抵抗素与炎症及动脉粥样硬化

【作者】 胡文兰；

【导师】 乔树宾；

【作者基本信息】 中国协和医科大学 ， 冠心病， 2007， 博士

【摘要】 第一部分心绞痛患者血浆抵抗素水平的研究研究背景：抵抗素是一种新的脂肪细胞因子，最初研究表明抵抗素可能是联系肥胖、胰岛素抵抗和2型糖尿病的细胞因子。但啮齿类动物抵抗素主要由脂肪细胞分泌，而人类抵抗素主要是由单核-巨噬细胞分泌，研究证实在小鼠和人的动脉粥样硬化斑块存在抵抗素蛋白，且抵抗素可能在动脉粥样硬化形成发展中具有潜在作用。抵抗素水平在冠心病患者显著升高，是动脉粥样硬化的一个独立的炎症标志物。但不同的心绞痛患者间，抵抗素水平是否存在变化国内尚无报道。目的：观察不同心绞痛患者循环抵抗素水平的变化，以及循环抵抗素水平与代谢指标、炎症指标等之间的相关性。方法：序贯入选因疑诊冠心病而在阜外医院行择期冠脉造影检查的患者114例，根据造影结果及临床表现，将研究对象分为不稳定心绞痛组(UAP，46例)、稳定心绞痛组(SAP，37例)和对照组(31例)。收集患者一般临床资料及生化指标，免疫比浊法检测高敏C反应蛋白(hs-CRP)水平；放免法检测血清胰岛素和内皮素-1(ET-1)水平；ELISA法检测不同人群血浆抵抗素、大内皮素的水平。结果：UAP组血浆抵抗素水平(12.09(8.40，18.08)ng／ml)显著高于SAP组(9.04(7.09，11.44)ng／ml)(P＜0.05)，以及对照组(8.71(6.58，11.56)ng／ml)(p＜0.001)；而SAP组和对照组之间无明显差异。我们证实了抵抗素与年龄(R~2=0.06，P=0.009)、白细胞计数(R~2=0.05，P=0.013)以及hs-CRP(R~2=0.05，P=0.014)之间呈正相关，而且还发现血浆抵抗素与大内皮素(R~2=0.05，P=0.013)以及FT-1(R~2=0.07，P=0.005)水平呈正相关。在校正年龄、性别和腰围后，除了大内皮素外，这些相关性仍存在。结论：血浆抵抗素水平在冠心病患者、尤其是UAP患者显著升高，而且与白细胞计数、hs-CRP及ET-1水平独立正相关。提示抵抗素可能通过影响系统的炎症反应和内皮活化而参与冠心病的发生发展。第二部分CRP对人单核细胞抵抗素表达影响的实验研究背景：动脉粥样硬化是一种慢性炎症过程，抵抗素是一种新的脂肪细胞因子，既往被认为与胰岛素抵抗相关。但近几年研究表明，抵抗素参与炎症反应和动脉粥样硬化的病理生理过程，且是动脉粥样硬化的一个独立的炎症标志物。C反应蛋白(CRP)是冠心病的一个重要的危险因素，同时还能调控参与动脉粥样硬化的多种基因的表达。CRP不仅是一个炎症标志物，还有可能直接参与炎症反应和动脉粥样硬化过程。多项研究表明循环抵抗素水平与CRP水平呈正相关，但CRP与抵抗素的因果关系如何，是系统低度炎症反应引起抵抗素分泌?还是抵抗素同IL-6等类似、是CRP的另一诱导因子，目前无相关的研究。目的：本部分主要研究CRP对培养的人外周血单核细胞抵抗素mRNA和蛋白表达的影响。方法：采用密度梯度离心法分离人外周血单核细胞。在培养基中分别加入不同浓度的CRP(0，5，10，25，50μg／ml)，培养24小时，以检测CRP影响抵抗素表达的剂量效应。另一组实验检测CRP影响抵抗素表达的时间效应，即在培养基中加入25μg／ml CRP，分别培养不同的时间(0，3，6，12，24小时)。分别用real time PCR和ELISA方法检测单核细胞抵抗素mRNA表达及细胞培养液中抵抗素的浓度。结果：CRP呈剂量依赖性诱导抵抗素mRNA表达，与空白对照组抵抗素mRNA水平相比，5μg／mlCRP刺激单核细胞24h，抵抗素mRNA表达显著增加2.1倍(p＜0.05)。当CRP浓度为25μg／ml，该效应最大，为9.8倍(p＜0.05)。对照组培养基中的抵抗素水平很低，甚至检测不出。但是与5μg／ml CRP孵育后，培养基中抵抗素水平为0.35±0.01ng／ml(p＜0.05)。当CRP为50μg／ml时，这种刺激效应最大，抵抗素是水平为1.19±0.30ng／ml(p＜0.05)。CRP诱导抵抗素的表达也呈时间依赖性。25μg／mLCRP与人单核细胞孵育不同的时间发现，抵抗素m RNA在CRP刺激3小时后即开始明显升高，为基础水平的2.9倍(P＜0.05)，并随刺激的时间延长而进一步增加，于24小时达峰值(10倍，P＜0.05)。而培养基中的抵抗素蛋白水平经CRP刺激3小时后仍未检测出，6小时后，抵抗素蛋白水平才显著升高，为0.21±0.12ng／ml(p＜0.05)，也于24小时达峰值，为0.89±0.16ng／ml(p＜0.05)。结论：促炎因子CRP可呈剂量依赖性和时间依赖性地诱导人单核细胞抵抗素mRNA和蛋白表达，可能是急性冠状动脉综合征患者循环中抵抗素水平升高的机制之一，也进一步证实了CRP在血管炎症和动脉粥样硬化的病理生理过程中的直接作用。第三部分他汀类药物对CRP诱导的抵抗素表达的调节作用背景：HMG-CoA还原酶抑制剂(他汀类药物)广泛用于心血管疾病的治疗，并能显著降低心血管相关的发病率和死亡率。近几年研究发现，他汀类药物除了传统的降脂外，还具有独立于降脂作用以外的抗炎作用。动脉粥样硬化是一种慢性炎症性疾病。抵抗素是一种新的脂肪细胞因子，也参与炎症反应和动脉粥样硬化的病理生理过程。本研究第二部分结果表明CRP可呈剂量依赖性和时间依赖性地诱导人单核细胞抵抗素mRNA和蛋白表达。他汀类药物是否能影响CRP诱导的单核细胞抵抗素表达，目前尚无相关的研究。目的：本研究主要比较辛伐他汀、洛伐他汀、血脂康几种他汀类药物对CRP诱导的人外周血单核细胞抵抗素表达的影响，并对相关的信号通路进行初步探讨。方法：采用密度梯度离心法分离人外周血单核细胞。单核细胞分别与不同浓度的辛伐他汀(0.1，1，10μM)、洛伐他汀(0.1，1，10μM)或血脂康(25，50，100μg／ml)预孵育2小时，再与25μg／ml CRP共同培养24小时，以检测药物对CRP诱导的抵抗素表达的影响。另一组信号通路实验中，单核细胞分别与不同他汀类药物以及甲羟戊酸(100μM)或法尼基焦磷酸(FPP)(10μM)或牻牛儿基栊牛儿基焦磷酸(GGPP)(10μM)预孵育2小时，再与25μg／ml CRP共同孵育24小时。分别用real time PCR和ELISA方法检测单核细胞抵抗素mRNA表达及细胞培养液中抵抗素的浓度。结果：辛伐他汀和洛伐他汀与单核细胞预孵育2小时能剂量依赖性抑制25μg／mlCRP诱导的抵抗素表达。1μM辛伐他汀和洛伐他汀均能显著抑制抵抗素m RNA水平表达，分别为CRP刺激组的55.4±4.2％，59.6±5.1％(P＜0.05)，以及蛋白水平表达[0.54±0.02ng／ml，0.58±0.08ng／ml，与CRP刺激组(0.89±0.16ng／ml)比较P＜0.05]。甲羟戊酸和GGPP几乎完全逆转两种他汀类药物对CRP诱导的抵抗素表达的抑制作用。50μg／ml血脂康亦显著抑制抵抗素m RNA水平表达，为CRP刺激组的34.9±5.4％(P＜0.01)；下调抵抗素蛋白水平表达，为0.47±0.07ng／ml(P＜0.01)。甲羟戊酸和GGPP只能部分逆转血脂康对CRP诱导的抵抗素表达的抑制作用。结论：三种他汀类药物均能完全或部分通过抑制类异戊二烯代谢及GGPP依赖的信号通路，不同程度地抑制CRP诱导的抵抗素的表达。进一步支持了他汀类药物的抗炎和抗动脉粥样硬化和作用。更多还原

【Abstract】 Part onePlasma Resistin Is Increased in Patients with Unstable AngniaBackground: Resistin, a novel adipokine , was originally regarded to link insulin resistance and obesity in rodents. In rodents, resistin is derived almost exclusively from adipose tissue, however subsequent studies produced disparate findings regarding the role of resistin in obesity and insulin resistance in human. In contrast to rodents, resistin is expressed primarily in inflammatory cells in humans, especially macrophages. Studies have shown a potential role of resistin in atherosclerosis ,and resistin mRNA and protein have been reported to be present in atherosclerotic lesions in both huaman and mice. Resistin levels increased in patients with coronary artery disease (CAD), and it was an independent inflammatory marker of atherosclerosis in human.While data concerning resistin in different stages of CAD in Chinese people are lacking.Objective: The aim of this study was to assess whether plasma concentrations of resistin differed between patients with unstable and stable angina pectoris in China, and if any correlations existed among resistin levels, other inflammatory markers andmetabolic parameters.Methods : One hundred and forteen consecutive patients hospitalized for elective diagnostic coronary angiography were studied, including 46 patients with unstable angina (UAP), 37 with stable angina (SAP) and 31 control subjects. Clinical and biochemical characteristics were collected. The levels of high-sensitivity C-reactive protein (hs-CRP) were determined by immunoturbidometry. The endothelin-1 (ET-1) and insulin concentrations were determined by radioimmunoassay .Plasma resistin and Big endothelin concentrations were measured by ELISA.Results: Plasma concentrations of resistin were significantly increased in UAP group 12.09 (8.40,18.08)) in comparison with SAP (9.04 (7.09,11.44)) and control groups (8.71 (6.58 ,11.56)). No differences in resistin levels were found between patients with SAP and controls. We confirmed prior findings of positive correlations of resistin with age(R~2 =0.06, P =0.009), leukocyte counts(R~2 =0.05, P =0.013)and hs-CRP(R~2 =0.05, P =0.014). We also found that plasma resistin positively correlated with big-endothelin(R~2 =0.05, P=0.013) and ET-1 (R~2 =0.07, P=0.005). All of these associations remained significant after adjusting for age, sex and waist, excpt for big-endothelin.Conclusions: These findings suggested that resistin may be involved in the development of CAD through systemic inflammation and endothelial activation. Part two Effects of C-reactive on resistin expression in cultured human monocytesBackground : Atherosclerosis is an inflammatory disease. Resistin, a novel adipokine, was originally proposed as a link between obesity and insulin resistance/diabetes. Currently accumulating evidence indicates that resistin is involved in inflammatory response and atherosclerosis, and resistin is an inflammatory marker of atherosclerosis. C-reactive protein (CRP) is an important risk factor for coronary heart disease, and also modifies the expression of genes involved in atherogenesis. Consequently, CRP may not only be a nonspecific marker of inflammation, it is also possible that CRP plays a direct role in the pathogenesis of inflammation/ atherosclerosis. Clinical studies have shown a positive correlation between CRP and resistin. However whether low grade inflammation induces resistin expression or resistin is another inducer of CRP is not yet known. It is not clear about the effect of CRP on resistin expression in human monocytes.Objective: The aim of the present study was to assess the possible effects of CRP on the expression of resistin by cultured human monocytes.Methods : Human peripheral blood monocytes were isolated from the whole blood of healthy volunteers by density gradient centrifugation. For dose-dependent study, cells were incubated with varying concentrations of CRP (0, 5, 10, 25 and 50μg/ml, n=6 for each) for 24 h. To evaluate time-dependent effects of CRP, human monocytes were incubated with 25μg/ml of CRP for different time (0, 3, 6, 12, 24, n=6 for each point respectively). Quantitative real-time reverse transcription- polymerase chain reaction analyses were performed to analyze resistin mRNA gene expression.The resistin in supernatants of cultured medium was measured by ELISA. Results : After stimulation with CRP for 24 hours, resistin mRNA expression increased significantly in a concentration-dependent manner. At a concentration of 5μg/ml CRP, resistin mRNA expression increased 2.1 fold (p＜0.05) compared to unstimulated monocytes. This effect was maximal in the presence of 25μg/ml CRP (9.8 fold, p＜0.05). The effect of CRP on resistin protein was also concentration- dependent. Resistin level in supernatant of unstimulated monocytes was undetectable. But 24-hour incubation even with 5μg/ml CRP showed a significant induction of resistin protein(0.35±0.01ng/ml, p＜0.05). These changes were maximal with the concentration of 50μg/ml CRP(1.19±0.30ng/ml, p＜0.05). In a 24-hour time course study, CRP at a concentration of 25μg/mL induced resistin mRNA expression significantly within 3 hour(2.9 fold, P＜0.001), with a maximal effect at 24 hours (10 fold, P＜0.05). Resistin protein in medium was not detectable until incubation of monocytes with C RP for 6 hours (0.21±0.12ng/ml; p＜0.05). Then re sistin secretion increased with the time, reached the peak at 24 hours (0.89±0.16ng/ml; p＜0.05).Conclusions: CRP induced the expression of resistin in cultured human monocytes in a dose and time-dependent manner. Our findings strengthen the role of CRP in the pathogenesis of vascular inflammation and atherosclerosis, and may partly explain the increased levels of resistin in patients of acute coronary syndrome. Background: Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase (statins) have been widely used in medical routine practice, and they are reported to reduce greatly cardiovascular-related morbidity and mortality in patients with and without coronary disease. Recent studies have shown that statins possess anti-inflammatory properties that are independent of their lipid-lowering action. Atherosclerosis is an inflammatory disease. Resistin, a novel adipokine, was reported to be involved inflammatory response and atherosclerosis. CRP, an important risk factor for coronary heart disease, could also induce the expression of resistin in cultured human monocytes in a dose and time-dependent manner. However whether statins could modulate resistin expression induced by CRP in cultured human monocytes is not yet known.Objective: The aim of the present study was to assess the possible effects of statins on the expression of resistin induced by CRP in cultured human monocytes, as well as the possible signal pathway.Methods: Human peripheral blood monocytes were isolated from the whole blood of healthy volunteers by density gradient centrifugation. To evaluate the effects of statins on resistin production subjected to CRP, cells were pretreated with simvastain (0.1,1,10μM), or lovastain (0.1,1,10μM), or xuezhikang (25, 50,100μg/ml) respectively for 2 h, and then co-incubated with 25μg/ml CRP for 24 h. In additional experiments, isoprenoids were used to identify the mode of statins’ action. Monocytes were incubated with different statins in the absence or presence of 100μM mevalonate or 10μM geranylgeranyl-pyrophosphate (GGPP) or 10μM farnesylpyrophosphate (FPP) for 2h, then co-incubated with CRP for 24 h. Quantitative real-time reverse transcription-polymerase chain reaction analyses were performed to analyze resistin mRNA gene expression. The resistin in supematants of cultured medium was measured using commercial assay kits by ELISAResults: Pre-incubation of monocytes with different concentrations of simvastatin and lovastatin suppressed the CRP-induced resistin expression in monocytes in a dose-dependent manner. 1μM statins significantly reduced the resistin mRNA levels (simvastain 55.4±4.2%, lovstatin 59.6±5.1% of CRP-treated cells, P＜0.05), and resistin protein in medium(simvastain 0.54±0.02ng/ml, lovstatin 0.58±0.08ng/ml, vs CRP-treated cells 0.89±0.16ng/ml, P＜0.05). Further co-treatment with 100μM mevalonate or 10μM GGPP, but not FPP almost reversed completely the inhibitory effects of statins on resistin expression. 50μg/ml xuezhikang also inhibited the resistin mRNA levels(34.9±5.4% of CRP-treated cells, P＜0.01) and protein(0.47±0.07ng/ml vs 0.89±0.16ng/ml, P＜0.01)markedly. While the inhibitory effect of xuezhikang on resistin expression only partly reversed by 100μM mevalonate and 10μM GGPP.Conclusions: Resistin expression induced by CRP were inhibited by different statins through interfering with cellular isoprenoid metabolism and GGPP-dependent processes. Our results contribute to the growing volume of evidence on the anti-inflammatory and anti-atherosclerotic effects of statins, and indicate that statins may control inflammatory responses partially by inhibiting expression of resistin.更多还原