阿托伐他汀对血管内皮功能及脂多糖刺激的巨噬细胞表达TNF-α的影响
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摘要
研究背景
脓毒症(Sepsis)是感染引起的全身炎症反应征候群,也是危重病人死亡的主要原因之一,临床主要表现为系统性炎性反应综合征(Systemic inflammatory response syndrome, SIRS)与多器官功能不全(Multiple organ dysfunction syndrome, MODS)。其病理过程复杂,涉及大量细胞、炎性介质与凝血系统的激活。革兰氏阴性菌(G-)感染是引起脓毒症的重要原因之一,其外膜的活性成分内毒素即脂多糖(Lipopolysaccharide, LPS)与宿主细胞膜上的模式识别受体TLR2和TLR4 (toll-like receptor, TLR)结合,启动一系列信号转导反应,引起多种细胞因子和炎症介质的表达和释放。内毒素可激活补体与凝血系统,促激肽产生,并刺激单核巨噬细胞生成白介素-1(Interleukin-1, IL-1)、IL-6和肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)等细胞因子,在脓毒症的病理过程中发挥重要的作用。细菌破裂时,LPS被释放出来,广泛作用于机体多组织器官,其中内皮细胞、单核巨噬细胞和中性粒细胞是最主要的效应细胞。在LPS刺激下,它们产生大量的细胞因子,即“细胞因子风暴”(Cytokin storm),众多实验研究证实前炎症细胞因子TNF-α是脓毒症和脓毒症休克发病的重要介质,巨噬细胞是脓毒症TNF-α产生的主要来源,大量TNF-α的产生可引起多种细胞因子和炎症介质的过度表达和释放,进而引起失控性的炎症级联反应,包括出血、白细胞侵润、血管扩张和血浆蛋白渗出、水肿等,导致微循环障碍、有效循环血量减少,最终引起内毒素休克、组织损伤和多器官功能障碍。因此,临床控制LPS引起的炎症反应成为脓毒症治疗的重要方面,也是该领域重要的研究课题。
动脉粥样硬化(Atherosclerosis, AS)是一种多因素疾病,多年来曾有多种学说如脂质浸润学说、受体缺失学说、内皮损伤反应学说等从不同角度来阐述其发病机制,根据不同的学说也孕育了动脉粥样硬化性疾病(Atherosclerotic diseases, ASD)不同的治疗方法。目前认为AS各种主要危险因素最终都通过损伤动脉内膜来启动和促进AS, AS也即是动脉对血管内膜损伤作出的炎症纤维增生性反应。越来越多的证据表明,炎症在AS的发生和发展过程中扮演着极为重要的角色,包括炎性细胞的浸润、泡沫细胞的形成、内皮功能的障碍、脂质条纹(斑块)的出现、乃至最终冠状动脉事件的发生等均和炎症密切相关。内皮是血管的重要保护屏障,血管内皮功能障碍作为AS病理过程中的一个早期全身性改变,和巨噬细胞等参与的炎症反应贯穿着AS发生、发展的全部过程,是动脉粥样硬化性疾病发生的核心环节,也是该类疾病防治的主要靶点之一。
心脑血管疾病目前已超越肿瘤性疾病成为人类的第一杀手,而动脉粥样硬化则是心脑血管疾病的最主要原因,虽然目前的治疗方法不断进展,但其发病率和住院率仍呈逐年上升趋势,而脓毒症患者存在动脉粥样硬化基础疾病或的动脉硬化性疾病合并脓毒症的患者在临床上也日益增多。脓毒症的治疗一直是危重病领域的研究重点和热点,而对于动脉粥样硬化性疾病患者出现脓毒症的治疗因为处于危重病和心血管疾病的学科交叉领域因而一直未受到特别的关注。尽管近年来脓毒症的治疗取得了一定的进展,但脓毒症的死亡率仍然居高不下,而动脉硬化性疾病基础上合并脓毒症则进一步增加了其死亡率。因此,如何在现有的治疗条件下探索新的治疗药物和方法以缩短该类患者的住院时间和降低其致残率和死亡率是一个值得深入研究的课题。然而,新药的研发需要漫长的研究过程和巨额的研究费用。因此,近年来,探索现有药物新的治疗用途也日益成为一个研究方向。
他汀类药物即羟甲戊二酰辅酶A (HMG-CoA)还原酶抑制剂是目前临床广泛使用也是最为重要的调脂药物,是降低血脂并降低心脑血管事件的关键药物之一。目前认为,动脉粥样硬化是一种炎症性疾病,它与脓毒症的发病机制有着很多相似之处。近年来的研究表明,他汀类药物不但具有降脂效应,还具有抗炎、抗氧化、抗栓等功能,后者被认为是他汀类药物的非降脂效应。有许多研究表明,他汀类药物对心脏的保护作用并非主要来自其降脂效应,其抗炎效应在其中扮演着极为重要的作用。因此,他汀类药物作为降脂药物的同时,也被视为抗炎药物的杰出代表,各领域对其研究也逐步深入。脓毒症作为一种炎症反应性疾病,而他汀类药物具有广泛的抗炎症效应,因此,他汀类药物通过调控炎症网络中相关基因表达,减轻炎症反应,从而达到抗脓毒症效应成为一种可能。多伦多一项新的回顾性分析证明,他汀类药物的多效性可能有助于预防心血管疾病患者发生脓毒症。近来有一些小规模临床研究也表明,他汀类药物对脓毒症患者、感染性休克患者预后具有重要保护作用。这进一步提示他汀类药物可能对合并动脉粥样硬化性疾病的脓毒症患者具有很好的治疗效果。但目前尚缺乏这方面的临床和基础研究。
血红素氧合酶-1 (Hemeoxygenase-1, HO-1)是一种血红素降解的催化酶,在蛋白二硫化物还原酶(Protein-disulfide reductase, NADPH)和细胞色素P-450还原酶及分子氧作用下,HO-1催化血红素降解为胆绿素、一氧化碳(Carbon monoxide, CO)和铁,前者还原成胆红素后具有很强的抗氧化能力,后者是一种重要的信使分子。近年研究发现,HO-1及其酶解产物胆红素、CO、转铁蛋白共同发挥着抗炎、抗氧化、抑制细胞凋亡和改善组织微循环等作用,广泛参与心、脑、肺、肝、肾等组织细胞的抗氧化应激损伤,是机体最重要的内源性保护体系之一,尤其是其组织器官的保护作用已逐渐成为目前研究的一个热点。HO-1是一种保护因子,其抗炎、抗凋亡、抗增生作用在内皮细胞、心肌细胞、平滑肌细胞和其他细胞研究中已得到证实。因此,HO-1通路介导的抗炎症效应也被认为是脓毒症治疗的有效途径。
本课题拟对阿托伐他汀对炎症相关的血管内皮功能障碍的影响及其对脂多糖介导的巨噬细胞炎症反应的干预效应进行研究,并探讨该效应是否由HO-1通路介导,为明确阿托伐他汀在动脉粥样硬化性疾病合并脓毒症治疗中的价值奠定一定的理论基础。
第一章阿托伐他汀对高脂饮食兔血管内皮功能障碍的影响目的：通过高脂饮食建立新西兰兔的高脂血症和血管内皮功能障碍模型,评价炎症在其中的作用和阿托伐他汀的干预效应。方法：16只新西兰兔给予高脂饮食,随机分为单纯高脂饮食组(CTL组,无药物干预)和阿托伐他汀组(ATV组,高脂饮食联合阿托伐他汀),分别测定总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、C-反应蛋白(CRP)、白介素-6 (IL-6)、一氧化氮(NO)、内皮素-1 (ET-1)、无创超声评价的血管内皮功能的基础水平。8周后再次复查上述参数并在ATV组同时撤去阿托伐他汀和高脂饮食,并分别于撤药1、4、7天后再次测定上述参数。结果：8周的高脂饮食可使血脂和炎症标记物(CRP,IL-6)的水平明显升高,导致ET-1/NO的失衡并引起内皮功能障碍。阿托伐他汀治疗能显着抑制该效应,但并不能将其控制到基线水平。阿托伐他汀撤药1、4、7天后未能引起明显的血脂水平变化,但4天和7天后炎症标志物水平明显升高,且内皮功能障碍加重,该效应独立于血脂水平的变化。结论：高脂饮食能引起内皮功能障碍及炎症反应的增强,阿托伐他汀能显著地反向调节该效应。终止阿托伐他汀治疗可导致炎症反应的反弹增强并使内皮功能障碍加重,该效应独立于血脂水平的变化而与炎症密切相关。
第二章阿托伐他汀对脂多糖刺激小鼠巨噬细胞TNF-α表达及分泌的影响
目的：了解阿托伐他汀对LPS刺激的小鼠巨噬细胞TNF-α表达及分泌的影响和可能机制。
方法：小鼠巨噬细胞株复苏后稳定传3代后作为实验用细胞,台盼蓝染色法观察细胞活力,酶联免疫吸附法(Enzyme-linked immunospecific assay, ELISA)测定细胞培养液中TNF-α的水平,定量PCR法检测细胞TNF-α、HO-1的mRNA表达,Westernblot检测HO-1蛋白表达水平。
结果：(1)阿托伐他汀能显著提高RAW 264.7细胞HO-1的mRNA和蛋白表达水平。(2)脂多糖能显著地升高TNF-α的mRNA表达及分泌水平,并呈时间和剂量依赖性,而阿托伐他汀能抑制该效应。但SnPP (HO-1抑制剂)能减弱阿托伐他汀的该抑制效应,而对脂多糖单独刺激的RAW264.7细胞TNF-α水平无明显影响。
结论：阿托伐他汀能减弱脂多糖刺激的小鼠巨噬细胞TNF-α的表达及分泌,该效应可能由HO-1通路介导。
Background:
Sepsis, an overwhelming systemic response to infection characterized by systemic inflammation and multiple organ dysfunction syndrome, is also the primary cause for the death of critical illness. The pathological process of sepsis is very complex, involving activation of generous cells, inflammatory mediator as well as blood coagulation system. Sepsis caused by Gram-negative bacteria, and lipopolysaccharide (LPS), released from the outer membrane of Gram-negative bacteria, are considered to be the major molecule responsible for the endotoxin sepsis. Endotoxin is properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens. It is in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria. It activates the cascade of complement and coagulation, promotes the production of kinin, stimulates macrophage to produce IL-1, IL-6 and tumor necrosis factor-α, as well as other cytokines and mediators, which play important role in the pathologic processes of sepsis. LPS is released and bound to the main effector cells including endothelial cells, monocytes, macrophages, and neutrophil cells to produce a great deal of cytokines, namely "cytokines storm", and then untrolled inflammatory cascade reaction including hemorrhage, leukocytes infiltration, vasodilatation, plasma protein extravasation and edema was originated, which leads to endotoxin shock, tissue damage and multiple organ dysfunction syndrome. Accordingly, it is very important to control inflammatory response in the therapy of sepsis, and also the key study project in this field.
Atherosclerosis is a multiple-factor disease. Over the years there have been many theories from different perspectives to elaborate its pathogenesis, including lipid infiltration theory, receptor deficiency theory and endothelial injury theory. According to different theories mentioned above, the different therapy strategies of atherosclerotic disease are developed. At present, it is well accepted that AS is originated and developed due to the endothelial injury caused by the major risk factors, and AS is thought to be the artery inflammatory fibrous proliferative response to vascular endothelial injury. Increasing evidences support that inflammation plays pivotal role in the pathogenesis and development of atherosclerosis. It has been demonstrated that an inflammatory process was involved in all stages of atherosclerosis including the infiltration of inflammatory cells, formation of foam cells, endothelial dysfunction, generation of fatty streak (plaque) and the final coronary events such as acute coronary syndromes (ACS). Vascular endothelium is an important protective barrier of blood vessel. Vascular endothelial dysfunction, as an early systemic change pathological process of atherosclerosis, as well as inflammatory response participate by macrophages through the whole process of occurrence and development of the AS. Vascular endothelial dysfunction and inflammatory response are core links of the course of atherogenesis, and become the main target of preventing and treating atherosclerotic diseases. Vascular endothelial is the key protective barrier. Vascular endothelial dysfunction is an early systemic change during the course of atherosclerosis, and it together with inflammatory response mediated by macrophages participates in all the courses of atherosclerosis, whuch is also the key parts of atheroselerotic diseasesand the main target of preventing and treating atherosclerotic diseases.
Cardiovascular and cerebrovascular diseases have already excessed cancer to become the top killer of mankind, while atherosclerosis is the most important basis of cardiovascular and cerebrovascular diseases. Despite of progress of atherosclerosis treatment, the morbility and hospitalization rates remain higher and seem to be increasing, as a result, the patients with sepsis and arteriosclerotic diseases are also increasing. The treatment of sepsis has been the research hot spot of critical illness, while the treatment of the patients with sepsis and arteriosclerotic disease has not been well concerned and studied due to the cross field between critical illness and arteriosclerotic disease. Although some progress was made in the treatment of sepsis in recent years, the mortality of sepsis is still high, and the mortality of the patients with sepsis and arteriosclerotic diseases are much higher. Accordingly, it is worthwhile to search the methods to shorten the duration of hospital and reduce mortality of these patients. However, the new drug research and development needs much longer time and huge amounts of money. Thus, exploring new therapeutic effects by using the existing drugs is becoming a trend in recent years.
Statins, namelyβ-hydroxy-β-mathylglutaryl-CoA (HMG-CoA) inhibitor, is one of the most important lipid-lowing drugs widely used in clinic, and it is one of the key drugs to reduce lipids and cardiovascular events. Recent studies showed atherosclerosis is an inflammatory disease, share many similarities with the pathogenesis of sepsis. Recent studies showed that statins could not only reduce blood lipids, but also antagonize inflammation, oxidation, thrombus, which is statins' non-lipid-lowing effects. Increasing studies demonstrated that the cardiac protective effects of statins are not only due to its lipid-lowing effects, but also anti-inflammatory effects. Accordingly, statins is accepted to be the outstanding representative of anti-inflammatory drugs, and the studies about statins in many fields become deeper and deeper. Sepsis being the inflammatory disease and statins possessing wide anti-inflammatory effects, consequently, it is possible that statins antagonizes sepsis by regulating the gene expression in inflammatory network and alleviating inflammatory response. A new retrospective analysis from Toronto showed that statins might help to prevent sepsis for patients with cardiovascular disease. Recent several small-scale clinical studies demonstrated that statins had important protective effects on the prognosis of patients with sepsis and septic shock, but the mechanisms are still unclear. So far, large-scale randomed control trials are lacking due to the characteristic of patients with sepsis and septic shock.
Hemeoxygenase-1 (HO-1) is a kind of kinase to degrade hemocrystallin. With the help of NADPH, cytochrome P-450 reductase, and dioxygen, hemocrystallin is degraded to biliverdine, carbon monoxide (CO), and iron ions by HO-l's catalysis. Biliverdine is reduced as hemocrystallin with very strong anti-oxidant ability, and CO is an important messenger molecule. Recent studies showed that the co-operation of HO-1 and its degradation products including hemocrystallin, CO, and transferrin resulted in the effects to antagonize imflammation response as well as oxidation stress, inhibit cell apoptosis and improve tissue microcirculation, participating in antagonizing oxidative stress injury of tissue cells of heart, brain, lung, liver, kidney, and so on. HO-1 and its degradation products is one of the most important endogeneous protective systems, recently, protection of tissues and organs gradually becomes the research hotspot of this field. HO-1 as the protective factor, the effects against inflammation, oxidative stress, apoptosis, hyperplasia have been demonstrated in endothelial cells, cardiomyocytes, smooth muscle cells, and the other cells. Accordingly, the present study aimed to evaluate the effects of atorvastatin on lipopolysaccharide (LPS)-mediated inflammatory response and the associated mechanisms to make certain the possible value of atorvastatin in the treatment of the patients with sepsis combined arteriosclerotic disease and establish the theory base in this field.
Part 1 Pivotal role of inflammation in vascular endothelial dysfunction of hyperlipidemic rabbit and atorvastatin's effects on it
Objective:To evaluate the role of inflammation in vascular endothelial function of hyperlipidemic rabbits and atorvastatin's effects on it.
Methods:16 rabbits were divided into single high-fat diet (CTL) and atorvastatin plus high-fat diet (ATV) group. Basic levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), C-reactive protein (CRP), interleukin-6 (IL-6), nitric oxide (NO), endothelin-1 (ET-1) and endothelial function were respectively measured when grouping. Eight weeks later, all above parameters were measured again and repeated at day 1,4 and 7 after atorvastatin withdrawal.
Results:Eight-week high-fat diet could significantly induce increased blood lipids, inflammatory markers, imbalance between ET-1 and NO, and direct endothelial dysfunction determined by transcutaneous noninvasive B-type ultrasound, which could be significantly improved by atorvastatin therapy but could not be well controlled to near baseline. Abrupt withdrawal of atorvastatin caused sharp increase of inflammatory markers, and endothelial dysfunction at day 4 and 7 after atorvastatin withdrawal independent of the changes of blood lipids.
Conclusions:High-fat diet could cause endothelial dysfunction associated with inflammation, and atorvastatin could counter-regulate it. Sudden withdrawal of statins could induce rebound of inflammatory response and endothelial dysfunction independent of changes of lipids, which may be responsible for increased cardiovascular events in patients with coronary artery disease after withdrawing statins.
Part 2 The effects of Atorvastatin attenuates TNF-alpha production in LPS-stimulated RAW264.7 macrophages
Objective:To evaluate the effects of atorvastatin on the expression and secretion of TNF-αin Lps stimulated RAW264.7 macrophages and related mechanisms.
Methods:Resuscitated well-being RAW264.7 macrophages were selected for experiment, and cells'activity was evaluated by trypanblau dying. The secretion of TNF-αwere measured by enzyme linked immunosorbent assay (ELISA), and the mRNA expressions of TNF-αand heme oxygenase-1 (HO-1) were detected by real-time RT-PCR. Protein expression of HO-1 was measured by westernblot.
Results:(1) HO-1 expression could be significantly increased by atorvastatin treatment. (2)LPS could significantly increase mRNA expression of TNF-αand its secretion in dose- and time-dependent manners, which could be significantly attenuated by atorvastatin. In addition, Suppressing HO-1 activity by SnPP could significantly attenuate attorvastatin's effects on TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages, while SnPP could not significantly attenuate TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages Conclusion:Atorvastatin can attenuate LPS-induced TNF-αexpression and secretion by activating HO-1.
脓毒症(Sepsis)是感染引起的全身炎症反应征候群,也是危重病人死亡的主要原因之一,临床主要表现为系统性炎性反应综合征(Systemic inflammatory response syndrome, SIRS)与多器官功能不全(Multiple organ dysfunction syndrome, MODS)。其病理过程复杂,涉及大量细胞、炎性介质与凝血系统的激活。革兰氏阴性菌(G-)感染是引起脓毒症的重要原因之一,其外膜的活性成分内毒素即脂多糖(Lipopolysaccharide, LPS)与宿主细胞膜上的模式识别受体TLR2和TLR4 (toll-like receptor, TLR)结合,启动一系列信号转导反应,引起多种细胞因子和炎症介质的表达和释放。内毒素可激活补体与凝血系统,促激肽产生,并刺激单核巨噬细胞生成白介素-1(Interleukin-1, IL-1)、IL-6和肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)等细胞因子,在脓毒症的病理过程中发挥重要的作用。细菌破裂时,LPS被释放出来,广泛作用于机体多组织器官,其中内皮细胞、单核巨噬细胞和中性粒细胞是最主要的效应细胞。在LPS刺激下,它们产生大量的细胞因子,即“细胞因子风暴”(Cytokin storm),众多实验研究证实前炎症细胞因子TNF-α是脓毒症和脓毒症休克发病的重要介质,巨噬细胞是脓毒症TNF-α产生的主要来源,大量TNF-α的产生可引起多种细胞因子和炎症介质的过度表达和释放,进而引起失控性的炎症级联反应,包括出血、白细胞侵润、血管扩张和血浆蛋白渗出、水肿等,导致微循环障碍、有效循环血量减少,最终引起内毒素休克、组织损伤和多器官功能障碍。因此,临床控制LPS引起的炎症反应成为脓毒症治疗的重要方面,也是该领域重要的研究课题。
动脉粥样硬化(Atherosclerosis, AS)是一种多因素疾病,多年来曾有多种学说如脂质浸润学说、受体缺失学说、内皮损伤反应学说等从不同角度来阐述其发病机制,根据不同的学说也孕育了动脉粥样硬化性疾病(Atherosclerotic diseases, ASD)不同的治疗方法。目前认为AS各种主要危险因素最终都通过损伤动脉内膜来启动和促进AS, AS也即是动脉对血管内膜损伤作出的炎症纤维增生性反应。越来越多的证据表明,炎症在AS的发生和发展过程中扮演着极为重要的角色,包括炎性细胞的浸润、泡沫细胞的形成、内皮功能的障碍、脂质条纹(斑块)的出现、乃至最终冠状动脉事件的发生等均和炎症密切相关。内皮是血管的重要保护屏障,血管内皮功能障碍作为AS病理过程中的一个早期全身性改变,和巨噬细胞等参与的炎症反应贯穿着AS发生、发展的全部过程,是动脉粥样硬化性疾病发生的核心环节,也是该类疾病防治的主要靶点之一。
心脑血管疾病目前已超越肿瘤性疾病成为人类的第一杀手,而动脉粥样硬化则是心脑血管疾病的最主要原因,虽然目前的治疗方法不断进展,但其发病率和住院率仍呈逐年上升趋势,而脓毒症患者存在动脉粥样硬化基础疾病或的动脉硬化性疾病合并脓毒症的患者在临床上也日益增多。脓毒症的治疗一直是危重病领域的研究重点和热点,而对于动脉粥样硬化性疾病患者出现脓毒症的治疗因为处于危重病和心血管疾病的学科交叉领域因而一直未受到特别的关注。尽管近年来脓毒症的治疗取得了一定的进展,但脓毒症的死亡率仍然居高不下,而动脉硬化性疾病基础上合并脓毒症则进一步增加了其死亡率。因此,如何在现有的治疗条件下探索新的治疗药物和方法以缩短该类患者的住院时间和降低其致残率和死亡率是一个值得深入研究的课题。然而,新药的研发需要漫长的研究过程和巨额的研究费用。因此,近年来,探索现有药物新的治疗用途也日益成为一个研究方向。
他汀类药物即羟甲戊二酰辅酶A (HMG-CoA)还原酶抑制剂是目前临床广泛使用也是最为重要的调脂药物,是降低血脂并降低心脑血管事件的关键药物之一。目前认为,动脉粥样硬化是一种炎症性疾病,它与脓毒症的发病机制有着很多相似之处。近年来的研究表明,他汀类药物不但具有降脂效应,还具有抗炎、抗氧化、抗栓等功能,后者被认为是他汀类药物的非降脂效应。有许多研究表明,他汀类药物对心脏的保护作用并非主要来自其降脂效应,其抗炎效应在其中扮演着极为重要的作用。因此,他汀类药物作为降脂药物的同时,也被视为抗炎药物的杰出代表,各领域对其研究也逐步深入。脓毒症作为一种炎症反应性疾病,而他汀类药物具有广泛的抗炎症效应,因此,他汀类药物通过调控炎症网络中相关基因表达,减轻炎症反应,从而达到抗脓毒症效应成为一种可能。多伦多一项新的回顾性分析证明,他汀类药物的多效性可能有助于预防心血管疾病患者发生脓毒症。近来有一些小规模临床研究也表明,他汀类药物对脓毒症患者、感染性休克患者预后具有重要保护作用。这进一步提示他汀类药物可能对合并动脉粥样硬化性疾病的脓毒症患者具有很好的治疗效果。但目前尚缺乏这方面的临床和基础研究。
血红素氧合酶-1 (Hemeoxygenase-1, HO-1)是一种血红素降解的催化酶,在蛋白二硫化物还原酶(Protein-disulfide reductase, NADPH)和细胞色素P-450还原酶及分子氧作用下,HO-1催化血红素降解为胆绿素、一氧化碳(Carbon monoxide, CO)和铁,前者还原成胆红素后具有很强的抗氧化能力,后者是一种重要的信使分子。近年研究发现,HO-1及其酶解产物胆红素、CO、转铁蛋白共同发挥着抗炎、抗氧化、抑制细胞凋亡和改善组织微循环等作用,广泛参与心、脑、肺、肝、肾等组织细胞的抗氧化应激损伤,是机体最重要的内源性保护体系之一,尤其是其组织器官的保护作用已逐渐成为目前研究的一个热点。HO-1是一种保护因子,其抗炎、抗凋亡、抗增生作用在内皮细胞、心肌细胞、平滑肌细胞和其他细胞研究中已得到证实。因此,HO-1通路介导的抗炎症效应也被认为是脓毒症治疗的有效途径。
本课题拟对阿托伐他汀对炎症相关的血管内皮功能障碍的影响及其对脂多糖介导的巨噬细胞炎症反应的干预效应进行研究,并探讨该效应是否由HO-1通路介导,为明确阿托伐他汀在动脉粥样硬化性疾病合并脓毒症治疗中的价值奠定一定的理论基础。
第一章阿托伐他汀对高脂饮食兔血管内皮功能障碍的影响目的：通过高脂饮食建立新西兰兔的高脂血症和血管内皮功能障碍模型,评价炎症在其中的作用和阿托伐他汀的干预效应。方法：16只新西兰兔给予高脂饮食,随机分为单纯高脂饮食组(CTL组,无药物干预)和阿托伐他汀组(ATV组,高脂饮食联合阿托伐他汀),分别测定总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、C-反应蛋白(CRP)、白介素-6 (IL-6)、一氧化氮(NO)、内皮素-1 (ET-1)、无创超声评价的血管内皮功能的基础水平。8周后再次复查上述参数并在ATV组同时撤去阿托伐他汀和高脂饮食,并分别于撤药1、4、7天后再次测定上述参数。结果：8周的高脂饮食可使血脂和炎症标记物(CRP,IL-6)的水平明显升高,导致ET-1/NO的失衡并引起内皮功能障碍。阿托伐他汀治疗能显着抑制该效应,但并不能将其控制到基线水平。阿托伐他汀撤药1、4、7天后未能引起明显的血脂水平变化,但4天和7天后炎症标志物水平明显升高,且内皮功能障碍加重,该效应独立于血脂水平的变化。结论：高脂饮食能引起内皮功能障碍及炎症反应的增强,阿托伐他汀能显著地反向调节该效应。终止阿托伐他汀治疗可导致炎症反应的反弹增强并使内皮功能障碍加重,该效应独立于血脂水平的变化而与炎症密切相关。
第二章阿托伐他汀对脂多糖刺激小鼠巨噬细胞TNF-α表达及分泌的影响
目的：了解阿托伐他汀对LPS刺激的小鼠巨噬细胞TNF-α表达及分泌的影响和可能机制。
方法：小鼠巨噬细胞株复苏后稳定传3代后作为实验用细胞,台盼蓝染色法观察细胞活力,酶联免疫吸附法(Enzyme-linked immunospecific assay, ELISA)测定细胞培养液中TNF-α的水平,定量PCR法检测细胞TNF-α、HO-1的mRNA表达,Westernblot检测HO-1蛋白表达水平。
结果：(1)阿托伐他汀能显著提高RAW 264.7细胞HO-1的mRNA和蛋白表达水平。(2)脂多糖能显著地升高TNF-α的mRNA表达及分泌水平,并呈时间和剂量依赖性,而阿托伐他汀能抑制该效应。但SnPP (HO-1抑制剂)能减弱阿托伐他汀的该抑制效应,而对脂多糖单独刺激的RAW264.7细胞TNF-α水平无明显影响。
结论：阿托伐他汀能减弱脂多糖刺激的小鼠巨噬细胞TNF-α的表达及分泌,该效应可能由HO-1通路介导。
Background:
Sepsis, an overwhelming systemic response to infection characterized by systemic inflammation and multiple organ dysfunction syndrome, is also the primary cause for the death of critical illness. The pathological process of sepsis is very complex, involving activation of generous cells, inflammatory mediator as well as blood coagulation system. Sepsis caused by Gram-negative bacteria, and lipopolysaccharide (LPS), released from the outer membrane of Gram-negative bacteria, are considered to be the major molecule responsible for the endotoxin sepsis. Endotoxin is properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens. It is in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria. It activates the cascade of complement and coagulation, promotes the production of kinin, stimulates macrophage to produce IL-1, IL-6 and tumor necrosis factor-α, as well as other cytokines and mediators, which play important role in the pathologic processes of sepsis. LPS is released and bound to the main effector cells including endothelial cells, monocytes, macrophages, and neutrophil cells to produce a great deal of cytokines, namely "cytokines storm", and then untrolled inflammatory cascade reaction including hemorrhage, leukocytes infiltration, vasodilatation, plasma protein extravasation and edema was originated, which leads to endotoxin shock, tissue damage and multiple organ dysfunction syndrome. Accordingly, it is very important to control inflammatory response in the therapy of sepsis, and also the key study project in this field.
Atherosclerosis is a multiple-factor disease. Over the years there have been many theories from different perspectives to elaborate its pathogenesis, including lipid infiltration theory, receptor deficiency theory and endothelial injury theory. According to different theories mentioned above, the different therapy strategies of atherosclerotic disease are developed. At present, it is well accepted that AS is originated and developed due to the endothelial injury caused by the major risk factors, and AS is thought to be the artery inflammatory fibrous proliferative response to vascular endothelial injury. Increasing evidences support that inflammation plays pivotal role in the pathogenesis and development of atherosclerosis. It has been demonstrated that an inflammatory process was involved in all stages of atherosclerosis including the infiltration of inflammatory cells, formation of foam cells, endothelial dysfunction, generation of fatty streak (plaque) and the final coronary events such as acute coronary syndromes (ACS). Vascular endothelium is an important protective barrier of blood vessel. Vascular endothelial dysfunction, as an early systemic change pathological process of atherosclerosis, as well as inflammatory response participate by macrophages through the whole process of occurrence and development of the AS. Vascular endothelial dysfunction and inflammatory response are core links of the course of atherogenesis, and become the main target of preventing and treating atherosclerotic diseases. Vascular endothelial is the key protective barrier. Vascular endothelial dysfunction is an early systemic change during the course of atherosclerosis, and it together with inflammatory response mediated by macrophages participates in all the courses of atherosclerosis, whuch is also the key parts of atheroselerotic diseasesand the main target of preventing and treating atherosclerotic diseases.
Cardiovascular and cerebrovascular diseases have already excessed cancer to become the top killer of mankind, while atherosclerosis is the most important basis of cardiovascular and cerebrovascular diseases. Despite of progress of atherosclerosis treatment, the morbility and hospitalization rates remain higher and seem to be increasing, as a result, the patients with sepsis and arteriosclerotic diseases are also increasing. The treatment of sepsis has been the research hot spot of critical illness, while the treatment of the patients with sepsis and arteriosclerotic disease has not been well concerned and studied due to the cross field between critical illness and arteriosclerotic disease. Although some progress was made in the treatment of sepsis in recent years, the mortality of sepsis is still high, and the mortality of the patients with sepsis and arteriosclerotic diseases are much higher. Accordingly, it is worthwhile to search the methods to shorten the duration of hospital and reduce mortality of these patients. However, the new drug research and development needs much longer time and huge amounts of money. Thus, exploring new therapeutic effects by using the existing drugs is becoming a trend in recent years.
Statins, namelyβ-hydroxy-β-mathylglutaryl-CoA (HMG-CoA) inhibitor, is one of the most important lipid-lowing drugs widely used in clinic, and it is one of the key drugs to reduce lipids and cardiovascular events. Recent studies showed atherosclerosis is an inflammatory disease, share many similarities with the pathogenesis of sepsis. Recent studies showed that statins could not only reduce blood lipids, but also antagonize inflammation, oxidation, thrombus, which is statins' non-lipid-lowing effects. Increasing studies demonstrated that the cardiac protective effects of statins are not only due to its lipid-lowing effects, but also anti-inflammatory effects. Accordingly, statins is accepted to be the outstanding representative of anti-inflammatory drugs, and the studies about statins in many fields become deeper and deeper. Sepsis being the inflammatory disease and statins possessing wide anti-inflammatory effects, consequently, it is possible that statins antagonizes sepsis by regulating the gene expression in inflammatory network and alleviating inflammatory response. A new retrospective analysis from Toronto showed that statins might help to prevent sepsis for patients with cardiovascular disease. Recent several small-scale clinical studies demonstrated that statins had important protective effects on the prognosis of patients with sepsis and septic shock, but the mechanisms are still unclear. So far, large-scale randomed control trials are lacking due to the characteristic of patients with sepsis and septic shock.
Hemeoxygenase-1 (HO-1) is a kind of kinase to degrade hemocrystallin. With the help of NADPH, cytochrome P-450 reductase, and dioxygen, hemocrystallin is degraded to biliverdine, carbon monoxide (CO), and iron ions by HO-l's catalysis. Biliverdine is reduced as hemocrystallin with very strong anti-oxidant ability, and CO is an important messenger molecule. Recent studies showed that the co-operation of HO-1 and its degradation products including hemocrystallin, CO, and transferrin resulted in the effects to antagonize imflammation response as well as oxidation stress, inhibit cell apoptosis and improve tissue microcirculation, participating in antagonizing oxidative stress injury of tissue cells of heart, brain, lung, liver, kidney, and so on. HO-1 and its degradation products is one of the most important endogeneous protective systems, recently, protection of tissues and organs gradually becomes the research hotspot of this field. HO-1 as the protective factor, the effects against inflammation, oxidative stress, apoptosis, hyperplasia have been demonstrated in endothelial cells, cardiomyocytes, smooth muscle cells, and the other cells. Accordingly, the present study aimed to evaluate the effects of atorvastatin on lipopolysaccharide (LPS)-mediated inflammatory response and the associated mechanisms to make certain the possible value of atorvastatin in the treatment of the patients with sepsis combined arteriosclerotic disease and establish the theory base in this field.
Part 1 Pivotal role of inflammation in vascular endothelial dysfunction of hyperlipidemic rabbit and atorvastatin's effects on it
Objective:To evaluate the role of inflammation in vascular endothelial function of hyperlipidemic rabbits and atorvastatin's effects on it.
Methods:16 rabbits were divided into single high-fat diet (CTL) and atorvastatin plus high-fat diet (ATV) group. Basic levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), C-reactive protein (CRP), interleukin-6 (IL-6), nitric oxide (NO), endothelin-1 (ET-1) and endothelial function were respectively measured when grouping. Eight weeks later, all above parameters were measured again and repeated at day 1,4 and 7 after atorvastatin withdrawal.
Results:Eight-week high-fat diet could significantly induce increased blood lipids, inflammatory markers, imbalance between ET-1 and NO, and direct endothelial dysfunction determined by transcutaneous noninvasive B-type ultrasound, which could be significantly improved by atorvastatin therapy but could not be well controlled to near baseline. Abrupt withdrawal of atorvastatin caused sharp increase of inflammatory markers, and endothelial dysfunction at day 4 and 7 after atorvastatin withdrawal independent of the changes of blood lipids.
Conclusions:High-fat diet could cause endothelial dysfunction associated with inflammation, and atorvastatin could counter-regulate it. Sudden withdrawal of statins could induce rebound of inflammatory response and endothelial dysfunction independent of changes of lipids, which may be responsible for increased cardiovascular events in patients with coronary artery disease after withdrawing statins.
Part 2 The effects of Atorvastatin attenuates TNF-alpha production in LPS-stimulated RAW264.7 macrophages
Objective:To evaluate the effects of atorvastatin on the expression and secretion of TNF-αin Lps stimulated RAW264.7 macrophages and related mechanisms.
Methods:Resuscitated well-being RAW264.7 macrophages were selected for experiment, and cells'activity was evaluated by trypanblau dying. The secretion of TNF-αwere measured by enzyme linked immunosorbent assay (ELISA), and the mRNA expressions of TNF-αand heme oxygenase-1 (HO-1) were detected by real-time RT-PCR. Protein expression of HO-1 was measured by westernblot.
Results:(1) HO-1 expression could be significantly increased by atorvastatin treatment. (2)LPS could significantly increase mRNA expression of TNF-αand its secretion in dose- and time-dependent manners, which could be significantly attenuated by atorvastatin. In addition, Suppressing HO-1 activity by SnPP could significantly attenuate attorvastatin's effects on TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages, while SnPP could not significantly attenuate TNF-αexpression and secretion in LPS-stimulated RAW264.7 macrophages Conclusion:Atorvastatin can attenuate LPS-induced TNF-αexpression and secretion by activating HO-1.
引文
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[25]Li JJ, Li YS, Chu JM, Zhang CY, Wang Y, Huang Y, et al. Changes of plasma inflammatory markers after withdrawal of statin therapy in patients with hyperlipidemia. Clin Chim Acta.2006;366:269-273.
[26]Devaraj S, Chan E, Jialal I. Direct demonstration of an antiinflammatory effect of simvastatin in subjects with the metabolic syndrome. J Clin Endocrinol Metab.2006;91:4489-4496.
[27]Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, et al. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation.1998;98:839-844.
[28]Sorrentino S, Landmesser U. Nonlipid-lowering effects of statins. Curr Treat Options Cardiovasc Med.2005;7:459-466.
[29]Hoffmann R, Haager P, Suliman H, Christott P, Radke P, Blindt R, et al. Effect of statin therapy before Q-wave myocardial infarction on myocardial perfusion. American Journal of Cardiology.2008;101:139-143.
[30]Gounari P, Tousoulis D, Antoniades C, Kampoli AM, Stougiannos P, Papageorgiou N, et al. Rosuvastatin but not ezetimibe improves endothelial function in patients with heart failure, by mechanisms independent of lipid lowering. International Journal of Cardiology.2009 Feb 4 [Epub ahead of print].
[31]Spencer FA, Fonarow GC, Frederick PD, Wright RS, Every N, Goldberg RJ, et al; National Registry of Myocardial Infarction. Early withdrawal of statin therapy in patients with non-ST-segment elevation myocardial infarction: national registry of myocardial infarction. Arch Intern Med. 2004;164:2162-2168.
[32]Schouten O, Hoeks SE, Welten GM, Davignon J, Kastelein JJ, Vidakovic R, et al. Effect of statin withdrawal on frequency of cardiac events after vascular surgery. Am J Cardiol.2007; 100:316-320.
[33]Puccetti L, Pasqui AL, Pastorelli M, Bova G, Di Renzo M, Leo A, et al. Platelet hyperactivity after statin treatment discontinuation. Thromb Haemost. 2003;90:476-482.
[1]Cavaillon JM, Annane D. Compartmentalization of the inflammatory response in sepsis and SIRS. J Endotoxin Res.2006;12:151-70.
[2]Matsuda N, Hattori Y. Systemic inflammatory response syndrome (SIRS): molecular pathophysiology and gene therapy. J Pharmacol Sci. 2006;101:189-198.
[3]Herzum I, Renz H. Inflammatory markers in SIRS, sepsis and septic shock. Current Medicinal Chemistry.2008;15:581-587.
[4]Winter V, Czeslick E, Sablotzki A. Sepsis and multiple organ dysfunctions--pathophysiology and the topical concepts of treatment. Anesteziologiia i Reanimatologiia.2007:66-72.
[5]Fabiano G, Pezzolla A, Filograna MA, Ferrarese F. Traumatic shock--physiopathologic aspects. G Chir.2008;29:51-57.
[6]Willis, D., Moore, A.R., Frederick, R., Willoughby, D.A., Heme oxygenase:a novel target for the modulation of the inflammatory response. Nature Medicine. 1996;2:87-90.
[7]T.S., Chau, L.Y. Heme oxygenase-1 mediates the anti-inflammatory effect of IL-10 in mice. Nature Medicine.2002;8:240-246.
[8]Otterbein, L.E., Choi, A.M. Heme oxygenase:colors of defense against cellular stress. American Journal of Physiology:Lung Cellular & Molecular Physiology. 2000;279:L1029-L1037.
[9]Hu, C.M., Chen, Y.H., Chiang, M.T., Chau, L.Y. Heme oxygenase-1 inhibits angiotensin II-induced cardiac hypertrophy in vitro and in vivo. Circulation. 2004; 110:309-316.
[10]Dilaveris P, Giannopoulos G, Riga M, Synetos A, Stefanadis C. Beneficial effects of statins on endothelial dysfunction and vascular stiffness. Curr Vasc Pharmacol.2007;5:227-37.
[11]Damas JK, Puccetti L, Aukrust P. Early anti-thrombotic and anti-inflammatory actions of statins and fibrates--time for adjuvant therapy in acute coronary syndromes? Thromb Haemost.2005;94:1-3.
[12]Sparrow CP, Burton CA, Hernandez M. Simvastatin has anti-inflammatory and anti-atherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol.2001;21:115-121.
[13]Patel TN, Shishehbor MH, Bhatt DL. A review of high-dose statin therapy: targeting cholesterol and inflammation in atherosclerosis. Eur Heart J. 2007;28:664-672.
[14]Hanefeld M, Marx N, Pfutzner A, Baurecht W, Lubben G, Karagiannis E, Stier U, Forst T. Anti-inflammatory effects of pioglitazone and/or simvastatin in high cardiovascular risk patients with elevated high sensitivity C-reactive protein: the PIOSTAT Study. J Am Coll Cardiol.2007;49:290-297.
[15]Schmidt H, Hennen R, Keller A, Russ M, Miiller-Werdan U, Werdan K, et al. Association of statin therapy and increased survival in patients with multiple organ dysfunction syndrome. Intensive Care Medicine.2006;32:1248-1251.
[16]Gao F, Linhartova L, Johnston AM, Thickett DR. Statins and sepsis. Br J Anaesth.2008;100:288-298.
[17]Donnino MW, Cocchi MN, Howell M, Clardy P, Talmor D, Cataldo L, et al. Statin therapy is associated with decreased mortality in patients with infection. Acad Emerg Med.2009;16:230-234.
[18]Dereli Y, Ege E, Kurban S, Narin C, Sarigul A, Yeniterzi M. Pre-operative atorvastatin therapy to decrease the systemic inflammatory response after coronary artery bypass grafting. Journal of International Medical Research. 2008;36:1248-1254.
[19]Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States:analysis of incidence, outcome, and associated costs of care. Crit Care Med.2001;29:1303-10.
[20]Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med.2003;348:1546-54.
[21]Melamed A, Sorvillo FJ. The burden of sepsis-associated mortality in the United States from 1999 to 2005:an analysis of multiple-cause-of-death data. Crit Care.2009;13:R28.
[22]Annane D, Aegerter P, Jars-Guincestre MC, Guidet B;CUB-Rea Network. Current epidemiology of septic shock:the CUB-Rea Network. Am J Respir Crit Care Med.2003;168:165-72.
[23]Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD 14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990;249:1431-1433.
[24]Berry MA, Pavord ID. Antagonism of tumour necrosis factor alpha in refractory asthma. Thorax.2008;63:571-572.
[25]Marshall JC. Sepsis:current status, future prospects. Curr Opin Crit Care.2004; 10:250-64.
[26]Bernard GR, Vincent JL, Laterre PF, et al. Efficiency and safety of recombinant human activated protein C for severe sepsis. N Engl J Med.2001; 344: 699-709.
[27]Polderman KH, Girbes AR. Drug intervention trials in sepsis:divergent results. Lancet.2004; 363:1721-23.
[28]Riedemann NC, Guo RF, Ward PA:Novel strategies for the treatment of sepsis. Nat Med.2003,9:517-524.
[29]Chung SW, Liu X, Macias AA, Baron RM, Perrella MA. Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J Clin Invest.2008;118:239-247.
[30]Maeda S, Nakatsuka I, Hayashi Y, Higuchi H, Shimada M, Miyawaki T. Heme oxygenase-1 induction in the brain during lipopolysaccharide-induced acute inflammation. Neuropsychiatr Dis Treat.2008;4:663-667.
[31]Tsoyi K, Lee TY, Lee YS, Kim HJ, Seo HG, Lee JH, Chang KC. Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo. Mol Pharmacol.2009;76:173-182.
[32]Ross R. Atherosclerosis-an infl ammatory disease. N Engl J Med.1999;340:115-26.
[33]Anon. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease:the Scandinavian Simvastatin Survival Study (4S). Lancet.1994; 344:1383-89.
[34]Sacks FM, Pfeff er MA, Moye LA, et al. The eff ect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med.1996; 335:1001-09.
[35]Serruys PW, de Feyter P, Macaya C, et al. Fluvastatin for prevention of cardiac events following successful fi rst percutaneous coronary intervention:a randomized controlled trial. JAMA.2002; 287:3215-22.
[36]Shepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med.1995; 333: 1301-07.
[37]Anon. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med.1998; 339:1349-57.
[38]Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20 536 high-risk individuals:a randomised placebo-controlled trial. Lancet.2002;360:7-22.
[39]Baigent C, Keech A, Kearney PM, et al. Effi cacy and safety of cholesterol-lowering treatment:prospective meta-analysis of data from 90 056 participants in 14 randomised trials of statins. Lancet.2005; 366:1267-78.
[40]Liao JK. Beyond lipid lowering:the role of statins in vascular protection. Int J Cardiol.2002; 86:5-18.
[41]Endres M, Laufs U, Hang Z, et of Scroke protection by 3-hydroxy-3methylglutaryl (HMG)-CoA reductase inhibitors(statins) unrelated to cholesterol lowering new concepts for cardiovascular disease. Proc Natl Acad Sci USA.1998;95:8880-5.
[42]Pruefer D, Makowski J, Schnell M, et al. Simvastatin inhibits inflammatory properties of Staphylococcus aureus alpha-Toxin. Circulation.2002; 106: 2104-10.
[43]Rice JB, Stoll LL, Li WG, et al. Low-level endotoxin induces potent inflammatory activation of human blood vessels inhibition by statins. Arterroscler Thromb Vasc Biol.2003;23:1576-82.
[44]Stalker TJ, Lefer AM, Scalia R. A new HMG-CoA reductase inhibitor iosuvaststin, exerts anti-inflammatory effects on the microvascular endothelium the role of mevalonic acid Br J Pharmacol.2001;133:406-12.
[45]Steiner S, Speidl WS, Pleiner J, et al. Simvastatin blunts endotoxin—induced tissue factor in vivo. Circulation.2005;111:1841-6.
[46]Niessner A, Steiner S, Spe idl WS, et al. Simvastatin suppresses endotoxin-induced upregulation of toll-like receptors 4 and 2 in vivo. Athemsclemsis.2006; 189:408-13.
[47]Hackam D, Mamdani M, Li P, Redelmeier D. Statins and sepsis in patients with cardiovascular disease:a population-based cohort analysis. Lancet.2006; 367: 413-8.
[48]Liappis AP, Kan VL, Rochester CG, Simon GL. The effect of statins on mortality in patients with bacteremia. Clin Infect Dis.2001; 33:1352-7.
[49]Almog Y, Shefer A, Novack V, et al. Prior statin therapy is associated with a decreased rate of severe sepsis. Circulation.2004;110:880-5.
[50]Mortensen EM, Restrepo MI, Anzueto A, Pugh J. The effect of prior statin use on 30-day mortality for patients hospitalized with community-acquired pneumonia. Respir Res.2005;6:82.
[51]Kruger P, Fitzsimmons K, Cook D, Jones M, Nimmo G. Statin therapy is associated with fewer deaths in patients with bacteraemia. Intensive Care Med. 2006;32:75-9.
[2]Ridker PM, Silvertown JD. Inflammation, C-reactive protein, and atherothrombosis. Journal of Periodontology.2008;79:1544-1551.
[3]Pereira I A, Borba EF. The role of inflammation, humoral and cell mediated autoimmunity in the pathogenesis of atherosclerosis. Swiss Med Wkly. 2008;138:534-539.
[4]Milioti N, Bermudez-Fajardo A, Penichet ML, Oviedo-Orta E. Antigen-induced immunomodulation in the pathogenesis of atherosclerosis. Clin Dev Immunol. 2008;2008:723539.
[5]Niessner A, Goronzy JJ, Weyand CM. Immune-mediated mechanisms in atherosclerosis:prevention and treatment of clinical manifestations. Current Pharmaceutical Design.2007;13:3701-3710.
[6]Shah PK. Inflammation and plaque vulnerability. Cardiovasc Drugs Ther. 2009;23:31-40.
[7]Waehre T, Halvorsen B, Damas JK, Yndestad A, Brosstad F, Gullestad L, et al. Inflammatory imbalance between IL-10 and TNFalpha in unstable angina potential plaque stabilizing effects of IL-10. European Journal of Clinical Investigation.2002;32:803-810.
[8]Alam SE, Nasser SS, Fernainy KE, Habib AA, Badr KF. Cytokine imbalance in acute coronary syndrome. Curr Opin Pharmacol.2004;4:166-170.
[9]Halvorsen B, Otterdal K, Dahl TB, Skjelland M, Gullestad L,φie E, et al. Atherosclerotic plaque stability--what determines the fate of a plaque? Prog Cardiovasc Dis.2008;51:183-194.
[10]Szalai AJ, van Ginkel FW, Wang Y, McGhee JR, Volanakis JE. Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component. J Immunol.2000;165:1030-1035.
[11]Weinhold B, Bader A, Poli V, Ruther U. Interleukin-6 is necessary, but not sufficient, for induction of the humanC-reactive protein gene in vivo. Biochem J.1997;325:617-621.
[12]Noto D, Cottone S, Baldassare Cefalu A, Vadala A, Barbagallo CM, Rizzo M, et al. Interleukin 6 plasma levels predict with high sensitivity and specificity coronary stenosis detected by coronary angiography. Thrombosis and Haemostasis.2007;98:1362-1367.
[13]Hartford M, Wiklund O, Mattsson Hulten L, Persson A, Karlsson T, Herlitz J, et al. C-reactive protein, interleukin-6, secretory phospholipase A2 group ⅡA and intercellular adhesion molecule-1 in the prediction of late outcome events after acute coronary syndromes. Journal of Internal Medicine.2007;262:526-536.
[14]Ferroni P, Rosa A, Di Franco M, Palmirotta R, Guadagni F, Davi G, et al. Prognostic significance of interleukin-6 measurement in the diagnosis of acute myocardial infarction in emergency department. Clin Chim Acta. 2007;381:151-156.
[15]Niccoli G, Biasucci LM, Biscione C, Fusco B, Porto I, Leone AM, et al. Independent prognostic value of C-reactive protein and coronary artery disease extent in patients affected by unstable angina. Atherosclerosis.2008; 196:779-785.
[16]Danesh J, Kaptoge S, Mann AG, Sarwar N, Wood A, Angleman SB, et al. Long-term interleukin-6 levels and subsequent risk of coronary heart disease: two new prospective studies and a systematic review. PLoS Med.2008;5:e78.
[17]Damas JK, Puccetti L, Aukrust P. Early anti-thrombotic and anti-inflammatory actions of statins and fibrates--time for adjuvant therapy in acute coronary syndromes? Thromb Haemost.2005;94:1-3.
[18]Hanefeld M, Marx N, Pfutzner A, Baurecht W, Lubben G, Karagiannis E, et al. Anti-inflammatory effects of pioglitazone and/or simvastatin in high cardiovascular risk patients with elevated high sensitivity C-reactive protein: the PIOSTAT Study. J Am Coll Cardiol.2007;49:290-297.
[19]Patel TN, Shishehbor MH, Bhatt DL. A review of high-dose statin therapy: targeting cholesterol and inflammation in atherosclerosis. Eur Heart J. 2007;28:664-672.
[20]Sparrow CP, Burton CA, Hernandez M. Simvastatin has anti-inflammatory and anti-atherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol.2001;21:115-121.
[21]Lai WT, Lee KT, Chu CS, Voon WC, Yen HW, Tsai LY, et al. Influence of withdrawal of statin treatment on proinflammatory response and fibrinolytic activity in humans:an effect independent on cholesterol elevation. Int J Cardiol. 2005;98:459-464.
[22]Puccetti L, Pasqui AL, Pastorelli M, Bova G, Di Renzo M, Leo A, et al. Platelet hyperactivity after statin treatment discontinuation. Thromb Haemost. 2003;90:476-482.
[23]Vecchione C, Brandes RP. Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors oxidative stress and induces endothelial dysfunction in mice. Circ Res.2002;91:173-179.
[24]Laufs U, Wassmann S, Hilgers S, Ribaudo N, Bohm M, Nickenig G. Rapid effects on vascular function after initiation and withdrawal of atorvastatin in healthy, normocholesterolemic men. American Journal of Cardiology. 2001;88:1306-1307.
[25]Li JJ, Li YS, Chu JM, Zhang CY, Wang Y, Huang Y, et al. Changes of plasma inflammatory markers after withdrawal of statin therapy in patients with hyperlipidemia. Clin Chim Acta.2006;366:269-273.
[26]Devaraj S, Chan E, Jialal I. Direct demonstration of an antiinflammatory effect of simvastatin in subjects with the metabolic syndrome. J Clin Endocrinol Metab.2006;91:4489-4496.
[27]Ridker PM, Rifai N, Pfeffer MA, Sacks FM, Moye LA, Goldman S, et al. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events (CARE) Investigators. Circulation.1998;98:839-844.
[28]Sorrentino S, Landmesser U. Nonlipid-lowering effects of statins. Curr Treat Options Cardiovasc Med.2005;7:459-466.
[29]Hoffmann R, Haager P, Suliman H, Christott P, Radke P, Blindt R, et al. Effect of statin therapy before Q-wave myocardial infarction on myocardial perfusion. American Journal of Cardiology.2008;101:139-143.
[30]Gounari P, Tousoulis D, Antoniades C, Kampoli AM, Stougiannos P, Papageorgiou N, et al. Rosuvastatin but not ezetimibe improves endothelial function in patients with heart failure, by mechanisms independent of lipid lowering. International Journal of Cardiology.2009 Feb 4 [Epub ahead of print].
[31]Spencer FA, Fonarow GC, Frederick PD, Wright RS, Every N, Goldberg RJ, et al; National Registry of Myocardial Infarction. Early withdrawal of statin therapy in patients with non-ST-segment elevation myocardial infarction: national registry of myocardial infarction. Arch Intern Med. 2004;164:2162-2168.
[32]Schouten O, Hoeks SE, Welten GM, Davignon J, Kastelein JJ, Vidakovic R, et al. Effect of statin withdrawal on frequency of cardiac events after vascular surgery. Am J Cardiol.2007; 100:316-320.
[33]Puccetti L, Pasqui AL, Pastorelli M, Bova G, Di Renzo M, Leo A, et al. Platelet hyperactivity after statin treatment discontinuation. Thromb Haemost. 2003;90:476-482.
[1]Cavaillon JM, Annane D. Compartmentalization of the inflammatory response in sepsis and SIRS. J Endotoxin Res.2006;12:151-70.
[2]Matsuda N, Hattori Y. Systemic inflammatory response syndrome (SIRS): molecular pathophysiology and gene therapy. J Pharmacol Sci. 2006;101:189-198.
[3]Herzum I, Renz H. Inflammatory markers in SIRS, sepsis and septic shock. Current Medicinal Chemistry.2008;15:581-587.
[4]Winter V, Czeslick E, Sablotzki A. Sepsis and multiple organ dysfunctions--pathophysiology and the topical concepts of treatment. Anesteziologiia i Reanimatologiia.2007:66-72.
[5]Fabiano G, Pezzolla A, Filograna MA, Ferrarese F. Traumatic shock--physiopathologic aspects. G Chir.2008;29:51-57.
[6]Willis, D., Moore, A.R., Frederick, R., Willoughby, D.A., Heme oxygenase:a novel target for the modulation of the inflammatory response. Nature Medicine. 1996;2:87-90.
[7]T.S., Chau, L.Y. Heme oxygenase-1 mediates the anti-inflammatory effect of IL-10 in mice. Nature Medicine.2002;8:240-246.
[8]Otterbein, L.E., Choi, A.M. Heme oxygenase:colors of defense against cellular stress. American Journal of Physiology:Lung Cellular & Molecular Physiology. 2000;279:L1029-L1037.
[9]Hu, C.M., Chen, Y.H., Chiang, M.T., Chau, L.Y. Heme oxygenase-1 inhibits angiotensin II-induced cardiac hypertrophy in vitro and in vivo. Circulation. 2004; 110:309-316.
[10]Dilaveris P, Giannopoulos G, Riga M, Synetos A, Stefanadis C. Beneficial effects of statins on endothelial dysfunction and vascular stiffness. Curr Vasc Pharmacol.2007;5:227-37.
[11]Damas JK, Puccetti L, Aukrust P. Early anti-thrombotic and anti-inflammatory actions of statins and fibrates--time for adjuvant therapy in acute coronary syndromes? Thromb Haemost.2005;94:1-3.
[12]Sparrow CP, Burton CA, Hernandez M. Simvastatin has anti-inflammatory and anti-atherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol.2001;21:115-121.
[13]Patel TN, Shishehbor MH, Bhatt DL. A review of high-dose statin therapy: targeting cholesterol and inflammation in atherosclerosis. Eur Heart J. 2007;28:664-672.
[14]Hanefeld M, Marx N, Pfutzner A, Baurecht W, Lubben G, Karagiannis E, Stier U, Forst T. Anti-inflammatory effects of pioglitazone and/or simvastatin in high cardiovascular risk patients with elevated high sensitivity C-reactive protein: the PIOSTAT Study. J Am Coll Cardiol.2007;49:290-297.
[15]Schmidt H, Hennen R, Keller A, Russ M, Miiller-Werdan U, Werdan K, et al. Association of statin therapy and increased survival in patients with multiple organ dysfunction syndrome. Intensive Care Medicine.2006;32:1248-1251.
[16]Gao F, Linhartova L, Johnston AM, Thickett DR. Statins and sepsis. Br J Anaesth.2008;100:288-298.
[17]Donnino MW, Cocchi MN, Howell M, Clardy P, Talmor D, Cataldo L, et al. Statin therapy is associated with decreased mortality in patients with infection. Acad Emerg Med.2009;16:230-234.
[18]Dereli Y, Ege E, Kurban S, Narin C, Sarigul A, Yeniterzi M. Pre-operative atorvastatin therapy to decrease the systemic inflammatory response after coronary artery bypass grafting. Journal of International Medical Research. 2008;36:1248-1254.
[19]Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States:analysis of incidence, outcome, and associated costs of care. Crit Care Med.2001;29:1303-10.
[20]Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med.2003;348:1546-54.
[21]Melamed A, Sorvillo FJ. The burden of sepsis-associated mortality in the United States from 1999 to 2005:an analysis of multiple-cause-of-death data. Crit Care.2009;13:R28.
[22]Annane D, Aegerter P, Jars-Guincestre MC, Guidet B;CUB-Rea Network. Current epidemiology of septic shock:the CUB-Rea Network. Am J Respir Crit Care Med.2003;168:165-72.
[23]Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD 14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990;249:1431-1433.
[24]Berry MA, Pavord ID. Antagonism of tumour necrosis factor alpha in refractory asthma. Thorax.2008;63:571-572.
[25]Marshall JC. Sepsis:current status, future prospects. Curr Opin Crit Care.2004; 10:250-64.
[26]Bernard GR, Vincent JL, Laterre PF, et al. Efficiency and safety of recombinant human activated protein C for severe sepsis. N Engl J Med.2001; 344: 699-709.
[27]Polderman KH, Girbes AR. Drug intervention trials in sepsis:divergent results. Lancet.2004; 363:1721-23.
[28]Riedemann NC, Guo RF, Ward PA:Novel strategies for the treatment of sepsis. Nat Med.2003,9:517-524.
[29]Chung SW, Liu X, Macias AA, Baron RM, Perrella MA. Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbial sepsis in mice. J Clin Invest.2008;118:239-247.
[30]Maeda S, Nakatsuka I, Hayashi Y, Higuchi H, Shimada M, Miyawaki T. Heme oxygenase-1 induction in the brain during lipopolysaccharide-induced acute inflammation. Neuropsychiatr Dis Treat.2008;4:663-667.
[31]Tsoyi K, Lee TY, Lee YS, Kim HJ, Seo HG, Lee JH, Chang KC. Heme-oxygenase-1 induction and carbon monoxide-releasing molecule inhibit lipopolysaccharide (LPS)-induced high-mobility group box 1 release in vitro and improve survival of mice in LPS- and cecal ligation and puncture-induced sepsis model in vivo. Mol Pharmacol.2009;76:173-182.
[32]Ross R. Atherosclerosis-an infl ammatory disease. N Engl J Med.1999;340:115-26.
[33]Anon. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease:the Scandinavian Simvastatin Survival Study (4S). Lancet.1994; 344:1383-89.
[34]Sacks FM, Pfeff er MA, Moye LA, et al. The eff ect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med.1996; 335:1001-09.
[35]Serruys PW, de Feyter P, Macaya C, et al. Fluvastatin for prevention of cardiac events following successful fi rst percutaneous coronary intervention:a randomized controlled trial. JAMA.2002; 287:3215-22.
[36]Shepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med.1995; 333: 1301-07.
[37]Anon. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med.1998; 339:1349-57.
[38]Heart Protection Study Collaborative Group. MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20 536 high-risk individuals:a randomised placebo-controlled trial. Lancet.2002;360:7-22.
[39]Baigent C, Keech A, Kearney PM, et al. Effi cacy and safety of cholesterol-lowering treatment:prospective meta-analysis of data from 90 056 participants in 14 randomised trials of statins. Lancet.2005; 366:1267-78.
[40]Liao JK. Beyond lipid lowering:the role of statins in vascular protection. Int J Cardiol.2002; 86:5-18.
[41]Endres M, Laufs U, Hang Z, et of Scroke protection by 3-hydroxy-3methylglutaryl (HMG)-CoA reductase inhibitors(statins) unrelated to cholesterol lowering new concepts for cardiovascular disease. Proc Natl Acad Sci USA.1998;95:8880-5.
[42]Pruefer D, Makowski J, Schnell M, et al. Simvastatin inhibits inflammatory properties of Staphylococcus aureus alpha-Toxin. Circulation.2002; 106: 2104-10.
[43]Rice JB, Stoll LL, Li WG, et al. Low-level endotoxin induces potent inflammatory activation of human blood vessels inhibition by statins. Arterroscler Thromb Vasc Biol.2003;23:1576-82.
[44]Stalker TJ, Lefer AM, Scalia R. A new HMG-CoA reductase inhibitor iosuvaststin, exerts anti-inflammatory effects on the microvascular endothelium the role of mevalonic acid Br J Pharmacol.2001;133:406-12.
[45]Steiner S, Speidl WS, Pleiner J, et al. Simvastatin blunts endotoxin—induced tissue factor in vivo. Circulation.2005;111:1841-6.
[46]Niessner A, Steiner S, Spe idl WS, et al. Simvastatin suppresses endotoxin-induced upregulation of toll-like receptors 4 and 2 in vivo. Athemsclemsis.2006; 189:408-13.
[47]Hackam D, Mamdani M, Li P, Redelmeier D. Statins and sepsis in patients with cardiovascular disease:a population-based cohort analysis. Lancet.2006; 367: 413-8.
[48]Liappis AP, Kan VL, Rochester CG, Simon GL. The effect of statins on mortality in patients with bacteremia. Clin Infect Dis.2001; 33:1352-7.
[49]Almog Y, Shefer A, Novack V, et al. Prior statin therapy is associated with a decreased rate of severe sepsis. Circulation.2004;110:880-5.
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