【作者】 毕秀萍；

【导师】 张薇；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 背景随着人类生活方式及饮食习惯的改变,代谢综合征（Metabolic syndrome,MS）已成为严重威胁人类健康的疾病。MS是一种介于正常代谢与2型糖尿病之间的状态,包括腹型肥胖、糖耐量异常、血脂异常、高血压以及胰岛素抵抗等一组心血管疾病危险因素的症候群。近半个世纪来,国内外学者对MS进行了大量研究,虽然取得了阶段性研究成果,但MS的发生机制仍未被完全阐明。MS的发病机制十分复杂,既与遗传易感基因有关,又涉及某些环境因素。目前大部分观点认为,MS的发生是复杂的遗传与环境因素相互作用的结果,而胰岛素抵抗是MS的病理生理基础与核心。但胰岛素抵抗又是如何发生的?它的始动因素是什么?至今尚无定论。最近有资料表明,胰岛素抵抗是一种慢性低水平的炎症状态,炎症在MS的发生过程中发挥了重要作用。而在炎症—胰岛素抵抗—MS这一病理生理过程中,脂肪组织的内分泌调节功能障碍扮演了重要角色。白介素-18（IL-18）是一种促炎症因子,在炎症反应链中起重要作用。临床研究发现,肥胖人群的IL-18水平升高,且随着体重的降低,IL-18、白介素6（IL-6）及C-反应蛋白（CRP）水平下降,脂联素水平上升;IL-18是2型糖尿病及非糖尿病患者胰岛素抵抗的标志。以上证据充分说明IL-18水平与MS危险性相关。最新的研究显示,人腹部及皮下脂肪细胞表达IL-18基因,肿瘤坏死因子α（TNF-α）能增强IL-18基因表达;脂肪细胞可以分泌IL-18,肥胖者较非肥胖者脂肪细胞分泌IL-18的能力强3倍;肥胖人群脂肪组织IL-18 mRNA水平升高且与胰岛素抵抗呈正相关。但以往有关IL-18与MS关系的研究仅限于临床,目前尚无系统的动物实验研究报道。MS已成为严重威胁人类健康的疾病,寻找能有效治疗和预防MS的药物显得尤为重要。炎症参与MS的发生,抑制慢性、亚临床性炎症已成为改善胰岛素抵抗、治疗MS的新靶点。目前已经被证实具有一定抗炎作用的药物包括原有的降糖药、调脂药、ACEI和ARB、大剂量阿斯匹林等,均可不同程度地改善胰岛素抵抗。相对上述药物,CCB类降压药在胰岛素抵抗和MS患者中的作用尚未受到应有的重视。多项大规模临床试验结果证实,CCB类降压药能有效降低伴糖尿病的高血压患者心血管事件死亡率,具有降压以外的心血管保护作用;CCB还能改善肥胖的2型糖尿病患者胰岛素抵抗,降低血TNF-α水平。但CCB能否通过抗炎作用抑制和延缓MS各代谢异常目前尚无定论。因此我们推测,IL-18可在脂肪组织表达,并通过脂肪组织在MS发病中发挥重要作用;非络地平可改善MS各代谢异常并下调IL-18在脂肪组织的表达,可能为CCB类降压药防治MS提供新的理论依据。目的1.探讨IL-18在果糖诱导的MS大鼠模型脂肪组织中的表达;2.探讨脂肪组织IL-18的表达和MS的关系;3.探讨非络地平对MS各代谢异常及IL-18在脂肪组织表达的影响。方法5周龄Wistar大鼠33只,随机分为两组:对照组（n=12只）,果糖组（n=21只）。对照组以标准大鼠饲料喂养及普通饮水,果糖组大鼠以标准大鼠饲料喂养,并给予10%（w/v）高果糖饮水。果糖喂养32周后,将果糖组大鼠再随机分为两组:果糖组（n=9只）,继续给予高果糖饮水并每天以生理盐水灌胃:非络地平组（n=9只）,继续给予高果糖饮水的同时,每天以非络地平（5mg/kg.d）灌胃。继续喂养及药物干预共持续6周,处死动物,留取脂肪标本备用。实验过程中,进行以下检测:（1）各组大鼠每隔2周测量体重及尾动脉血压一次。（2）分别于喂养前、喂养32周及干预后6周抽血测定空腹血脂、血糖、胰岛素以及血清IL-18,并计算稳态模型胰岛素抵抗指数（HOMA-IR）。（3）脂肪组织病理学检测。（4）免疫组织化学染色法进行脂肪组织IL-18定位。（5）实时定量RT-PCR法检测IL-18 mRNA的表达。（6）Western-blot法检测IL-18蛋白含量的表达。结果1.实验动物基本情况:实验过程中共有3只大鼠死亡,均为果糖喂养组大鼠,在果糖喂养10～32周内死亡,而在干预期6周内无大鼠死亡。共30只大鼠完成实验,其中对照组12只,果糖组9只,非络地平组9只。2.果糖喂养32周两组大鼠代谢指标比较果糖喂养前,两组大鼠体重、尾动脉收缩压、血脂、血糖、空腹胰岛素及HOMA-IR均无差异（P＞0.05）。高果糖喂养32周后,与对照组比较果糖组大鼠体重、尾动脉收缩压、血甘油三酯、血胰岛素及HOMA-IR升高,差异有显著性意义（P＜0.01～0.0001）;而两组大鼠血糖及血总胆固醇无显著差异。表明经32周果糖喂养,已成功建立了大鼠MS模型。3.非络地平干预后对大鼠各代谢指标的影响非络地平药物干预前,与对照组比较果糖喂养各组大鼠体重、尾动脉血压、甘油三酯、血胰岛素及HOMA-IR均明显升高,表明已经成功建立MS模型。非络地平干预后6周,与果糖组比较,非络地平组尾动脉血压（P＜0.0001）、血胰岛素（P＜0.0001）及HOMA-IR（P＜0.05）显著降低;而体重、甘油三酯在非络地平组与果糖组间无显著差异。与干预前比较,非络地平组在干预后尾动脉血压（P＜0.01）、血胰岛素（P＜0.05）及HOMA-IR（P＜0.05）降低,差异有显著性意义;而体重和甘油三酯在非络地平干预前后则无显著差异。4.非络地平干预前后各组大鼠血清IL-18水平比较非络地平干预前,果糖喂养各组大鼠血清IL-18水平较对照组明显升高;干预后果糖组与非络地平组大鼠血清IL-18水平仍明显高于对照组（P＜0.0001）。与干预前比较,非络地平组在干预后血清IL-18水平显著降低（P＜0.01）;果糖组干预前后无明显变化。5.脂肪组织病理观察HE染色显示:对照组脂肪细胞体积较小,大小均一,饱满,排列较整齐;果糖组脂肪细胞体积变大,大小不均一,形态不规则,排列紊乱;细胞壁不完全清楚,细胞相交处可见融合,有合成大细胞的趋势。非络地平组脂肪细胞介于对照组与果糖组之间。6.脂肪组织IL-18免疫组织化学染色检测染色阳性信号为棕色,定位于细胞间质及细胞胞浆内。对照组细胞间质呈现浅棕色,胞浆内偶可见浅棕色颗粒;果糖组细胞间质呈现深棕色,胞浆内可见较浓密的深棕色颗粒;非络地平组细胞间质则介于两组之间,胞浆内偶见浅棕色的颗粒。7.实时定量RT-PCR检测脂肪组织中IL-18 mRNA表达与对照组相比,果糖组大鼠脂肪组织IL-18 mRNA表达明显升高（0.072±0.016vs0.019±0.006.P＜0.01）;与果糖组相比,非络地平组大鼠脂肪组织IL-18 mRNA表达水平显著下降（0.016±0.007 vs 0.072±0.016,P＜0.01）。相关性分析显示,果糖组IL-18 mRNA表达与HOMA-IR呈明显的正相关（r=0.74,P＜0.01）。8.Western-blot检测脂肪组织IL-18蛋白表达与对照组相比,果糖组大鼠脂肪组织IL-18蛋白表达水平显著升高（1.010±0.149vs0.531±0.085.P＜0.01）;与果糖组相比,非络地平组脂肪组织IL-18蛋白表达则显著下降（0.579±0.124vs1.010±0.149,P＜0.01）。相关性分析显示,果糖组IL-18蛋白表达与HOMA-IR呈明显的正相关（r=0.82,P＜0.01）。结论1.应用高果糖饮水并适当延长喂养时间,成功地建立了符合人类特征的大鼠MS模型,为MS发病机制的研究奠定了基础。2.从蛋白、基因水平均证实IL-18在脂肪组织中表达。3.果糖喂养大鼠血清和脂肪组织中IL-18表达升高,且与HOMA-IR呈明显正相关,提示脂源性炎症因子IL-18参与了大鼠MS的病变过程。4.非络地平可降低MS大鼠血压,改善胰岛素抵抗,降低血清和脂肪组织IL-18表达,提示非络地平可通过抗炎作用改善胰岛素抵抗,从而防治MS的发生。背景代谢综合征（MS）的发病机制十分复杂,既与遗传易感基因有关,又涉及某些环境因素,为多种因素综合作用的结果。目前大部分观点认为,MS的发生是复杂的遗传与环境因素相互作用的结果,而胰岛素抵抗（IR）是MS的病理生理基础与核心。随着分子生物学的发展,人们逐渐认识到胰岛素信号转导障碍可引起IR。发生IR的分子机制是靶细胞胰岛素受体（InsR）后信号传导通路的缺陷,其中IRS-1、PI-3K及PKB/Akt信号传递分子受损最为关键。越来越多的实验资料证实炎症和IR/MS之间存在相关和因果关系。研究发现,InsR后的信号通路与炎症因子的信号传导存在交叉作用,非特异性炎症所产生的炎症因子干扰胰岛素IRS/PI-3K信号转导通路,是炎症导致IR的主要分子机制。目前许多证据还表明脂肪组织是IR产生的始发部位,尤其是内脏脂肪组织在IR/MS的发生和发展过程中起着非常重要的作用。脂肪组织既是能量储存中心,又具有强大的内分泌功能。肥胖时,特别是腹部肥胖,脂肪组织表达的脂肪因子谱发生改变,细胞因子能够阻止InsR信号在脂肪和其他胰岛素敏感组织的转导,从而直接参与了IR的形成。本研究的第一部分结果显示,IL-18在脂肪组织的表达升高与IR相关,提示IL-18可能通过脂肪组织参与了IR/MS的病理生理过程。最新研究表明,IL-18能通过NF-κB依赖性途径参与炎症反应,增加多种细胞因子及炎症因子的表达。而InsR后的信号通路与炎症因子的信号传导存在交叉作用。因此,我们提出IL-18可能通过抑制脂肪组织IRS-1/PI-3K/Akt胰岛素信号通路促进IR,从而导致MS的发生。另一方面,由于Akt在胰岛素刺激的葡萄糖摄取中起重要作用,是目前胰岛素信号通路中最为人知的下游成分。TRB3是晚近在果蝇体内发现一种抑制有丝分裂的蛋白激酶,资料显示TRB3可通过直接与Akt结合,阻断该激酶的活化以中断胰岛素信号,在肝脏诱导IR。以上提示TRB3可能通过抑制Akt活化导致IRfMS的发生,此外,TRB3的基因水平可能被细胞内各种应激如炎症刺激上调。因此我们推断IL-18可诱导TRB3在脂肪组织的上调,进而抑制Akt活化,导致了IR/MS的发生。综上所述,我们提出如下假说:IL-18通过抑制脂肪组织IRS-1/PI-3K胰岛素信号转导,诱导脂肪组织TRB3表达上调,共同导致Akt活化抑制,促进IR/MS的发生。深入研究此信号通路的作用及其调控机制,可能为防治MS提供新的思路。目的1.明确脂肪组织IRS-1/PI-3K/Akt及TRB3/Akt信号转导障碍在MS大鼠IR发病中的作用:2.构建IL-18腺病毒载体并转染MS大鼠,探讨IL-18对果糖诱导的MS大鼠各代谢异常的影响;3.探讨脂肪组织IL-18在IR发病中的作用及其信号转导机制。方法5周龄Wistar大鼠48只,随机分为两组:对照组（n=12只）,果糖组（n=36只）。对照组以标准大鼠饲料喂养,果糖组大鼠以标准大鼠饲料喂养,并给予10%（w/v）高果糖饮水。果糖喂养32周后,将果糖组大鼠再随机分为三组:果糖组（n=9只）,继续给予果糖饮水;空载体组（n=9只）,继续给予果糖饮水的同时,通过尾静脉注射1×10¹⁰pfu含GFP的腺病毒载体;IL-18载体组（n=12只）,继续给予果糖饮水的同时通过尾静脉注射1×10¹⁰pfu含IL-18的腺病毒载体;对照组及果糖组同时经尾静脉注射等体积的生理盐水作对照。腺病毒载体转染后继续喂养6周。实验过程中,进行以下检测:（1）各组大鼠每隔2周测量体重及尾动脉血压一次。（2）分别于喂养前、喂养32周及转染后6周抽血测定空腹血脂、血糖、胰岛素,并计算稳态模型胰岛素抵抗指数（HOMA-IR）。（3）载体转染0,1,2,3,4,6周抽血测定血清IL-18浓度。（4）实时定量RT-PCR法检测IL-18及TRB3mRNA的表达。（5）Western-blot法检测IL-18及IL-18后的信号通路各关键分子蛋白含量的表达。结果1.实验动物基本情况:实验过程中共有6只大鼠死亡,均为果糖喂养组大鼠,在果糖喂养10～32周内死亡,而在腺病毒载体转染后6周内无大鼠死亡。共42只大鼠完成实验,其中对照组12只,其中对照组12只,果糖组9只,空载体组9只,IL-18载体组12只。2.IL-18腺病毒载体转染前两组大鼠体重、血压及生化指标比较果糖喂养前,两组大鼠体重、尾动脉收缩压、血脂、血糖、空腹胰岛素等指标均无差异（P＞0.05）。高果糖喂养32周后,与对照组比较果糖组大鼠体重、尾动脉收缩压、血甘油三酯、血胰岛素及HOMA-IR升高,差异有显著性意（P＜0.01～0.0001）;而两组大鼠血糖及血总胆固醇无显著差异。表明经32周果糖喂养,已成功建立了大鼠MS模型。3.IL-18腺病毒载体转染后各组大鼠血清IL-18水平变化IL-18腺病毒载体转染0～6周,对照组、果糖组及空载体组血清IL-18水平均无明显改变,IL-18载体组则随时间呈现比较显著的变化趋势;转染后各个时间点,果糖喂养各组大鼠血清IL-18水平均较对照组明显升高（P＜0.001）。IL-18腺病毒转染0周,IL-18载体组大鼠与空载体组比较,血清IL-18水平未见明显升高;转染后1周,载体组较之空载体组血清IL-18水平显著升高（P＜0.0001）:载体组于转染后2周血清IL-18水平达到最高,约为空载体组的4倍（P＜0.0001）;载体组血清IL-18水平于转染后3周开始降低,但仍明显高于空载体组（P＜0.0001）;4周、6周时载体组血清IL-18水平已与空载体组水平相当,差异无统计学意义。4.IL-18腺病毒载体转染后对大鼠各代谢指标的影响IL-18腺病毒载体转染前,与对照组比较果糖喂养各组大鼠体重、尾动脉血压、甘油三酯、血胰岛素及HOMA-IR均明显升高,表明MS模型已经成功建立。IL-18腺病毒载体转染6周后,与对照组比较果糖喂养各组大鼠体重、尾动脉血压、甘油三酯、血胰岛素及HOMA-IR均明显升高;与果糖及空载体组比较,载体组血胰岛素水平（P＜0.001）及HOMA-IR（P＜0.05）显著升高,而体重、血压、甘油三酯在载体组与空载体组间无显著差异。与转染前比较,IL-18载体组血胰岛素水平（P＜0.001）及HOMA-IR（P＜0.05）显著性升高,而体重、血压、甘油三酯在载体组与空载体组间无显著性差异。5.实时定量RT-PCR检测脂肪组织IL-18和TRB3 mRNA表达水平与对照组比较,果糖喂养各组大鼠脂肪组织IL-18 mRNA表达均明显上调（果糖组:P＜0.01;空载体组:P＜0.01;载体组:P＜0.001）;与果糖组、空载体组比较,IL-18载体组大鼠脂肪组织IL-18 mRNA表达明显增加（P＜0.01）;而果糖组与空载体组大鼠脂肪组织IL-18 mRNA表达无显著性差异。与对照组比较,果糖喂养各组大鼠脂肪组织TRB3 mRNA表达均明显上调（果糖组:P＜0.01;空载体组:P＜0.01;载体组:P＜0.001）;与果糖组、空载体组比较,IL-18载体组大鼠脂肪组织TRB3 mRNA表达明显增加（P＜0.01）:而果糖组与空载体组大鼠脂肪组织TRB3 mRNA表达无显著性差异。6.Western-blot检测脂肪组织IL-18及IL-18后的信号通路中各关键分子蛋白表达水平（1）各组大鼠脂肪组织IL-18蛋白表达水平比较:与对照组比较,果糖喂养大鼠脂肪组织IL-18蛋白表达显著升高（P＜0.01）;与果糖组、空载体组比较,载体组IL-18蛋白表达明显升高（P＜0.01）;而果糖组与空载体组IL-18蛋白表达则未见显著性差异。（2）各组大鼠脂肪组织IRS-1蛋白表达水平比较:与对照组比较,果糖喂养大鼠脂肪组织IRS-1蛋白表达明显降低（果糖组:P＜0.05;空载体组:P＜0.01;载体组:P＜0.01）;与果糖组、空载体组比较,IL-18载体组IRS-1蛋白表达显著降低（P＜0.01）;而果糖组与空载体组IRS-1蛋白表达则未见显著性差异。（3）各组大鼠脂肪组织p85/P-p85蛋白表达水平比较:四组大鼠脂肪组织p85总蛋白表达水平比较,均未见显著性差异。与对照组比较,果糖喂养大鼠脂肪组织P-p85蛋白表达显著降低（P＜0.01）;与果糖组、空载体组比较,IL-18载体组P-p85蛋白表达显著降低（P＜0.01）:而果糖组与空载体组P-p85蛋白表达则未见显著性差异。（4）各组大鼠脂肪组织Akt/P-Akt蛋白表达水平比较:四组大鼠脂肪组织Akt总蛋白表达水平比较,均未见显著性差异。与对照组比较,果糖喂养大鼠脂肪组织P-Akt（Ser473）蛋白表达明显降低（P＜0.01）;与果糖组、空载体组比较,IL-18载体组P-Akt（Ser473）蛋白表达显著降低（P＜0.01）;而果糖组与空载体组P-Akt（Ser473）蛋白表达则未见显著性差异。（5）各组大鼠脂肪组织TRB3蛋白表达水平比较:与对照组比较,果糖喂养大鼠脂肪组织TRB3蛋白表达明显上调（P＜0.01）;与果糖组、空载体组比较,IL-18载体组TRB3蛋白表达上调更加显著（P＜0.01）;而果糖组与空载体组TRB3蛋白表达则未见显著性差异。结论1.果糖诱导的MS大鼠脂肪组织IRS-1/PI-3K信号抑制,TRB3表达上调,共同导致Akt活化抑制,提示脂肪组织IRS-1/PI-3K/Akt,TRB3/Akt信号转导障碍可导致IR;2.构建IL-18腺病毒载体并转染MS大鼠,血液中和脂肪组织中IL-18水平显著增加,同时血胰岛素及HOMA-IR进一步增加,提示IL-18可促进果糖诱导的MS大鼠IR的进展;3.IL-18载体组脂肪组织胰岛素信号转导进一步抑制,TRB3进一步上调,Akt活化抑制更加明显。提示IL-18通过抑制脂肪组织IRS-1/PI-3K胰岛素信号通路和诱导TRB3表达上调,共同导致Akt活化抑制,促进IR,从而导致MS的发生。更多还原

【Abstract】 BackgroundMetabolic syndrome(MS) has become one of the major public health challenges worldwide accompanied with changes in human lifestyle and dietary habits.MS consists of the combined presentation of multiple cardiovascular risk factors that include abdominal obesity,glucose intolerance,dyslipidemia,hypertension and insulin resistance.In recent half a century,a great of studies on MS have been conducted,but development mechanisms of MS remains largely unclear.Recently,MS has been considered to be usually due to the interaction between genetic and environmental factors,and insulin resistance has been discussed as major pathophysiological mechanism for the development of MS.However,the molecular mechanisms triggering insulin resistance are not fully understood.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS.Adipose tissue is now viewed as an active endocrine organ that plays a crucial role in the pathophysiological process of inflammation-insulin resistance-metabolic syndrome.MS often accompanies the elevated inflammatory factors such as TNF-a,Interleukin (IL)-6,Interleukin(IL)-18 and so on.IL-18,a particularly important cytokines is recently thought as a risk predictor for MS.Circulating levels of IL-18 are elevated in obesity and reduced by weight loss.Increased plasma IL-18 is a marker of insulin resistance in type 2 diabetic and non-diabetic humans.Elevated IL-18 levels are associated with the MS independent of obesity and insulin resistance.New data show that IL-18 is expressed in human adipose tissue and strongly upregulated by TNFa in human adipocytes,and IL-18 release from adipocytes from obese donors was about 3-fold higher compared to adipocytes from non-obese donors.Adittionally,Adipose tissue(AT) IL-18 mRNA content was higher in the obese group than in the non-obese group and positively correlated with IR.Previous studies on the relationship between MS and IL-18 were limited in clinical trials,and animal experiments were never reported.There is a great significance to search effective drugs of preventing and treating MS due to its risk of cardiovascular disease.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS,and suppression of inflammation has already become a novel strategy to improve MS. There is some evidence that these drugs have anti-inflammatory actions,including hypoglycemic agent,lipid regulating agent,large dose aspirin,angiotensin-converting enzyme inhibitor(ACEI) and angiotensin receptor antagonist(ARB).Compared with these drugs,the effects of CCB on subjects with insulin resistance and MS were not thought highly.Several large-scale clinical tests showed that calcium-channel blocker (CCB) were safe and effective in reducing most types of cardiovascular morbidity and mortality in diabetic hypertensive patients,and thus have cardiovascular protection besides reducing blood pressure(BP).Besides reducing BP,amlodipine seemed to improve insulin resistance and decrease tumor necrosis factor-alpha(TNF-a) levels in obese hypertensive type 2 diabetic patients.Whether CCB can inhibit and delay MS by anti-inflammatory actions is not still conclusive.Objective1.To investigate the expression of IL-18 at both the mRNA and protein levels in adipose tissue of Rats with Fructose-Induced MS.2.To evaluate the correlation between the adipose tissue expression of IL-18 and MS in Fructose-fed rats.3.To investigate the effects of Filodipine on a cluster of metabolic abnormalities and adipose tissue expression of IL-18 in fructose-fed rats.MethodsThirty three male Wistar rats were divided randomly into two groups:a control group (n=12) in which the rats were fed the normal Rodent Diet and water,and a fructose-fed group(n=21) in which the rats were fed the normal Rodent Diet plus fructose in the drinking water as a 10%(w/v) solution.At the end of the 32th weeks, the fructose-fed rats were subdivided randomly into two groups:fructose group(n= 9) was continued with a fructose diet,and filodipine(5mg/kg/d) was administered by gavage daily for 6 weeks to filodipine group(n=9) with a fructose diet.All animals were sacrificed by cervical decapitation and the epididymal fat pads were stored at -80℃for analysis.The follwing parameters were measured during the study:(1) All the rats have their body weight and tail blood pressure measured once per 2 week.(2) Blood was collected from jugular vein at 0 week,the end of 32 weeks,and 6 weeks after treatment respectively.Plasm lipid,glucose,insulin and IL-18 were determined using routine method,and the HOMA-IR expressed as an index of insulin resistance was calculated.(3) Pathological study of adipose tissue.(4) Immunohistochemistry for the location of IL-18 in adipose tissue.(5) The mRNA expression of IL-18 was measured by quantification real-time RT-PCR.(6) The protein expression of IL-18 was analyzed by western blot.Results1.The experimental animalsThree rats of fructose group died in the entire experiment,none of control group died. A total of 30 rats finished the study,12 rats in control group,9 rats in fructose group and 9 rats infelodipine group.2.Comparison of metabolic variables between control and fructose group after fructose treatmentThere is no significant difference in terms of body weight,tail blood pressure,glucose, lipids and insulin before fructose treatment.Chronic administration of fructose during 32 weeks induced a cluster of metabolic abnormalities.Fructose-fed rats had higher body weight,systolic BP,fasting levels of plasma triglycerides,plasma insulin,and HOMA-R than the Con rats.There were significant differences in total cholesterol and glucose,both of which were although slightly increased.3.Effect of felodipine on metabolic variables in ratsAfter treatment with felodipine for 6 weeks,systolic BP,insulin and HOMA were significantly decreased in felodipine group compared with fructose group,other parameters showed no significant difference.Systolic BP,insulin and HOMA were significantly decreased in felodipine group after 6 weeks treatment compared with those before treatment;other parameters showed no significant difference.4.Comparison of Serum IL-18 levels between pro-treatment and post-treatment with felodipineSerum IL-18 levels were significantly higher in fructose and filodipine group than those in control group.6 weeks treatment with filodipine significantly decreased serum IL-18 levels compared with those before treatment.5.Pathological findingsHE staining slides under optical microscopy showed that in control group,cell size of adipocytes was smaller,uniform,and array was regular;In fructose group,adipocytes size was greater,not uniform,cell form was irregular,and cell arrange was disorder; Cell wall was not clear and cell fusion was observed at intersection of cell. Adipocytes infelodipine group lied between control and fructose group.6.Location of IL-18 by immunohistochemistryThe positive reaction of IL-18 protein was stained brown,and localized in intercellular substance and adipocyte cytoplasm.Intercellular substance was stained weak brown and weak brown granules were seldom distributed in cytoplasm in control group,whereas intercellular substance was stained deep brown and deep brown granules were thickly distributed in cytoplasm in fructose group.Staining in felodipine group lied between control and fructose group.7.IL-18 mRNA expression by RT-PCRIn the fructose-fed groups,the mRNA expression levels of IL-18 in adipose tissue were significantly increased compared with those in the control group.IL-18 mRNA levels in felodipine group were markedly lower than in fructose group.In addition, IL-18 mRNA levels showed a significant positive correlation with HOMA-IR in the fructose group(r=0.74,P＜0.01).8.IL-18 protein expression by westein blotCompared to control group,the groups fed on a fructose diet showed a significant rise in IL-18 protein content in adipose tissue.IL-18 protein content in the felodipine group was significantly lower than in fructose group.Moreover,IL-18 protein content showed a significant positive correlation with HOMA-IR in the fructose group (r=0.82,P＜0.01).Conclusions1.Rats model mimicked human MS can be achieved by kepting rats on a high-fructose diet for up to 32 week.This lays the foundation for further studying the mechanism of MS.2.IL-18 is confirmed to be expressed in asipose tissue at both the mRNA and protein levels.3.IL-18 expression is increased in serum and adipose tissue and showed a significant positive correlation with HOMA-IR in fructose group,which suggests that IL-18 is involved in the pathogenesis of MS via adipose tissue.4.Filodipine down regulates serum and adipose tissue expression of IL-18,with blood pressure reduced,and insulin resistance improved,showing that Filodipine may improve insulin resistance,prevent and delay MS by anti-inflammatory effect. BackgroundThe mechanisms of metabolic syndrome(MS) are very complex,usually due to the interaction between genetic and environmental factors.Insulin resistance has been discussed as major pathophysiological mechanism for the development of MS.Insulin resistance,the inability of target tissues to adequately increase glucose transport in response to a physiological level of insulin,results from defects of insulin signaling behind insulin receptor.Of the total,It is crucial for impaired IRS-1,PI-3K and PKB/Akt.A growing number of studies indicate that low-grade chronic inflammation is involved in the pathogenesis of MS.There exist intersections between signaling pathway behind insulin receptor and signaling transduction of inflammatory cytokines. Inflammatory cytokines are involved in the molecular mechanism that inflammation leads to insulin resistance by interfering with IRS-1/PI-3K insulin signaling pathway. Studies demonstrate that adipose tissue is considered as dominant sites affecting systemic insulin resistance and especially visceral adipose tissue plays an important role in metabolic syndrome.Adipose tissue is not only an energy storage organ,but an active endocrine organ.Abdominal obesity results in changes of adipocytokines which are involved in the development of insulin resistance by blocking insulin receptor signaling transduction in adipose tissue and other insulin-sensitive tissues. In part 1 of our study,results showed adipose tissue expression of IL-18 was higher in fructose-induced MS group than in the control group and positively correlated with IR, which suggests that IL-18 is involved in the pathogenesis of MS via adipose tissue. Recent studies indicate that IL-18 regutes inflammatory reaction through NF-κB -dependent signal transduction.Additionally,there exist intersections between signaling pathway behind insulin receptor and signaling transduction of inflammatory cytokines.Thereby we suggested that IL-18 promoted IR via defects of IRS-1/PI-3K/Akt insulin signaling pathway,and inducing the development of MS. On the other hand,Akt stands as the furthest known downstream component of the insulin-signaling pathway that leads to metabolic regulation for the pivotal role of this kinase in the linkage between the insulin signal and the control of glucose uptake.At present,much interest has recently been focused on Ttibble3(TRB3) because it is involved in insulin resistance.The current studies suggest that TRB3 disrupts insulin signaling by binding directly to Akt and blocking activation of the kinase,thereby inducing insulin resistance in liver.Furthermore,the expression of TRB3 mRNA level can be upregulated by various cellular stresses such as inflammatory stimulation.For the above reasons,we suggested that IL-18 promotes insulin resistance through TRB3 upregulation and TRB3-mediated inhibition of Akt in adipose tissue.Taken together,the hypothesis is proposed that IL-18 promotes insulin resistance through suppression of Akt activation via IRS/PI-3K and TRB3 signaling pathway in adipose tissue,thus leading to the development of MS.Objective1.To investigate the protein expression of key signaling molecule in IRS/PI-3K/Akt and TRB3/Akt pathway in adipose tissue of Rats with Fructose-Induced MS.2.To investigate the effects of IL-18 on a cluster of metabolic abnormalities in fructose-fed rats used IL-18 adenovirus administered.3.To investigate the role of IL-18 on development of IR:involvement of signalling pathway in adipose tissue.MethodsForty-eight male Wistar ratswere divided randomly into two groups:the control group (Con,n=12),which received a standard rodent chow and water,and the fructose-fed group(Fru,n=36) which received the standard rodent chow plus fructose in the drinking water as a 10%(w/v) solution.At the end of the 32th weeks,the fructose-fed rats were subdivided randomly into three groups:the fructose-fed group(Fru,n=9), the adenovirus-GFP control group(Ad-Con,n=9),and the adenovirus-IL-18 group (Ad-IL-18,n=12).The three groups were treated with PBS,Ad-GFP(1×1010pfu in 0.4ml PBS) and Ad-IL-18(1×1010pfu in 0.4ml PBS) via tail vein injection, respectively,and continued on a fructose diet for 6 weeks.All animals were sacrificed by cervical decapitation and the epididymal fat pads were stored at -80℃for analysis. The follwing parameters were measured during the study:(1) All the rats have their body weight and tail blood pressure measured once per 2 week.(2) Blood was collected fi’om jugular vein at 0 week,the end of 32 weeks,and 6 weeks after adenovirus transfection respectively.Plasm lipid,glucose,insulin were determined using routine method,and the HOMA-IR expressed as an index of insulin resistance was calculated.(3) Serum IL-18 levels of four groups were determined by ELISA at the end of 1st,2nd,3rd,4th,and 6th weeks after adenovirus injection.(4) The mRNA expression of IL-18 and TRB3 were measured by quantification real-time RT-PCR.(6) The protein expression of IL-18 and IL-18-induced signaling pathway in adipose tissue were analyzed by western blot.Results1.The experimental animalsThree rats of fructose group died in the entire experiment,none of control group died. A total of 42 rats finished the study,12 rats in Con group,9 rats in Fru group,9 rats in Ad-Con group and 12 rats in Ad-IL-18 group.2.Comparison of metabolic variables between Con and fructose-fed group before adenovirus transfectionChronic administration of fructose as a 10%drinking solution during 32 weeks induced a cluster of metabolic abnormalities.Fructose-fed rats had higher body weight and systolic BP than the Con rats.Compared with the Con group,three other variables including fasting levels of plasma triglycerides,plasma insulin,and HOMA-R were significantly increased in fructose-fed group,without significant differences in total cholesterol and glucose,both of which were although slightly increased.3.Changes of serum IL-18 after adenoviral transfectionAd-IL-18 group showed a significant change with time,and the other three groups had no changes in serum IL-18 levels after adenovirus transfection.Fructose-fed rats had higher serum IL-18 levels than Con rats.Serum IL-18 levels were higher in Ad-IL-18 group than in Ad-Con group at 1st week, without significant difference at 0 week after transfection.Serum IL-18 levels,which reached to peak value at 2nd week,were about 4-fold higher in the Ad-IL-18 group than in the Ad-Con group.At 3rd week,Ad-IL-18 group showed a significant decline tendency,but a significant increase in serum IL-18 levels compared with the Ad-Con group.There was no significant difference in IL-18 levels between the two groups at 4th and 6th week.4.Effect of IL-18 on metabolic parameters in ratsAfter IL-18 transfection for 6 weeks,insulin and HOMA were significantly increased in IL-18 vector group compared to vehicle group;other parameters showed no significant difference.Insulin and HOMA-IR were significantly increased in Ad-IL-18 group after transfection for 6 weeks compared to the levels of insulin and HOMA-IR before transfection;other parameters showed no significant difference.5.IL-18 and TRB3 mRNA expression by RT-PCRIn the fructose-fed groups,the mRNA expression levels of IL-18 and TRB3 in adipose tissue were significantly increased compared with those in the Con group.IL-18 and TRB3 mRNA levels in Ad-IL-18 group were markedly higher than in both Fru group and Ad-Con group,without significant differences between the Fru and Ad-Con groups6.Protein expression of IL-18 and IL-18 induced signaling pathway by westein blot(1) Comparison of IL-18 protein expression in adipose tissue of rats Compared to Con group,the three fructose-fed groups showed a significant rise in IL-18 protein content in adipose tissue.IL-18 protein content in the Ad-IL-18 group was significantly higher than in both the Fru group and the Ad-Con group.IL-18 protein content in the Fru group was similar to that in the Ad-Con group.(2) Comparison of IRS-1 protein expression in adipose tissue of ratsWestern blot analysis showed that IRS-1 protein expression was significantly decreased in adipose tissue of fructose-fed rats compared with the Con rats.The rats treated with Ad-IL-18 had significantly lower IRS-1 protein content in comparison with Fru and Ad-Con rats.But IRS-1 protein had no significant difference between the Fru group and the Ad-Con group.(3) Comparison of p85/P-p85 protein expression in adipose tissue of ratsIn adipose tissue,there was no significant difference among the 4 groups in the p85 regulatory subunit of PI3K(T-p85) protein expression.Phospho-p85(P-p85) protein expression was markedly reduced in adipose tissue of fructose-fed rats compared with that of Con rats.The P-p85 protein expression of the Ad-IL-18 group was significantly reduced compared with that of the Fru and the Ad-Con groups,without significant difference between Fru group and Ad-Con group.(4) Comparison of Akt/P-Akt protein expression in adipose tissue of ratsIn adipose tissue,total-Akt(T-Akt) protein expression was similar among the 4 groups.In adipose tissue,phospho-Akt(Ser473) protein expression was markedly lower in fructose-fed groups than Con group.Rats treated with Ad-IL-18 had markedly lower P-Akt protein expression than both the Fru and Ad-Con rats.P-Akt protein expression in the Fru group was similar to that in the Ad-Con group.(5) Comparison of TRB3 protein expression in adipose tissue of rats In the fructose-fed groups,the mRNA expression levels of TRB3 in adipose tissue were significantly increased compared with those in the Con group.TRB3 mRNA levels in the Ad-IL-18 group were markedly higher than in both Fru group and Ad-Con group.However,there were no significant differences in mRNA levels of TRB3 between the Fru and the Ad-Con group.Conclusion 1.Inhibition of IRS-1/PI-3K signal and upregulation of TRB3 expression induce suppression of Akt activation in adipose tissue of fructose-fed rats,suggesting defects in IRS-1/PI-3K/Akt and TRB3/Akt pathway in adipose tissue are involved in the development of insulin resistance.2.After IL-18-adenovirus was administered,serum and adipose tissue expression of IL-18 were high,and serum insulin and HOMA-IR were further increased,which suggests that IL-18 promotes insulin resistance in fructose-induced MS rats.3.Ad-IL-18 group showed marked inhibition of IRS-1/PI-3K signal and upregulation of TRB3 expression and more significant suppression of Akt activation in adipose tissue,which suggests that IL-18 promotes insulin resistance through suppression of Akt activation via suppression of PI-3K signaling pathway and upregulation of TRB3 in adipose tissue.更多还原