to the above method, and then were randomized into two groups: DM-Vector group and DM-siRNA group.12weeks after successful modeling of DM, DM-siRNA group rats were injected via the jugular vein with2.5x1010PFU of pAdxsi-TRIB3-shRNA, while DM-Vector group rats were injected with2.5x1010PFU of pAdxsi.Adenovirus transfection was repeated after two weeks. Four weeks after first adenovirus injection, rats were sacrificed.
3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT):Glucose tolerance was assessed by IPGTT after12-hour diet fasting without water fasting. A bolus of glucose (lg/kg, ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60and120min and tested for glucose. Blood glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The area under the curve of glucose (AUCg) was calculated. To evaluate insulin tolerance, IPITT was performed after4-hour diet fasting without water fasting. A bolus of insulin (1unit/kg, ip) was injected, and blood samples were taken for glucose measurement as described above.
4. Serum biochemical parameter analysis:blood glucose and serum insulin were measured to calculate the insulin sensitivity index (ISI). Furthermore, serum total cholesterol (TC), triglycerides(TG) and free fatty acids (FFA) were also measured.
5. Measurement of blood pressure:Heart rate (HR) and blood pressure (BP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan).
6. Ultrasonography of abdominal aorta:Successive observation of the changes of abdominal aorta in the initiation and progression of diabetic macroangiopathy by2-D, pulsed-wave Doppler and ultrasonic integrated backscatter technique.
7. Pathological analysis:HE staining, Masson trichrome staining, Sirius red staining and Verhoeff staining were preformed on rat aorta. The aortic media thickness, perivascular collagen area/luminal area (PVCA/LA), aortic collagen percentage per medial area, aortic elastin percentage per medial area and aortic collagen/elastin ratio were determined with a computerized image analysis system. The average aortic wall architecture score was graded on Verhoeff-stained sections.
8. Immunohistochemical staining for collagen Ⅰ and Ⅲ:The collagen Ⅰ and Ⅲ depositions of aorta were viewed by immunohistochemical staining under a light microscope. The aortic Collagen Ⅰ and Ⅲ content were determined with a computerized image analysis system.
9. Hydroxyproline analysis:The collagen content of rat aortic tissues was determined by Hydroxyproline assay. The rat aortic tissues dried to constant weight were weighed, and then hydrolyzed in6mol/L HC1at110℃for16hours. The Hydroxyproline content of the hydrolysates was detected by an ELISA kit. Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%Hydroxyproline.
10. Real-time quantitative RT-PCR:Total RNA of the fresh rat aortic tissues was extracted by Trizol. The mRNA of TRIB3was quantified by Real-time Fluorescent Quantitative RT-PCR.
11. Western blot analysis:The proteins of TRIB3, collagen Ⅰ and Ⅲ, phospho-PI3K/PI3K,phospho-Akt/Akt,phospho-MKK4/MKK4and phospho-JNK/JNK were detected by western blotting.
Results
1. General characteristics of rats in each group at the end of the experiment
The rats in Chow+STZ group had obviously higher values of water intake, food intake and24h urine volume compared with Control group (P<0.05). The rats in HF group had increased values of water intake, food intake and24h urine volume, and significantly increased body weight compared with Control group (P<0.05). The rats in DM group had markedly increased values of water intake, food intake and24h urine volume compared with Control group (P<0.01). The rats in Chow+STZ group had significantly increased values of water intake and24h urine volume compared with HF group (P<0.05～0.001). The rats in DM group had significantly increased values of water intake, food intake and24h urine volume compared with HF group and Chow+STZ group (P<0.001, P<0.001).
2. Results of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)
(1) Results of IPGTT
After a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than that at baseline at Omin,30min,60min,120min in IPGTT. The area under the curve of glucose (AUCg) increased significantly at week4than at baseline (P<0.05).
Results of IPGTT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at30min and60min in IPGTT compared with the Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Control groups. The DM group had significantly elevated blood glucose levels at15min and60min in IPGTT compared with Control group (P<0.01); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Chow+STZ groups. The DM group had significantly elevated blood glucose level at15min in IPGTT compared with Chow+STZ group (P<0.05), and also at120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM and Chow+STZ groups in IPGTT increased significantly compared with Control group (P<0.05～0.01).
Results of IPGTT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at60min and120min in IPGTT compared with Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and control group. The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPGTT compared with Control group (P<0.01); The DM group had significantly elevated blood glucose levels at15min and30min in IPGTT compared with Chow+STZ group (P<0.05), and also at60min and120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPGTT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group increased significantly compared with HF group (P<0.05).
(2) Results of IPITT
After a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than at baseline at Omin,15min,30min,60min,120min in IPITT. The area under the curve of glucose (AUCg) in IPITT increased significantly at week4than at baseline (P<0.01).
Results of IPITT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The HF group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The DM group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Chow+STZ group, and between DM group and Chow+STZ group. The DM group had significantly elevated blood glucose levels at Omin,15min,30min and60min in IPITT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of the DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05～P<0.01). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).
Results of IPITT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at15min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Control group; The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and DM group, and between DM group and Chow+STZ group. The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).
3. Serum biochemical parameters analysis:
The serum biochemical parameters were of no statistical difference among each group before the experiment. 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.3. Blood pressures and heart rate:There were no differences in SBP, DBP and MAP among four groups during the experimental process. At the end of the experiment, HR was higher in DM group compared with control and chow+STZ groups, but the difference was not statistically significant.
4. Ultrasonography of abdominal aorta
At the end of the experiment, compared to Control group, the HF group showed no significant differences in ultrasonic measurements; In Chow+STZ group, the Peterson's elastic modulus (Ep) and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the peak systolic velocity (PSV) and cross-sectional distensibility (CD) were deceased (P<0.05～0.001) compared with Control group; In DM group, systolic diameter (Ds), diastolic diameter (Dd), Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the end diastolic velocity (EDV), peak systolic velocity (PSV) and crosssectional distensibility (CD) were decreased (P<0.05～0.001) compared with Control group; In Chow+STZ group, the stiffness index of abdominal aorta was significantly increased compared with HF group (P<0.01); In DM group, the Dd, Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the EDV and PSV were decreased (P<0.05～0.001) compared with HF group; In DM group, the Dd and Ds of abdominal aorta were significantly increased compared with Chow+STZ group (P<0.05～0.001).
At the end of the experiment, in Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with Control group (P<0.01); In Chow+STZ, HF and DM groups, cyclic variation of integrated backscatter (CVIB) of abdominal aorta was significantly decreased compared with Control group (P<0.001); In Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with HF group (P<0.05～0.01); Cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM group was significantly decreased (P<0.01).
6. Pathological characteristics of aorta
(1) HE staining:Compared with Control group, in DM group, the endothelial cells adhering to inner elastic plates showed arrangement disorder, and the vascular smooth muscle cells (VSMCs) of tunica media arranged irregularly, with irregular nucleus and uneven cytoplasm staining. In Chow+STZ, HF, and DM groups, the media thickness of aorta was significantly increased compared with Control group (72.73.09±2.12vs.58.09±0.97μm, P<0.01;62.69±0.67vs.58.09±0.97μm, P<0.05;72.38±2.30vs.58.09±0.97μm, P<0.001). In Chow+STZ and DM groups, the media thickness of aorta was increased compared with HF group (P<0.05). In DM group, the media thickness of aorta was increased compared with Chow+STZ group, but the difference was not statistically significant.
(2) Masson staining:The VSMCs in aorta were stained red, while the collagen fibers were stained bluish green. In control group with mild perivascular fibrosis, collagen fibers were thin, intact and appropriately arranged. While in DM group, the collagen content increased significantly, thick collagen fibers connected into reticular or mass structure arranged irregularly.
Sirius red staining:In control group with mild perivascular fibrosis, the collagen fibers in aorta were thin and uniform under conventional microscope, and the tunica media showed no staining, tunica adventitia was mainly composed of red collagen Ⅰ and green collagen Ⅲ under polarization microscope. While in the DM group, collagen fibers in aorta increased significantly, arranged irregularly and was disrupted markedly under conventional microscope. Under polarization microscope, collagen Ⅰ and collagen Ⅲ in tunica media and tunica adventitia of DM group both increased significantly. Semi-quantitative analysis showed that compared with Control group, the PVCA/LA in Chow+STZ, HF, and DM groups increased significantly (0.06±0.0029vs.0.03±0.0015, P<0.001;0.04±0.0016vs.0.03±0.0015, P<0.05;0.10±0.007vs.0.03±0.0015,P<0.01), the CVF increased significantly (4.42±0.48vs.1.12±0.14, P<0.001;2.45±0.22vs.1.12±0.14, P<0.01;5.70±0.31vs.1.12±0.14, P<0.001); Compared with HF group, the PVCA/LA and collagen percentage per medial area of Chow+STZ and DM groups increased significantly (P<0.01-0.001); Compared with Chow+STZ group, the PVCA/LA of DM group slightly increased (P<0.05); Compared with Chow+STZ group, the collagen percentage per medial area of DM group slightly increased, but the difference was not statistically significant.
(3) Verhoeff elastin staining:In Control group, the VSMCs in aorta were stained yellow or Yellowish red, and the elastin fibers stained black blue were intact and uniform, and arranged appropriately. While in DM group, the VSMCs were stained red, the elastin fibers showed arrangement disorder, sparsity, nonuniformity, and had significant disruptions. Semi-quantitative analysis showed that compared with Control group, the elastin volume fraction of abdominal aorta in Chow+STZ, HF, and DM groups decreased significantly(5.19±0.76vs.11.96±0.75, P<0.001;8.83±0.98vs.11.96±0.75, P<0.05;4.70±0.36vs.11.96±0.75, P<0.001); The elastin percentage per medial area in DM group decreased significantly compared with HF group(P<0.05); Compared with Chow+STZ group, the elastin percentage per medial area in DM group slightly decreased, but the difference was not statistically significant; Compared with Control group, the aortic collagen/elastin ratio in Chow+STZ, HF, and DM groups, increased significantly(0.57±0.09vs.0.05±0.007, P<0.01;0.17±0.0024vs.0.05±0.007, P<0.01;0.71±0.075vs.0.05+0.007, P<0.001); Compared with HF group, the aortic collagen/elastin ratio in Chow+STZ and DM groups increased significantly (P<0.05～0.001); Compared with Chow+STZ group, the aortic collagen/elastin ratio of DM group slightly increased, but the difference was not statistically significant; Compared with Control group, the average aortic wall architecture score of Chow+STZ, HF and DM groups increased significantly (p<0.001); Compared with HF group, the average aortic wall architecture score of Chow+STZ and DM groups increased significantly (p<0.01～0.001); Compared with Chow+STZ group, the average aortic wall architecture score of DM group slightly increased (p<0.05).
(4) Determination of collagen content in rat aorta by Hydroxyproline assay.
Compared with Control group, the collagen content in Chow+STZ, HF and DM groups increased significantly (13.34±0.37vs.7.01±0.21ug/mg dry weight, P<0.001;8.47±0.21vs.7.01±0.21ug/mg dry weight, P<0.01;15.34±0.47vs.7.01±0.21ug/mg dry weight, P<0.001); Compared with HF group, the collagen content in Chow+STZ and DM groups increased significantly (P<0.001); Compared with Chow+STZ group, the collagen content in DM group increased significantly (P<0.001).
(5) Results of Immunohistochemical staining:Compared with Control group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ group markedly increased, the protein expression of collagen Ⅰ and Ⅲ in HF group slightly increased, while the expression of collagen Ⅰ and Ⅲ in DM group increased significantly. Compared with HF group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ and DM groups both increased markedly. Compared with Chow+STZ group, the protein expression of collagen Ⅰ and Ⅲ of DM group increased markedly.
(6) Western blot results of collagen Ⅰ and Ⅲ:Compared with Control group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ group markedly increased (collagen Ⅰ:0.82±0.05vs.0.51±0.05, P<0.001; collagen Ⅲ:0.62±0.03vs.0.31±0.02, P<0.001), the protein expression of collagen Ⅰ and Ⅲ in HF group slightly increased (collagen Ⅰ:0.60±0.03vs.0.51±0.05, P<0.01; collagen Ⅲ:0.47±0.03vs.0.31±0.02, P<0.05), while the expression of collagen Ⅰ and Ⅲ in DM group increased significantly (collagen Ⅰ:1.04±0.06vs.0.51±0.05, P<0.001; collagen Ⅲ:0.95±0.04vs.0.31±0.02, P<0.001). Compared with HF group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ and DM groups both increased markedly (P<0.01-0.001, P<0.001). Compared with Chow+STZ group, the protein expression of collagen Ⅰ and Ⅲ in DM group increased markedly (P<0.05).
(7) Western blot results of TRIB3:Compared with Control group, the protein expression of TRIB3in Chow+STZ group increased in1.33folds (P<0.001), the protein expression of TRIB3in HF group had a increase of2.3372%(P<0.001), the protein expression of TRIB3in DM group increased in1.78folds (P<0.001); Compared with HF group, the protein expression of TRIB3in Chow+STZ group had a increase of35%(P<0.001), the protein expression of TRIB3in DM group had a increase of61%(P<0.001); Compared with Chow+STZ group, the protein expression of TRIB3in DM group had a increase of19%(P<0.001).
(8) Western blot results of the key signal molecules in Akt and MAPK pathway
①The phospho-PI3K/PI3K ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.97,0.67,0.75,0.45. Compared with Control group, the phosphorylation level of PI3K in Chow+STZ, HF and DM groups declined significantly (P<0.001for all); Compared with HF group, the phosphorylation level of PI3K in Chow+STZ and DM groups declined significantly (P<0.001); Compared with Chow+STZ group, the phosphorylation level of PI3K in DM group declined markedly (P<0.01).
②The phospho-Akt/Akt ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.90,0.48,0.61,0.29. Compared with Control group, the phosphorylation level of Akt in Chow+STZ, HF and DM groups declined significantly (P<0.01～P<0.001), and among them the DM group had the Lowest phosphorylation level of Akt, which was the32%of Control group (P<0.001); Compared with HF group, the phosphorylation level of Akt in Chow+STZ and DM groups declined significantly, and between the two groups the DM group had Lower phosphorylation level of Akt, which was the47%of Control group (P<0.001); Compared with Chow+STZ group, the phosphorylation level of Akt in DM group declined slightly, but the difference was not statistically significant.
③The phospho-MKK4/MKK4ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.59,0.85,0.64,1.03. Compared with Control group, the phosphorylation level of MKK4in Chow+STZ, HF and DM groups increased significantly (P<0.05～P<0.001), and among them the DM group had the highest phosphorylation level of MKK4, which was1.86times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of MKK4in Chow+STZ and DM groups increased significantly (P<0.001for both); Compared with Chow+STZ group, the phosphorylation level of MKK4in DM group increased markedly (P<0.01).
④The phospho-JNK/JNK ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.45,0.78,0.60,0.97. Compared with Control group, the phosphorylation level of JNK in Chow+STZ, HF and DM groups increased significantly (P<0.01～P<0.001), and among them the DM group had the highest phosphorylation level of JNK, which was2.15times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of JNK in DM group increased significantly (P<0.001), while the phosphorylation level of JNK in Chow+STZ group slightly increased, but the difference was not statistically significant; Compared with Chow+STZ group, the phosphorylation level of JNK in DM group increased slightly, but the difference was not statistically significant.
7. The mRNA and protein expression of TRIB3after TRIB3gene silencing
The mRNA and protein expression of TRIB3in aorta:The results of RT-PCR showed that Compared with DM-Vector group, the mRNA expression of aortic TRIB3in DM-siRNA group declined by66%(P<0.001). The results of Western blot showed that compared with DM-Vector group, the protein expression of aortic TRIB3in DM-siRNA group declined by67%(P<0.001).
8. TRIB3gene silencing ameliorated the general characteristics of type2diabetic rat
Compared with DM-Vector group, the DM-siRNA group rats had obviously lower values of water intake (P<0.05) and24h urine volume (P<0.05) after TRIB3gene silencing. The DM-siRNA group rats had lower values of food take compared with DM-Vector group, but the difference was not statistically significant. The body weight, Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) of DM-siRNA group had no statistical difference compared with DM-Vector group after TRIB3gene silencing.
9. The effects of TRIB3gene silencing on IPGTT and IPITT
(1) Results of IPGTT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at30min and120min in IPGTT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.041) in IPGTT after TRIB3gene silencing.
(2) Results of IPITT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at Omin and120min in IPITT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.030) in IPITT after TRIB3gene silencing.
10. The effects of TRIB3gene silencing on serum biochemical parameters
4weeks after TRIB3gene silencing, serum TG level of DM-siRNA group was markedly decreased compared with DM-Vector group (4.98±1.62vs2.57±0.62mmol/L, P<0.05). Serum TC level of DM-siRNA group was markedly decreased compared with DM-Vector group (3.96±1.01vs.7.74±2.15mmol/L, P<0.05). Serum FFD level of DM-siRNA group was markedly decreased compared with DM-Vector group (355.85±26.24vs.464.69±31.23μmol/L, P<0.05). The fasting blood-glucose level of DM-siRNA group was remarkably decreased compared with DM-Vector group (15.63±2.04vs.24.58±3.04mmol/L, P<0.05). The ISI of DM-siRNA group was markedly increased compared with DM-Vector group (-5.17±0.14vs.-5.67±0.14, P<0.05). The fasting insulin level of DM-siRNA had no statistical difference compared with DM-Vector group.
11. The effects of TRIB3gene silencing on ultrasonography of abdominal aorta
Four weeks after TRIB3gene silencing, The Dd of abdominal aorta in DM-siRNA group was markedly decreased compared with DM-Vector group (P<0.05); The EDV and PSV of DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The Peterson's elastic modulus (Ep) and crosssectional distensibility (CD) of abdominal aorta in DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The stiffness index of abdominal aorta in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.001);
4weeks after TRIB3gene silencing, IBS%of abdominal aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (1.76±0.14vs1.42±0.15, P<0.001); The cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM-siRNA group was significantly increased compared with DM-Vector group (4.17±0.81vs5.21±0.57, P<0.01).
12. TRIB3gene silencing ameliorated diabetic arterial remodeling
(1) HE staining:In DM-siRNA group there were only some of the endothelial cells of aorta showed disruption and structure abnormity compared with DM-Vector group. The endothelial cells in DM-siRNA group arranged more regularly, and had a looser adhesion to inner elastic plates compared with DM-Vector group. The VSMCs of abdominal aorta in DM-siRNA group with regular nucleus and even cytoplasm staining were fusiform shaped and arranged more irregularly compared with DM-Vector group. Results showed that the media thickness of aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (74.88±1.21μm vs.69.38±1.11μm,P<0.01).
(2) Masson staining:The collagen content of aorta in DM-siRNA group was decreased significantly, and the collagen fibers were thinner, arranged more regularly and distributed more evenly around the vascular smooth muscle cells compared with DM-Vector group. Sirius red staining:The collagen fibers of aorta in DM-siRNA group decreased significantly arranged more irregularly and showed less disruption under conventional microscope compared with DM-Vector group. The yellowish red collagen Ⅰ and the yellowish green collagen Ⅲ in tunica media and tunica adventitia of aorta in DM-siRNA group decreased significantly, and had less disruption under polarization microscope compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the PVCA/LA of aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.10±0.005vs.0.06±0.006,P<0.001). The aortic collagen percentage per medial area in DM-siRNA group was decreased markedly compared with DM-Vector group (3.77±0.36vs.2.48±0.22,P<0.01).
(3) Verhoeff elastin staining:The elastin fibers of aorta in DM-siRNA group arranged more regularly and had less disruptions compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the aortic elastin percentage per medial area in DM-siRNA group was increased significantly(5.29±0.36vs.7.79±0.72, P<0.01); The aortic collagen/elastin ratio of abdominal aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.75±0.09vs.0.36±0.07, P<0.01); The average aortic wall architecture score in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.05).
(4) Determination of collagen content in rat aorta by Hydroxyproline assay: Compared with DM-Vector group, the collagen content of aorta in DM-siRNA group was decr
糖尿病的发病率逐年增高,在发达国家已被列为继心血管疾病及肿瘤之后的第三大疾病。研究结果显示,糖尿病患者的医疗服务使用是非糖尿病患者的3至4倍(包括门诊和住院次数)；糖尿病患者的医疗支出是同年龄、同性别无糖尿病患者的9倍。另一项研究显示,我国糖尿病患者医疗花费中,只有不到20%是用于控制血糖,超过80%的费用是治疗糖尿病引起的并发症。目前糖尿病对人类健康危害最大的是在动脉硬化及微血管病变基础上产生的多种慢性并发症。糖尿病患者的血管病变发病率是非糖尿病患的2至4倍。现有研究证实,糖尿病血管病变的基本病理基础为动脉粥样硬化及血管平滑肌细胞和成纤维细胞大量增殖所致的动脉中膜、外膜不规则增厚、纤维化和钙化。UKPDS研究证实,严格控制血糖可以明显降低糖尿病患者微血管病变的发生率。由此,奠定了糖尿病血管并发症的防治基础。然而,2008年相继揭晓的三项重要强化降糖试验(ACCORD.ADVANCE及VADT)显示,积极控制血糖可以降低糖尿病患者微血管病变的发生率,但却未能证实强化降糖治疗可降低2型糖尿病患者大血管事件发生率。因此,亟需深入探索糖尿病患者心血管并发症风险增高的深层机制,奠定糖尿病患者心血管事件源头性防治的理论基础。
胰岛素抵抗贯穿于糖尿病的整个病程。UKPDS资料表明,在血糖未明显升高以前,单胰岛素抵抗就可引起大血管病变。胰岛素抵抗是糖尿病代谢性异常和心血管疾病的原始动因和致病基础。胰岛素抵抗的选择性使血管平滑肌和成纤维细胞增殖增强。“选择性胰岛素抵抗”的研究指出,在胰岛素抵抗状态下,MAPK增殖通路活性明显增强,而Akt代谢通路显著受损。TRIB3作为Akt和MAPK共同的调节蛋白,与“选择性胰岛素抵抗”密切相关。因此,我们推测TRIB3不仅通过Akt信号途径引起胰岛素抵抗和参与糖尿病发病,并可能通过调控Akt及MAPK信号途径而影响糖尿病大血管病变的发生和进展。因此,本课题将建立2型糖尿病大血管病变大鼠模型,采用RNAi技术,构建能够在哺乳动物细胞中表达siRNA的腺病毒表达载体,诱导TRIB3基因沉默,研究TRIB3在糖尿病大血管病变发生发展中的作用,综合运用各种病理学组织学染色技术研究TRIB3基因沉默后大血管形态结构的变化。通过分析上述实验结果,探讨TRIB3在糖尿病大血管病变中的作用及其可能机制。这些结果将为糖尿病心血管并发症患者的早期防治提供新靶点,具有重要的学术理论意义和潜在的临床应用价值。
研究目的
1.探讨高脂饮食联合小剂量链脲佐菌素建立2型糖尿病大血管病变大鼠动物模型的可行性；
2.2型糖尿病大血管病变大鼠体内转染TRIB3-shRNA腺病毒,观察糖尿病大血管病变改善情况；
3.在细胞分子水平、组织学水平和整体功能水平研究TRIB3基因沉默改善2型糖尿病大血管病变的机制；研究方法
1.5周龄健康雄性SD (Sprague-Dawley)大鼠60只,行腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT)后随机分为4组：对照组(Control组)、普通饮食STZ组(Chow+STZ组)、高脂高糖饮食组(HF组)、DM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,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,留取静脉血,测定空腹血糖(FBG)和空腹胰岛素(FINS),计算胰岛素敏感指数(ISI, ISI=Ln(FINS×FBG)-1)。连续两次空腹血糖≥11.1mmol/L,胰岛素敏感指数降低且有多尿、多饮、多食现象的大鼠作为成模大鼠纳入实验；
2.5周龄健康雄性SD (Sprague-Dawley)大鼠30只,糖尿病造模过程同上,造模成功后随机分为DM-Vector组和DM-siRNA组。糖尿病成模12周后,DM-siRNA组大鼠经颈静脉注射pAdxsi-TRIB3-shRNA腺病毒2.5×1010PFU/只,DM-Vector组注射pAdxsi空病毒载体2.5×1010PFU/只,2周后补充注射一次,剂量同第一次,腺病毒注射4周后处死动物留取标本。
3.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(禁食不禁水),断尾取血测定空腹血糖,继而给予腹腔注射20%葡萄糖(1g葡萄糖/kg体重),于注射葡萄糖后15min、30min、60min、120min按时从尾静脉取血测定血糖水平。采用强生稳步血糖仪和试纸条测定血糖。通过IPGTT结果计算血糖曲线下面积(AUC of glucose, AUCg).大鼠禁食4h后(禁食不禁水)行IPITT,给予腹腔注射胰岛素(1U胰岛素/kg体重),余同IPGTT；
4.血液生化指标的测定：检测血清总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)的水平,测定血糖和血清胰岛素,计算胰岛素敏感指数；
5.血流动力学监测：所有实验动物通过大鼠尾动脉血压测量仪监测血压和心率；
6.腹主动脉超声：采用二维、脉冲多普勒和背向散射积分等超声技术,连续观察2型糖尿病大血管病变发生和发展过程中腹主动脉结构和功能的变化；
7.病理学检测：对大鼠主动脉组织分别进行HE染色、Masson染色、天狼猩红染色以及Verhoeff染色,应用图象分析仪测定主动脉中层厚度,主动脉血管周围胶原面积/管腔面积,主动脉中膜胶原比例,主动脉中膜弹力纤维比例,主动脉胶原与弹力纤维的比值,计算平均主动脉壁结构积分；
8.免疫组织化学染色：应用免疫组织化学染色法,光镜下观察大鼠主动脉间质的Ⅰ型胶原、Ⅱ工型胶原分布情况；
9.大鼠主动脉羟脯氨酸含量测定：将干燥至恒重的大鼠主动脉组织称重后置于6mol/L HCl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主动脉干重)；
10.实时定量RT-PCR检测：取新鲜大鼠主动脉组织,Trizol法提取RNA,实时荧光定量RT-PCR技术检测TRIB3mRNA相对表达量；
11Western blot检测：取新鲜大鼠主动脉组织,提取蛋白,Western blot检测主动脉组织内TRIB3、胶原Ⅰ、胶原Ⅲ、磷酸化及非磷酸化的MKK4、JNK、PI3K、Akt的蛋白表达量。
结果
1、实验末各组大鼠的一般状况比较：与Control组大鼠相比,Chow+STZ组大鼠日饮水量、日摄食量及24h尿量均明显增加(P<0.05)；与Control组大鼠相比,HF组大鼠体重明显增加,日饮水量、日摄食量及24h尿量均有所增加(P<0.05)；与Control组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均显著增加(P<0.01)。与HF组大鼠相比,Chow+STZ组大鼠日饮水量、24h尿量明显增多(P<0.05～0.001)；与HF组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均显著增加(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠日饮水量、日摄食量及24h尿量均明显增加(P<0.001)。
2、实验期间各组大鼠腹腔葡萄糖耐量试验(IPGTT)及腹腔胰岛素耐量试验(IPITT)结果分析：
(1)IPGTT结果分析：
DM组大鼠高脂高糖饮食4周后与高脂高糖饮食前IPGTT结果显示：与高脂高糖饮食前相比,DM组大鼠高脂高糖饮食4周后在IPGTT试验Omin、30min、60min、120min的血糖值均明显升高,差异具有统计学意义(P均<0.05)；血糖曲线下面积(AUCg)明显增大(P
(2)IPITT结果分析：
DM组大鼠高脂高糖饮食4周后与高脂高糖饮食前IPITT结果显示：与高脂高糖饮食前相比,DM组大鼠高脂高糖饮食4周后在IPITT试验Omin、15min、30min、60min、120min的血糖值均显著升高(P<0.05)；血糖曲线下面积明显增大(P<0.01)。
2型糖尿病糖尿病成模后12周各组大鼠IPITT结果显示：与Control组大鼠相比,Chow+STZ组大鼠IPITT试验15min、30min、60min及120min的血糖值明显升高(P<0.05～P<0.01)；与Control组大鼠相比,HF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Control组大鼠相比,DM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Chow+STZ组相比,HF组大鼠在IPITT试验各时间点血糖值差异无统计学意义；与Chow+STZ组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与HF组相比,DM组大鼠在IPITT试验Omin、15min、30min、60min的血糖值均显著升高(P<0.05)。与Control组相比,Chow+STZ组、HF组及DM组的血糖曲线下面积均明显增大(P<0.05～P<0.01)；与HF组大鼠相比,DM组血糖曲线下面积明显增大(P<0.05)。
实验末IPITT试验结果显示：与Contro1组大鼠相比,Chow+STZ组大鼠IPITT试验15min的血糖值明显升高(P<0.05)；与Control组大鼠相比,HF组大鼠在IPITT试验各时间点血糖值差异无统计学意义；与Control组大鼠相比,DM组大鼠在IPITT试验15min、60min、120min的血糖值均明显升高(P<0.05)。与Chow+STZ组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与HF组相比,DM组大鼠在IPITT试验各时间点血糖值差异无统计学意义。与Control组相比,Chow+STZ组、HF组及DM组的血糖曲线下面积均明显增大(P<0.05)。与HF组大鼠相比,DM组血糖曲线下面积明显增大(P<0.05)。
3、生化指标的测定：实验前各组大鼠生化指标无明显差异；高脂饮食4周后,与Control相比,HF组及DM组血清TG、FFA及FINS均明显升高(P<0.05),ISI明显降低(P<0.05),FBG无显著变化,提示注射STZ前DM组大鼠已存在胰岛素抵抗；STZ注射后一周,与Control相比,Chow+STZ组及DM组FBG明显升高(P<0.05),ISI显著降低(P<0.05),HF组及DM组血清TG、FFA明显升高(P<0.05),FINS无显著差异；糖尿病成模后12周,与Control相比,DM组FBG明显升高(P<0.05),ISI显著降低(P<0.05),DM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14周,与Control相比,DM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Control相比,DM组血清TC、TG、FFA及FBG均显著升高(P<0.05),ISI明显降低(P<0.05)。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。
4、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Control组大鼠及Chow+STZ组大鼠相比,DM组大鼠心率略有增加,但是差异没有统计学意义。
5、腹主动脉超声监测
实验结束时,与Control组大鼠比较,HF组大鼠各项指标变化均无统计学意义；与Control组大鼠相比,Chow+STZ组大鼠腹主动脉Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～0.001),舒张末期流速、横断面扩张系数均降低,差异具有统计学意义(P<0.05～0.001,)；与Control组大鼠相比,DM组大鼠腹主动脉收缩期内径、舒张期内径、Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～P<0.001),收缩期峰值流速、舒张末期流速、横断面扩张系数均降低,差异具有统计学意义(P<0.05～0.001)。与HF组大鼠相比,Chow+STZ组大鼠腹主动脉僵硬度指数明显增加,差异具有统计学意义(P<0.01)；与HF组大鼠相比,DM组大鼠腹主动脉舒张期内径、Peterson弹性模量、僵硬度指数均明显增加,差异具有统计学意义(P<0.05～P<0.001),收缩期峰值流速、舒张末期流速均降低,差异具有统计学意义(P<0.05～P<0.001)。与Chow+STZ组大鼠相比,DM组大鼠腹主动脉收缩期内径和舒张期内径明显增加,差异具有统计学意义(P<0.05～0.001)。余各组大鼠指标比较,差异均无统计学意义。
实验结束时,超声背向散射积分结果显示,与Control组大鼠比较,Chow+STZ组和DM组大鼠腹主动脉校正平均背向散射积分(IBS%)均明显增加(P<0.01)；与Control组大鼠比较,Chow+STZ组、HF组和DM组大鼠腹主动脉背向散射积分周期变化幅度(CVIB)均明显降低(P<0.001)；与HF组比较,Chow+STZ组和DM组大鼠腹主动脉IBS%明显增加(P<0.05～0.01),DM组大鼠腹主动脉CVIB明显降低(P<0.01)。
6、主动脉病理学分析
(1)主动脉HE染色,与Control组大鼠相比,DM组大鼠内皮细胞排列紊乱、致密,仍紧贴于内弹力板上。血管平滑肌细胞肥大、扭曲、排列紊乱,层数增多,细胞核大小不一,细胞膜及核膜欠清晰、完整,胞浆染色不均,可见大量肌纤维断裂。定量分析结果显示：与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉中膜厚度明显增加,差异具有统计学意义(72.73.09±2.12vs.58.09±0.97μ m,P<0.01；62.69±0.67vs.58.09±0.97μ m,P<0.05；72.38±2.30vs.58.09±0.97μ m,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉中膜厚度略有增加,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜厚度略有增加,差异无统计学意义。
(2)Masson染色显示,大鼠主动脉血管平滑肌细胞及弹力纤维呈红色,间质胶原纤维呈蓝绿色。Control组大鼠主动脉胶原纤维分布均匀、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；与Control组相比,DM组大鼠主动脉胶原组织明显增多,排列紊乱,分布不匀,血管周围纤维化明显。
天狼猩红染色：在普通显微镜下,Control组大鼠主动脉胶原纤维均匀纤细,血管周围有极少量胶原；偏振光显微镜下,Control组大鼠主动脉中膜未见明显显色,外膜以Ⅰ型及Ⅲ型胶原纤维为主。在普通显微镜下,DM组大鼠主动脉间质胶原纤维明显增多,迂曲,排列紊乱,断裂明显增多；偏振光显微镜下,DM组大鼠主动脉中膜和外膜Ⅰ型及Ⅲ型胶原明显增加,胶原纤维断裂明显。半定量分析结果显示,与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉管周胶原面积/管腔面积(PVCA/LA)均有明显增加,差异具有统计学意义(0.06±0.0029vs.0.03±0.0015,P<0.001；0.04±0.0016vs.0.03±0.0015,P<0.05；0.10±0.007vs.0.03±0.0015,P<0.01),中膜胶原比例均有明显增加,差异具有统计学意义(4.42±0.48vs.1.12±0.14,P<0.001；2.45±0.22vs.1.12±0.14,P<0.01；5.70±0.31vs.1.12±0.14,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉中膜胶原比例及管周胶原面积/管腔面积(PVCA/LA)均有明显增加,差异具有统计学意义(P<0.01～0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉管周胶原面积/管腔面积(PVCA/LA)有所增加,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜胶原比例有所增加,但差异没有统计学意义。
(3)Verhoeff弹力纤维染色显示：Control组大鼠主动脉弹力纤维分布均匀、整齐,完整无断裂；DM组大鼠主动脉弹力纤维迂曲,排列紊乱、疏松,分布欠均匀,可见明显断裂。半定量分析结果显示,与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉中膜弹力纤维比例均明显降低,差异具有统计学意义(5.19±0.76vs.11.96±0.75,P<0.001；8.83±0.98vs.11.96±0.75,P<0.05；4.70±0.36vs.11.96±0.75,P<0.001)；与HF组大鼠相比,DM组大鼠主动脉中膜弹力纤维比例明显降低,差异具有统计学意义(P<0.05)；与Chow+STZ组大鼠相比,DM组大鼠主动脉中膜弹力纤维比例略有降低,差异没有统计学意义。与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉胶原/弹力纤维比值均明显增加,差异具有统计学意义(0.57±0.09vs.0.05±0.007,P<0.01；0.17±0.0024vs.0.05±0.007,P<0.01；0.71±0.075vs.0.05±0.007,P<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉胶原/弹力纤维比值均明显增加,差异具有统计学意义(P<0.05～0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉胶原/弹力纤维比值略有增加,差异没有统计学意义。与Control组大鼠相比,Chow+STZ组、HF组、DM组大鼠主动脉平均主动脉壁结构积分均明显增加,差异具有统计学意义(P均<0.001)；与HF组大鼠相比,Chow+STZ组、DM组大鼠主动脉平均主动脉壁结构积分均明显增加,差异具有统计学意义(P<0.01、0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉平均主动脉壁结构积分略有增加,差异具有统计学意义(P<0.05)。
(4)采用羟脯氨酸法测定大鼠主动脉胶原含量。结果显示：与Control组相比,Chow+STZ组、HF组、DM组大鼠主动脉胶原含量均明显增加,差异具有统计学意义(13.34±0.37vs.7.01±0.21ug/mg血管干重,P<0.001；8.47±0.21vs.7.01±0.21ug/mg血管干重,P<0.01；15.34±0.47vs7.01±0.21ug/mg血管干重,P<0.001)；与HF组大鼠相比,Chow+STZ组和DM组大鼠主动脉胶原含量明显增加,差异具有统计学意义(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠主动脉组织胶原含量明显增加,差异具有统计学意义(P<0.001)。
(5)免疫组化结果显示：与Control组相比,Chow+STZ组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达明显增多,HF组大鼠主动脉内collagen Ⅰ、collagen Ⅲ表达有所增加,DM组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达显著增多；与HF组相比,Chow+STZ组、DM组大鼠主动脉内collagen Ⅰ、collagen Ⅲ阳性表达均明显增多；与Chow+STZ组相比,DM组大鼠主动脉collagenⅠ、collagen Ⅲ阳性表达均明显增多。
(6)Collagen Ⅰ、collagen Ⅲ的Western blot结果显示：与Control组相比,Chow+STZ组大鼠主动脉collagen Ⅰ、collagen Ⅲ蛋白表达明显增加,差异具有统计学意义(collagen Ⅰ:0.82±0.05vs.0.51±0.05,P<0.001：collagen Ⅲ:0.62±0.03vs.0.31±0.02,P<0.001),HF组主动脉collagen Ⅰ、collagen Ⅲ蛋白含量略有增加,差异具有统计学意义(collagen Ⅰ:0.60±0.03vs.0.51±0.05,P<0.01；collagen Ⅲ：0.47±0.03vs.0.31±0.02,P
(8)Western blot检测Akt和MAPK通路各关键信号分子
①Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化P13K与总P13K比值分别为：0.97,0.67,0.75,0.45。与Control组大鼠相比,Chow+STZ组、HF组及DM组大鼠主动脉P13K磷酸化水平均明显降低,差异具有统计学意义(P均<0.001)；与HF组大鼠相比较,Chow+STZ组及DM组大鼠主动脉P13K磷酸化水平均明显降低(P<0.001)；与Chow+STZ组大鼠相比,DM组大鼠P13K磷酸化水平明显降低(P<0.01)
②Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化Akt与总Akt的比值分别为：0.90,0.48,0.61,0.29。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉Akt的磷酸化水平均明显降低,差异具有统计学意义(P<0.01～P<0.001),其中DM组大鼠主动脉Akt的磷酸化水平最低,为Control组的32%(P<0.001)；与HF组相比,Chow+STZ组及DM组大鼠主动脉Akt的磷酸化水平均有下降,以DM组降低最为显著,为HF组的47%(P<0.001)；与Chow+STZ组相比,DM组大鼠主动脉Akt的磷酸化水平有所下降,但差异无统计学意义。
③Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化MKK4与总MKK4比值分别为：0.59,0.85,0.64,1.03。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉MKK4磷酸化水平均明显升高,差异具有统计学意义(P<0.05～0.001),其中DM组大鼠主动脉MKK4的磷酸化水平最高,为Control组的1.86倍(P<0.001)；与HF组比较,Chow+STZ组、DM组大鼠主动脉MKK4磷酸化水平明显升高,差异具有统计学意义(P均<0.001)；与Chow+STZ组相比,DM组大鼠主动脉MKK4磷酸化水平明显升高,差异具有统计学意义(P<0.01)
④Control组、Chow+STZ组、HF组及DM组大鼠主动脉磷酸化JNK与总JNK比值分别为：0.45,0.78,0.60,0.97。与Control组相比,Chow+STZ组、HF组及DM组大鼠主动脉JNK磷酸化水平均明显升高,差异具有统计学意义(P<0.01、0.001),其中DM组大鼠主动脉JNK的磷酸化水平最高,为Control组的2.15倍(P<0.001)；与HF组比较,DM组大鼠主动脉JNK磷酸化水平明显升高,差异具有统计学意义(P<0.001),Chow+STZ组大鼠主动脉JNK磷酸化水平略有升高,差异没有统计学意义；与Chow+STZ组相比,DM组大鼠主动脉JNK磷酸化水平略有升高,差异没有统计学意义。
7、TRIB3基因沉默后主动脉TRIB3mRNA及蛋白的表达
主动脉TRIB3mRNA及蛋白的表达：RT-PCR结果显示：与糖尿病空载体(DM-Vector)组相比,糖尿病siRNA (DM-siRNA)组大鼠主动脉TRIB3mRNA表达降低了66%,差异具有统计学意义(P
经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠日饮水量显著减少,差异具有统计学意义(P<0.05),同时DM-siRNA组大鼠24h尿量也显著减少,差异具有统计学意义(P<0.05)。DM-siRNA组大鼠日摄食量较DM-Vector组均有所降低,但未达到统计学意义。DM-siRNA组大鼠体重、收缩压、舒张压、平均动脉压及心率较DM-Vector组无变化。
9、TRIB3基因沉默对IPGTT及IPITT的改变
(1)IPGTT结果显示：经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠在IPGTT试验30min、120min的血糖值均明显降低(Ｐ均<0.05),血糖曲线下面积明显减少(P=0.041),差异具有统计学意义。
(2)IPITT结果显示：经TRIB3腺病毒干预后,与DM-Vector组大鼠相比,DM-siRNA组大鼠在IPITT试验0min、120min的血糖值均明显降低(P均<0.05),血糖曲线下面积明显减少(P=0.030),差异具有统计学意义。
10、TRIB3基因沉默对血清生化指标的影响
经TRIB3腺病毒干预4周后,与DM-Vector组大鼠相比,DM-siRNA组大鼠血清总胆固醇明显降低,差异具有统计学意义(4.98±1.62vs2.57±0.62mmol/L,P<0.05)；DM-siRNA组大鼠血清甘油三酯明显降低,差异具有统计学意义(3.96±1.01vs.7.74±2.15mmol/L;P<0.05)；DM-siRNA组大鼠血清游离脂肪酸明显降低,差异具有统计学意义(355.85±26.24vs.464.69±31.23μmol/L,P<0.05)；DM-siRNA组大鼠血清空腹血糖明显降低,差异具有统计学意义(15.63±2.04vs.24.58±3.04mmol/L,P<0.05)；DM-siRNA组大鼠胰岛素敏感指数明显升高,差异具有统计学意义(-5.17±0.14vs.-5.67±0.14,P<0.05)；DM-siRNA组大鼠空腹胰岛素水平较空载体组略有降低,但差别没有统计学意义。
11、TRIB3基因沉默对腹主动脉超声指标的影响
经TRIB3腺病毒干预4周后,与DM-Vector组大鼠相比,DM-siRNA组大鼠腹主动脉舒张期内径明显减小,差异具有统计学意义(P<0.05)；DM-siRNA组大鼠收缩期峰值流速和舒张末期流速均明显增加,差异具有统计学意义(P
12、TRIB3基因沉默改善糖尿病大鼠主动脉重构
(1)主动脉HE染色可见,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉仅有部分内皮细胞部分突起、断裂,结构不完整,排列趋于整齐。血管平滑肌细胞呈长梭形,平行排列,层数减少,细胞核大小基本均一,细胞膜及核膜清晰、完整,胞浆染色均匀,肌纤维断裂明显减少。分析结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜厚度明显减少(74.88±1.21μ m vs.69.38±1.11μ m,P<0.01),差异具有统计学意义；
(2)Masson染色显示,与DMVector组大鼠相比,DM-siRNA组大鼠主动脉胶原纤维明显减少,胶原纤维变细,排列区域规整,分布基本均匀。天狼猩红染色,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉间质胶原纤维明显减少,排列较为整齐,断裂明显减少；偏振光显微镜下,DM-siRNA组大鼠主动脉中膜和外膜Ⅰ型及Ⅲ型胶原纤维均明显减少,胶原纤维断裂减少。半定量分析结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉血管周围胶原面积/管腔面积(PVCA/LA)明显减少,差异具有统计学意义(0.10±0.005vs.0.06±0.006,P<0.001)；与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜胶原比例明显减少,差异具有统计学意义(3.77±0.36vs.2.48±0.22,P<0.01)
(3)Verhoeff弹力纤维染色染色显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉弹力纤维趋于均匀,整齐,断裂明显减少。半定量分析结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉中膜弹力纤维比例均明显增加,差异具有统计学意义(5.29±0.36vs.7.79±0.72,P<0.01)；DM-siRNA组大鼠主动脉胶原/弹力纤维比值明显降低,差异具有统计学意义(0.75±0.09vs.0.36±0.07,P<0.01)；DM-siRNA组大鼠主动脉平均主动脉壁结构积分有所降低,差异具有统计学意义(P<0.05)
(4)采用羟脯氨酸法测定大鼠主动脉胶原含量,结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉胶原含量明显减少,差异具有统计学意义(14.16±0.30vs.10.70±0.19μ g/mg血管干重,P<0.001)
(5)TRIB3基因沉默降低了主动脉Ⅰ、Ⅲ型胶原的表达
免疫组化结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagen Ⅰ、collagen Ⅲ阳性表达均有所减少。Western blot结果显示：与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagenⅠ、collagen Ⅲ蛋白表达有所降低。定量分析Western blot结果显示,与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉collagen Ⅰ、 collagen Ⅲ蛋白表达明显降低,差异具有统计学意义(P<0.001),其中collagen Ⅰ蛋白表达降至DM-Vector组大鼠的61%(P<0.01),collagen Ⅲ蛋白表达下降至DM-Vector组大鼠的59%(P<0.01)。
(6)TRIB3基因沉默后主动脉PI3K/Akt及MKK4/JNK通路各关键分子的表达
①与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉PI3K的磷酸化水平明显升高,其蛋白表达为DM-Vector组大鼠的1.79倍(P<0.001)。
②与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉Akt的磷酸化水平明显升高,其蛋白表达为DM-Vector组大鼠的1.94倍(P<0.001)。
③与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉MKK4的磷酸化水平明显降低,其蛋白表达降为DM-Vector组大鼠的37%(P<0.001)
④与DM-Vector组大鼠相比,DM-siRNA组大鼠主动脉JNK的磷酸化水平明显降低,其蛋白表达下降至DM-Vector组大鼠的66%(P<0.001)
结论
(1)高糖高脂高热量饮食喂养联合小剂量链脲佐菌素诱发的2型糖尿病状态稳定,符合2型糖尿病的临床特点,能够出现糖尿病大血管病变,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；
(2)2型糖尿病大鼠主动脉重构以血管硬化为主,主要表现为血管壁内胶原含量明显增加,主动脉中膜厚度增加,主动脉内径增加,主动脉僵硬度指数明显增加；
(3)2型糖尿病状态下,TRIB3表达上调,进而激活MMK4及JNK信号传导途径引起Ⅰ、Ⅲ型胶原合成明显增加,最终导致血管间质纤维化,是2型糖尿病大血管病变发生的重要分子机制；
(4)2型糖尿病大鼠发生主动脉重构的同时,主动脉组织TRIB3表达明显升高,PI3K/Akt信号通路活性减弱,而MAPK信号通路明显增强,提示选择性胰岛素抵抗的信号通路参与糖尿病大血管病变的发病；
(5)TRIB3基因沉默能够通过减少主动脉内胶原含量,降低主动脉中膜厚度,调整主动脉内胶原/弹性纤维比值,从而减轻主动脉硬化,延缓了主动脉重构的进展；
(6)TRIB3基因沉默延缓2型糖尿病大鼠主动脉重构的同时,主动脉组织TRIB3表达明显降低,PI3K/Akt信号通路活性随之增加,而MAPK信号通路明显降低,进一步证实以TRIB3为核心的选择性胰岛素抵抗参与了糖尿病大血管病变的发生和发展；
(7)TRIB3基因沉默能够改善2型糖尿病大鼠胰岛素抵抗和糖脂代谢异常,有效地减少了糖尿病大血管病变发生的危险因素；
(8)TRIB3基因沉默主要通过增加主动脉组织内Akt的磷酸化水平,阻断主动脉组织内MMK4及JNK的活化,尤其是通过对MMK4的抑制,从分子水平有效改善选择性胰岛素抵抗,纠正代谢性信号通路与增殖性信号通路的失衡,延缓了糖尿病主动脉重构的进展,改善了大血管的功能。
Background
Diabetes mellitus, the incidence of which increasing year by year, has been listed as the third mighty disease following cardiovascular disease and tumor in the Developed Countries. A research result shows that the times of clinical service and hospitalization in diabetic patients increased obviously, and the use of medical services of diabetic patients was two to four times that of non-diabetic patients, the medical costs of diabetic populations were nine times that of non-diabetic populations with the same age and sex. Another research indicates that for diabetic patients in china less than20%of the diabetic medical expenses were spent in blood-glucose control, and more than80%of that spent in the treatment of diabetic complications. The serious harm of diabetes mellitus to human health is the chronic diabetic complications developed from arteriosclerosis and microangiopathy. The incidence of angiopathy in diabetic patients is three to four times that of non-diabetics patients. It has been suggested that the fundamental pathological characteristics of diabetic angiopathy were atherosclerosis and the anomaly thickening, fibrosis and calcification of tunica media and tunica adventitia. The UKPDS shows conclusively that the incidence of diabetic microangiopathy could be obviously reduced by good glycemia control. However, the three important trials in intensive glucose lowering (ACCORD, ADVANCE and VADT) published in2008show that intensive blood-glucose control could reduce the rate of diabetic microangiopathy, but did not reduce the rate of major CVD events in patients with type2diabetes. So it is desiderated to further explore the mechanisms of the high rate of cardiovascular diseases in patients with type2diabetes, in order to find the theoretical basis of upstream treatment strategies for cardiovascular complications in patients with type2diabetes.
Insulin resistance runs through the whole course of type2diabetes. The UKPDS shows that insulin resistance could cause macroangiopathy before the onset of hyperglycemia. Insulin resistance is the primitive cause and pathogenic basis of metabolic abnormality and cardiovascular complications in type2diabetes. Selective insulin resistance could enhance the proliferation of vascular smooth muscle cell and fibroblasts. Studies on selective insulin resistance indicate that in insulin-resistant states, the metabolic Akt pathway of insulin effect is impaired obviously, while the proliferative MAPK pathway is enhanced. TRIB3, served as the common regulatory protein of the Akt and MAPK pathway, is closely related to selective insulin resistance. So we hypothesize that TRIB3not only participates in pathogenesis of type2diabetes by inducing insulin resistance through the metabolic Akt pathway, but also participates in the initiation and progression of diabetic macroangiopathy through the proliferative MAPK pathway. So in this study, we aim to establish the type2diabetic model, determine the effects of TRIB3on the initiation and progression of diabetic macroangiopathy through inducing in vivo TRIB3gene silencing by RNAi technique, and investigate the morph metric and structural changes of great vessels after TRIB3gene silencing by various pathological and histological staining techniques. We aim to explore the effects of TRIB3on diabetic macroangiopathy and its possible mechanisms. The results will provide potential new targets for the early treatment of cardiovascular complications in type2diabetes, and have important academic theoretical significance and potential clinical application perspective.
Objectives
1.To confirm the feasibility of establishing diabetic macroangiopathy rat model induced by a high fat and sugar diet combined with a small dose of STZ;
2.To investigate the improvement of diabetic macroangiopathy by transfection of pAdxsi-TRIB3-shRNA into type2diabetic rat;
3. To explore the mechanism by which TRIB3silencing would ameliorate diabetic macroangiopathy in molecular, histological and functional levels.
Methods
1. Sixty5-week-old male SD (Sprague-Dawley) rats were randomized into Four groups after intraperitoneal glucose tolerance test(IPGTT) and intraperitoneal insulin tolerance test (IPITT):Control, chow+streptozotocin (Chow+STZ), high-fat and sugar diet (HF) and diabetes mellitus (DM).Control and Chow+STZ groups rats were fed on normal chow (20%crude protein,3%crude fat,3%crude fiber, others74%,including carbohydrates, microelements, etc). HF and DM groups rats received high-fat and sugar diet (34.5%fat,17.5%protein and48%carbohydrates). Four weeks later, IPGTT and IPITT were performed again, rats with insulin resistant in DM group were injected once with low dose of STZ (intraperitoneal at27.5mg/kg), and meanwhile Chow+STZ group rats received same dose of STZ. Control and HF groups rats received same dose of citrate buffer. One week after STZ administration, blood was sampled through the jugular vein. Fasting blood glucose (FBG) and insulin (FINS) were measured, and the insulin sensitivity index(ISI, ISI=Ln(FINS×FBG)-1)was calculated. Rats with FBG>11.1mmol/1in two consecutive analyses, lower insulin sensitivity and characterized by polyuria, polydipsia and polyphagia were considered the diabetic rat model.
2.Thirty5-week-old male SD (Sprague-Dawley) rats were made into diabetic rat model according to the above method, and then were randomized into two groups: DM-Vector group and DM-siRNA group.12weeks after successful modeling of DM, DM-siRNA group rats were injected via the jugular vein with2.5x1010PFU of pAdxsi-TRIB3-shRNA, while DM-Vector group rats were injected with2.5x1010PFU of pAdxsi.Adenovirus transfection was repeated after two weeks. Four weeks after first adenovirus injection, rats were sacrificed.
3. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT):Glucose tolerance was assessed by IPGTT after12-hour diet fasting without water fasting. A bolus of glucose (lg/kg, ip) was injected, and blood samples were collected sequentially from the tail vein at0,15,30,60and120min and tested for glucose. Blood glucose was measured with a One-Touch Glucometer (LifeScan, Milpitas, CA). The area under the curve of glucose (AUCg) was calculated. To evaluate insulin tolerance, IPITT was performed after4-hour diet fasting without water fasting. A bolus of insulin (1unit/kg, ip) was injected, and blood samples were taken for glucose measurement as described above.
4. Serum biochemical parameter analysis:blood glucose and serum insulin were measured to calculate the insulin sensitivity index (ISI). Furthermore, serum total cholesterol (TC), triglycerides(TG) and free fatty acids (FFA) were also measured.
5. Measurement of blood pressure:Heart rate (HR) and blood pressure (BP) were measured with a noninvasive tail-cuff system (Softron BP-98A, Tokyo, Japan).
6. Ultrasonography of abdominal aorta:Successive observation of the changes of abdominal aorta in the initiation and progression of diabetic macroangiopathy by2-D, pulsed-wave Doppler and ultrasonic integrated backscatter technique.
7. Pathological analysis:HE staining, Masson trichrome staining, Sirius red staining and Verhoeff staining were preformed on rat aorta. The aortic media thickness, perivascular collagen area/luminal area (PVCA/LA), aortic collagen percentage per medial area, aortic elastin percentage per medial area and aortic collagen/elastin ratio were determined with a computerized image analysis system. The average aortic wall architecture score was graded on Verhoeff-stained sections.
8. Immunohistochemical staining for collagen Ⅰ and Ⅲ:The collagen Ⅰ and Ⅲ depositions of aorta were viewed by immunohistochemical staining under a light microscope. The aortic Collagen Ⅰ and Ⅲ content were determined with a computerized image analysis system.
9. Hydroxyproline analysis:The collagen content of rat aortic tissues was determined by Hydroxyproline assay. The rat aortic tissues dried to constant weight were weighed, and then hydrolyzed in6mol/L HC1at110℃for16hours. The Hydroxyproline content of the hydrolysates was detected by an ELISA kit. Data were expressed as micrograms collagen per milligram dry weight, with the assumption that collagen contains an average of13.5%Hydroxyproline.
10. Real-time quantitative RT-PCR:Total RNA of the fresh rat aortic tissues was extracted by Trizol. The mRNA of TRIB3was quantified by Real-time Fluorescent Quantitative RT-PCR.
11. Western blot analysis:The proteins of TRIB3, collagen Ⅰ and Ⅲ, phospho-PI3K/PI3K,phospho-Akt/Akt,phospho-MKK4/MKK4and phospho-JNK/JNK were detected by western blotting.
Results
1. General characteristics of rats in each group at the end of the experiment
The rats in Chow+STZ group had obviously higher values of water intake, food intake and24h urine volume compared with Control group (P<0.05). The rats in HF group had increased values of water intake, food intake and24h urine volume, and significantly increased body weight compared with Control group (P<0.05). The rats in DM group had markedly increased values of water intake, food intake and24h urine volume compared with Control group (P<0.01). The rats in Chow+STZ group had significantly increased values of water intake and24h urine volume compared with HF group (P<0.05～0.001). The rats in DM group had significantly increased values of water intake, food intake and24h urine volume compared with HF group and Chow+STZ group (P<0.001, P<0.001).
2. Results of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)
(1) Results of IPGTT
After a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than that at baseline at Omin,30min,60min,120min in IPGTT. The area under the curve of glucose (AUCg) increased significantly at week4than at baseline (P<0.05).
Results of IPGTT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at30min and60min in IPGTT compared with the Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Control groups. The DM group had significantly elevated blood glucose levels at15min and60min in IPGTT compared with Control group (P<0.01); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and Chow+STZ groups. The DM group had significantly elevated blood glucose level at15min in IPGTT compared with Chow+STZ group (P<0.05), and also at120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM and Chow+STZ groups in IPGTT increased significantly compared with Control group (P<0.05～0.01).
Results of IPGTT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at60min and120min in IPGTT compared with Control group (P<0.05); The blood glucose levels at all time points in IPGTT had no statistical difference between HF and control group. The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPGTT compared with Control group (P<0.01); The DM group had significantly elevated blood glucose levels at15min and30min in IPGTT compared with Chow+STZ group (P<0.05), and also at60min and120min in IPGTT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPGTT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group increased significantly compared with HF group (P<0.05).
(2) Results of IPITT
After a4-week high-fat and sugar diet, the levels of blood glucose in DM group were significantly higher than at baseline at Omin,15min,30min,60min,120min in IPITT. The area under the curve of glucose (AUCg) in IPITT increased significantly at week4than at baseline (P<0.01).
Results of IPITT in each group at12weeks after the onset of diabetes (at week17):The Chow+STZ group had significantly elevated blood glucose levels at15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The HF group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The DM group had significantly elevated blood glucose levels at Omin,15min,30min,60min and120min in IPITT compared with Control group (P<0.05～P<0.01); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Chow+STZ group, and between DM group and Chow+STZ group. The DM group had significantly elevated blood glucose levels at Omin,15min,30min and60min in IPITT compared with HF group (P<0.05); The area under the curve of glucose (AUCg) of the DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05～P<0.01). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).
Results of IPITT in each group at the end of the experiment:The Chow+STZ group had significantly elevated blood glucose levels at15min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and Control group; The DM group had significantly elevated blood glucose levels at15min,60min and120min in IPITT compared with Control group (P<0.05); The blood glucose levels at all time points in IPITT had no statistical difference between HF group and DM group, and between DM group and Chow+STZ group. The area under the curve of glucose (AUCg) of DM group, HF group and Chow+STZ group in IPITT increased significantly compared with Control group (P<0.05). The area under the curve of glucose (AUCg) of DM group in IGITT increased significantly compared with HF group (P<0.05).
3. Serum biochemical parameters analysis:
The serum biochemical parameters were of no statistical difference among each group before the experiment. 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.3. Blood pressures and heart rate:There were no differences in SBP, DBP and MAP among four groups during the experimental process. At the end of the experiment, HR was higher in DM group compared with control and chow+STZ groups, but the difference was not statistically significant.
4. Ultrasonography of abdominal aorta
At the end of the experiment, compared to Control group, the HF group showed no significant differences in ultrasonic measurements; In Chow+STZ group, the Peterson's elastic modulus (Ep) and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the peak systolic velocity (PSV) and cross-sectional distensibility (CD) were deceased (P<0.05～0.001) compared with Control group; In DM group, systolic diameter (Ds), diastolic diameter (Dd), Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the end diastolic velocity (EDV), peak systolic velocity (PSV) and crosssectional distensibility (CD) were decreased (P<0.05～0.001) compared with Control group; In Chow+STZ group, the stiffness index of abdominal aorta was significantly increased compared with HF group (P<0.01); In DM group, the Dd, Ep and stiffness index of abdominal aorta were significantly increased (P<0.05～0.001), while the EDV and PSV were decreased (P<0.05～0.001) compared with HF group; In DM group, the Dd and Ds of abdominal aorta were significantly increased compared with Chow+STZ group (P<0.05～0.001).
At the end of the experiment, in Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with Control group (P<0.01); In Chow+STZ, HF and DM groups, cyclic variation of integrated backscatter (CVIB) of abdominal aorta was significantly decreased compared with Control group (P<0.001); In Chow+STZ and DM groups, IBS%of abdominal aorta was significantly increased compared with HF group (P<0.05～0.01); Cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM group was significantly decreased (P<0.01).
6. Pathological characteristics of aorta
(1) HE staining:Compared with Control group, in DM group, the endothelial cells adhering to inner elastic plates showed arrangement disorder, and the vascular smooth muscle cells (VSMCs) of tunica media arranged irregularly, with irregular nucleus and uneven cytoplasm staining. In Chow+STZ, HF, and DM groups, the media thickness of aorta was significantly increased compared with Control group (72.73.09±2.12vs.58.09±0.97μm, P<0.01;62.69±0.67vs.58.09±0.97μm, P<0.05;72.38±2.30vs.58.09±0.97μm, P<0.001). In Chow+STZ and DM groups, the media thickness of aorta was increased compared with HF group (P<0.05). In DM group, the media thickness of aorta was increased compared with Chow+STZ group, but the difference was not statistically significant.
(2) Masson staining:The VSMCs in aorta were stained red, while the collagen fibers were stained bluish green. In control group with mild perivascular fibrosis, collagen fibers were thin, intact and appropriately arranged. While in DM group, the collagen content increased significantly, thick collagen fibers connected into reticular or mass structure arranged irregularly.
Sirius red staining:In control group with mild perivascular fibrosis, the collagen fibers in aorta were thin and uniform under conventional microscope, and the tunica media showed no staining, tunica adventitia was mainly composed of red collagen Ⅰ and green collagen Ⅲ under polarization microscope. While in the DM group, collagen fibers in aorta increased significantly, arranged irregularly and was disrupted markedly under conventional microscope. Under polarization microscope, collagen Ⅰ and collagen Ⅲ in tunica media and tunica adventitia of DM group both increased significantly. Semi-quantitative analysis showed that compared with Control group, the PVCA/LA in Chow+STZ, HF, and DM groups increased significantly (0.06±0.0029vs.0.03±0.0015, P<0.001;0.04±0.0016vs.0.03±0.0015, P<0.05;0.10±0.007vs.0.03±0.0015,P<0.01), the CVF increased significantly (4.42±0.48vs.1.12±0.14, P<0.001;2.45±0.22vs.1.12±0.14, P<0.01;5.70±0.31vs.1.12±0.14, P<0.001); Compared with HF group, the PVCA/LA and collagen percentage per medial area of Chow+STZ and DM groups increased significantly (P<0.01-0.001); Compared with Chow+STZ group, the PVCA/LA of DM group slightly increased (P<0.05); Compared with Chow+STZ group, the collagen percentage per medial area of DM group slightly increased, but the difference was not statistically significant.
(3) Verhoeff elastin staining:In Control group, the VSMCs in aorta were stained yellow or Yellowish red, and the elastin fibers stained black blue were intact and uniform, and arranged appropriately. While in DM group, the VSMCs were stained red, the elastin fibers showed arrangement disorder, sparsity, nonuniformity, and had significant disruptions. Semi-quantitative analysis showed that compared with Control group, the elastin volume fraction of abdominal aorta in Chow+STZ, HF, and DM groups decreased significantly(5.19±0.76vs.11.96±0.75, P<0.001;8.83±0.98vs.11.96±0.75, P<0.05;4.70±0.36vs.11.96±0.75, P<0.001); The elastin percentage per medial area in DM group decreased significantly compared with HF group(P<0.05); Compared with Chow+STZ group, the elastin percentage per medial area in DM group slightly decreased, but the difference was not statistically significant; Compared with Control group, the aortic collagen/elastin ratio in Chow+STZ, HF, and DM groups, increased significantly(0.57±0.09vs.0.05±0.007, P<0.01;0.17±0.0024vs.0.05±0.007, P<0.01;0.71±0.075vs.0.05+0.007, P<0.001); Compared with HF group, the aortic collagen/elastin ratio in Chow+STZ and DM groups increased significantly (P<0.05～0.001); Compared with Chow+STZ group, the aortic collagen/elastin ratio of DM group slightly increased, but the difference was not statistically significant; Compared with Control group, the average aortic wall architecture score of Chow+STZ, HF and DM groups increased significantly (p<0.001); Compared with HF group, the average aortic wall architecture score of Chow+STZ and DM groups increased significantly (p<0.01～0.001); Compared with Chow+STZ group, the average aortic wall architecture score of DM group slightly increased (p<0.05).
(4) Determination of collagen content in rat aorta by Hydroxyproline assay.
Compared with Control group, the collagen content in Chow+STZ, HF and DM groups increased significantly (13.34±0.37vs.7.01±0.21ug/mg dry weight, P<0.001;8.47±0.21vs.7.01±0.21ug/mg dry weight, P<0.01;15.34±0.47vs.7.01±0.21ug/mg dry weight, P<0.001); Compared with HF group, the collagen content in Chow+STZ and DM groups increased significantly (P<0.001); Compared with Chow+STZ group, the collagen content in DM group increased significantly (P<0.001).
(5) Results of Immunohistochemical staining:Compared with Control group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ group markedly increased, the protein expression of collagen Ⅰ and Ⅲ in HF group slightly increased, while the expression of collagen Ⅰ and Ⅲ in DM group increased significantly. Compared with HF group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ and DM groups both increased markedly. Compared with Chow+STZ group, the protein expression of collagen Ⅰ and Ⅲ of DM group increased markedly.
(6) Western blot results of collagen Ⅰ and Ⅲ:Compared with Control group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ group markedly increased (collagen Ⅰ:0.82±0.05vs.0.51±0.05, P<0.001; collagen Ⅲ:0.62±0.03vs.0.31±0.02, P<0.001), the protein expression of collagen Ⅰ and Ⅲ in HF group slightly increased (collagen Ⅰ:0.60±0.03vs.0.51±0.05, P<0.01; collagen Ⅲ:0.47±0.03vs.0.31±0.02, P<0.05), while the expression of collagen Ⅰ and Ⅲ in DM group increased significantly (collagen Ⅰ:1.04±0.06vs.0.51±0.05, P<0.001; collagen Ⅲ:0.95±0.04vs.0.31±0.02, P<0.001). Compared with HF group, the protein expression of collagen Ⅰ and Ⅲ in Chow+STZ and DM groups both increased markedly (P<0.01-0.001, P<0.001). Compared with Chow+STZ group, the protein expression of collagen Ⅰ and Ⅲ in DM group increased markedly (P<0.05).
(7) Western blot results of TRIB3:Compared with Control group, the protein expression of TRIB3in Chow+STZ group increased in1.33folds (P<0.001), the protein expression of TRIB3in HF group had a increase of2.3372%(P<0.001), the protein expression of TRIB3in DM group increased in1.78folds (P<0.001); Compared with HF group, the protein expression of TRIB3in Chow+STZ group had a increase of35%(P<0.001), the protein expression of TRIB3in DM group had a increase of61%(P<0.001); Compared with Chow+STZ group, the protein expression of TRIB3in DM group had a increase of19%(P<0.001).
(8) Western blot results of the key signal molecules in Akt and MAPK pathway
①The phospho-PI3K/PI3K ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.97,0.67,0.75,0.45. Compared with Control group, the phosphorylation level of PI3K in Chow+STZ, HF and DM groups declined significantly (P<0.001for all); Compared with HF group, the phosphorylation level of PI3K in Chow+STZ and DM groups declined significantly (P<0.001); Compared with Chow+STZ group, the phosphorylation level of PI3K in DM group declined markedly (P<0.01).
②The phospho-Akt/Akt ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.90,0.48,0.61,0.29. Compared with Control group, the phosphorylation level of Akt in Chow+STZ, HF and DM groups declined significantly (P<0.01～P<0.001), and among them the DM group had the Lowest phosphorylation level of Akt, which was the32%of Control group (P<0.001); Compared with HF group, the phosphorylation level of Akt in Chow+STZ and DM groups declined significantly, and between the two groups the DM group had Lower phosphorylation level of Akt, which was the47%of Control group (P<0.001); Compared with Chow+STZ group, the phosphorylation level of Akt in DM group declined slightly, but the difference was not statistically significant.
③The phospho-MKK4/MKK4ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.59,0.85,0.64,1.03. Compared with Control group, the phosphorylation level of MKK4in Chow+STZ, HF and DM groups increased significantly (P<0.05～P<0.001), and among them the DM group had the highest phosphorylation level of MKK4, which was1.86times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of MKK4in Chow+STZ and DM groups increased significantly (P<0.001for both); Compared with Chow+STZ group, the phosphorylation level of MKK4in DM group increased markedly (P<0.01).
④The phospho-JNK/JNK ratio of rat aorta in Control, Chow+STZ, HF and DM groups were respectively0.45,0.78,0.60,0.97. Compared with Control group, the phosphorylation level of JNK in Chow+STZ, HF and DM groups increased significantly (P<0.01～P<0.001), and among them the DM group had the highest phosphorylation level of JNK, which was2.15times that of Control group (P<0.001); Compared with HF group, the phosphorylation level of JNK in DM group increased significantly (P<0.001), while the phosphorylation level of JNK in Chow+STZ group slightly increased, but the difference was not statistically significant; Compared with Chow+STZ group, the phosphorylation level of JNK in DM group increased slightly, but the difference was not statistically significant.
7. The mRNA and protein expression of TRIB3after TRIB3gene silencing
The mRNA and protein expression of TRIB3in aorta:The results of RT-PCR showed that Compared with DM-Vector group, the mRNA expression of aortic TRIB3in DM-siRNA group declined by66%(P<0.001). The results of Western blot showed that compared with DM-Vector group, the protein expression of aortic TRIB3in DM-siRNA group declined by67%(P<0.001).
8. TRIB3gene silencing ameliorated the general characteristics of type2diabetic rat
Compared with DM-Vector group, the DM-siRNA group rats had obviously lower values of water intake (P<0.05) and24h urine volume (P<0.05) after TRIB3gene silencing. The DM-siRNA group rats had lower values of food take compared with DM-Vector group, but the difference was not statistically significant. The body weight, Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) of DM-siRNA group had no statistical difference compared with DM-Vector group after TRIB3gene silencing.
9. The effects of TRIB3gene silencing on IPGTT and IPITT
(1) Results of IPGTT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at30min and120min in IPGTT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.041) in IPGTT after TRIB3gene silencing.
(2) Results of IPITT:Compared with DM-Vector group, the DM-siRNA group had significantly decreased blood glucose levels at Omin and120min in IPITT (P<0.05for both), and significantly decreased area under the curve of glucose (AUCg)(P=0.030) in IPITT after TRIB3gene silencing.
10. The effects of TRIB3gene silencing on serum biochemical parameters
4weeks after TRIB3gene silencing, serum TG level of DM-siRNA group was markedly decreased compared with DM-Vector group (4.98±1.62vs2.57±0.62mmol/L, P<0.05). Serum TC level of DM-siRNA group was markedly decreased compared with DM-Vector group (3.96±1.01vs.7.74±2.15mmol/L, P<0.05). Serum FFD level of DM-siRNA group was markedly decreased compared with DM-Vector group (355.85±26.24vs.464.69±31.23μmol/L, P<0.05). The fasting blood-glucose level of DM-siRNA group was remarkably decreased compared with DM-Vector group (15.63±2.04vs.24.58±3.04mmol/L, P<0.05). The ISI of DM-siRNA group was markedly increased compared with DM-Vector group (-5.17±0.14vs.-5.67±0.14, P<0.05). The fasting insulin level of DM-siRNA had no statistical difference compared with DM-Vector group.
11. The effects of TRIB3gene silencing on ultrasonography of abdominal aorta
Four weeks after TRIB3gene silencing, The Dd of abdominal aorta in DM-siRNA group was markedly decreased compared with DM-Vector group (P<0.05); The EDV and PSV of DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The Peterson's elastic modulus (Ep) and crosssectional distensibility (CD) of abdominal aorta in DM-siRNA group were increased markedly compared with DM-Vector group (P<0.01); The stiffness index of abdominal aorta in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.001);
4weeks after TRIB3gene silencing, IBS%of abdominal aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (1.76±0.14vs1.42±0.15, P<0.001); The cyclic variation of integrated backscatter (CVIB) of abdominal aorta in DM-siRNA group was significantly increased compared with DM-Vector group (4.17±0.81vs5.21±0.57, P<0.01).
12. TRIB3gene silencing ameliorated diabetic arterial remodeling
(1) HE staining:In DM-siRNA group there were only some of the endothelial cells of aorta showed disruption and structure abnormity compared with DM-Vector group. The endothelial cells in DM-siRNA group arranged more regularly, and had a looser adhesion to inner elastic plates compared with DM-Vector group. The VSMCs of abdominal aorta in DM-siRNA group with regular nucleus and even cytoplasm staining were fusiform shaped and arranged more irregularly compared with DM-Vector group. Results showed that the media thickness of aorta in DM-siRNA group was significantly decreased compared with DM-Vector group (74.88±1.21μm vs.69.38±1.11μm,P<0.01).
(2) Masson staining:The collagen content of aorta in DM-siRNA group was decreased significantly, and the collagen fibers were thinner, arranged more regularly and distributed more evenly around the vascular smooth muscle cells compared with DM-Vector group. Sirius red staining:The collagen fibers of aorta in DM-siRNA group decreased significantly arranged more irregularly and showed less disruption under conventional microscope compared with DM-Vector group. The yellowish red collagen Ⅰ and the yellowish green collagen Ⅲ in tunica media and tunica adventitia of aorta in DM-siRNA group decreased significantly, and had less disruption under polarization microscope compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the PVCA/LA of aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.10±0.005vs.0.06±0.006,P<0.001). The aortic collagen percentage per medial area in DM-siRNA group was decreased markedly compared with DM-Vector group (3.77±0.36vs.2.48±0.22,P<0.01).
(3) Verhoeff elastin staining:The elastin fibers of aorta in DM-siRNA group arranged more regularly and had less disruptions compared with DM-Vector group. Semi-quantitative analysis showed that compared with DM-Vector group, the aortic elastin percentage per medial area in DM-siRNA group was increased significantly(5.29±0.36vs.7.79±0.72, P<0.01); The aortic collagen/elastin ratio of abdominal aorta in DM-siRNA group was decreased significantly compared with DM-Vector group (0.75±0.09vs.0.36±0.07, P<0.01); The average aortic wall architecture score in DM-siRNA group was decreased markedly compared with DM-Vector group (P<0.05).
(4) Determination of collagen content in rat aorta by Hydroxyproline assay: Compared with DM-Vector group, the collagen content of aorta in DM-siRNA group was decr
引文
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2.中国糖尿病治疗费用每年1734亿人民币(250亿美元).糖尿病天地：教育刊.2010；(12)：38.
3.潘长玉,田慧,刘国良,李秀钧,傅祖植,陈家伟.中国城市中心医院糖尿病健康管理调查.中华内分泌代谢杂志.2004；20(5)：420-424.
4. Grundy SM, Howard B, Smith S Jr, Eckel R, Redberg R, Bonow RO. Prevention Conference VI:Diabetes and Cardiovascular Disease:executive summary: conference proceeding for healthcare professionals from a special writing group of the American Heart Association. Circulation.2002 May 7;105(18):2231-9.
5. Howard BV, Rodriguez BL, Bennett PH, Harris MI, Hamman R, Kuller LH, Pearson TA, Wylie-Rosett J. Prevention Conference VI:Diabetes and Cardiovascular disease:Writing Group I:epidemiology. Circulation.2002 May 7;105(18):e132-7.
6. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet.1998 Sep 12;352(9131):837-53
7. Schernthaner G. Diabetes and Cardiovascular Disease:Is intensive glucose control beneficial or deadly? Lessons from ACCORD, ADVANCE, VADT, UKPDS, PROactive, and NICE-SUGAR. Wien Med Wochenschr.2010 Jan;160(1-2):8-19.
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