可溶性环氧化物水解酶抑制剂在心血管领域中的研究进展

在哺乳动物系统中的可溶性环氧化物水解酶属于α/β水解酶类折叠家族的一个成员,它对环氧脂肪酸具有高度的选择性。环氧花生四烯酸或环氧二十碳三烯酸是其特异性底物。环氧二十碳三烯酸是内皮衍生超极化因子的主要组成成分。因此,环氧二十碳三烯酸具有舒张血管及降低血压作用,同时还具有抗炎、促进纤溶、调节血管生长等多种强大生物学效应,其水解酶的抑制剂—可溶性环氧化物水解酶抑制剂可以通过减少环氧二十碳三烯酸降解、增加环氧二十碳三烯酸水平从而模拟这些作用。因此,可溶性环氧化物水解酶抑制剂作为具有治疗高血压、动脉粥样硬化和炎症性疾病可能性的药物被广泛研究。     

可溶性环氧化物水解酶抑制剂TPPU改善阿尔茨海默病的作用和机制

目的 利用小鼠模型探讨新型可溶性环氧化物水解酶(sEH)抑制剂(TPPU)改善阿尔茨海默病(AD)的分子机制。方法 将5xFAD转基因小鼠随机分为WT溶剂组、5xFAD溶剂组和5xFAD-TPPU干预,5xFAD-TPPU干预组每日腹腔注射1.5 mg/kg体质量TPPU,连续注射8 w。免疫荧光染色观察β淀粉样蛋白水平和小胶质细胞活化。Y迷宫检测小鼠认知功能。逆转录聚合酶链反应(RT-PCR)检测白细胞介素(IL)-6、IL-β和肿瘤坏死因子(TNF)-α三种炎症因子mRNA水平。高效液相和质谱联用技术检测环氧四烯酸(EETs)水平。Western印迹检测给药前后Toll样受体(TLR)2及其下游信号分子表达。结果 外源性给予TPPU可显著抑制小胶质细胞的活化和炎症因子的释放,并且能够改善AD小鼠病理症状。与WT溶剂组相比,5xFAD溶剂组TLR2相关信号通路异常,给予TPPU后,可有效抑制TLR2信号通路的激活。结论 sEH抑制剂TPPU可能通过TLR2信号通路抑制小胶质细胞的过度激活,从而改善AD的病理症状。     

可溶性环氧化物水解酶抑制剂t-AUCB对巨噬细胞脂质代谢的影响

目的观察可溶性环氧化物水解酶抑制剂t-AUCB对小鼠巨噬细胞脂质摄取及降解的影响,并探明其可能机制。方法培养小鼠巨噬细胞RAW264.7,分别用不同浓度t-AUCB(1、10、50及100μmol/L)干预24 h,或在100μmol/L t-AUCB干预前1 h加入PPARγ拮抗剂GW9662 5μmol/L,采用t-AUCB 0μmol/L干预组作为空白对照。采用125Ⅰ-ox-LDL放射配基法测定小鼠巨噬细胞对氧化型低密度脂蛋白(ox-LDL)的摄取及降解,实时荧光定量PCR和Western blot分别测定小鼠巨噬细胞CD36 mRNA和蛋白表达。结果 t-AUCB呈剂量依赖性地增加巨噬细胞125Ⅰ-ox-LDL摄取量和降解量,0、1、10、50和100μmol/L t-AUCB干预时,摄取量分别为414.96±46.71μg/g、519.54±47.7μg/g、629.04±37.97μg/g、720.66±48.58μg/g和881.57±68.44μg/g,降解量分别为16180.23±967.28μg/g、17369.62±478.34μg/g、21794.85±689.36μg/g、27883.03±712.25μg/g和30194.61±635.71μg/g,与空白对照组比较差异显著(P<0.05),加入GW9662后100μmol/L组摄取量降至467.80±51.98μg/g,降解量降至16326.19±735.95μg/g,与单独加入t-AUCB组比较差异具有显著性(P<0.05);t-AUCB可呈剂量依赖性的增加小鼠巨噬细胞CD36 mRNA和蛋白的表达,而加入GW9662后明显抑制上述作用。结论 t-AUCB可通过上调PPARγ-CD36信号通路分子表达增加小鼠巨噬细胞摄取和降解ox-LDL。     

RNA干扰下调小鼠心肌细胞可溶性环氧化物水解酶的表达

目的构建含有可溶性环氧化物水解酶基因特异性的RNA干扰序列pSUPER retro neo质粒载体,选择性下调小鼠心肌细胞可溶性环氧化物水解酶的表达,筛选出抑制效果最明显的表达质粒。方法构建2条靶向可溶性环氧化物水解酶基因的特异性小干扰RNA质粒载体(EH-1和EH-2),以含非特异性小干扰RNA编码序列的质粒载体为阴性对照(pCN组),用FuGENE HD将质粒转染入原代培养的小鼠心肌细胞,并设空白对照组,转染后通过半定量RT-PCR和Western Blotting法,检测可溶性环氧化物水解酶的mRNA和蛋白表达情况。结果与空白对照组、阴性对照组和EH-1组相比,质粒EH-2使心肌细胞可溶性环氧化物水解酶mRNA和蛋白的相对表达量明显下调(分别为0.202±0.017和0.212±0.029,P<0.01)。结论构建了特异性小干扰RNA质粒表达载体,利用RNA干扰技术成功下调了原代培养的心肌细胞中可溶性环氧化物水解酶的表达,为进一步进行心肌细胞的RNA干扰研究奠定了基础。     

RNA干扰下调可溶性环氧化物水解酶表达对异丙肾上腺素诱导心肌细胞凋亡的影响

目的:构建含有可溶性环氧化物水解酶(sEH)基因特异性的RNA干扰序列质粒,选择性下调小鼠心肌细胞sEH的表达,筛选出抑制sEH效果最明显的表达质粒;观察其对异丙基肾上腺素(ISO)诱导的心肌细胞的凋亡及相关基因的影响.方法:构建2条靶向sEH基因的特异性小干扰RNA(siRNA)质粒 EH-1和 EH-2,以含非特异性siRNA编码序列的质粒(PCN)为阴性对照,用FuGENE HD转染原代培养的心肌细胞,通过半定量RT-PCR和Western blot法检测sEH的mRNA和蛋白的表达情况,筛选出抑制sEH效果最明显的表达质粒,命名为EH-R.采用浓度为10μmol*L-1ISO诱导心肌凋亡.分为正常对照组、ISO组、PCN+ISO组和EH-R+ISO组.利用EH-R下调心肌细胞sEH基因的表达,观察其对ISO诱导的心肌细胞凋亡的影响,流式细胞仪检测各组细胞凋亡的发生率,Western blot法检测各组Bcl-2和Bax蛋白的表达变化.结果:质粒EH-R组心肌细胞sEH的mRNA和蛋白质表达较其余组明显降低,差异有统计学意义(P<0.01).与正常对照组相比,应用ISO各组,心肌细胞凋亡率明显升高,心肌细胞的Bax表达升高,而Bcl-2表达下降(P<0.01).然而EH-R+ISO组与ISO组和PCN+ISO组相比心肌细胞凋亡率明显降低;心肌Bax表达降低,Bcl-2表达升高(P<0.01).结论:构建了特异性小干扰RNA质粒,利用RNAi技术成功下调了原代培养的心肌细胞中sEH的表达,从而增加了抑凋亡基因Bcl-2的表达,减轻了ISO诱导心肌细胞的凋亡.为进一步进行心肌细胞的RNA干扰研究奠定了基础.     

可溶性环氧化物水解酶抑制剂通过过氧化体增殖物激活型受体γ调节内皮祖细胞功能

目的研究可溶性环氧化物水解酶抑制剂t-AUCB对小鼠来源内皮祖细胞(EPC)功能的调节及相关机制。方法密度梯度离心法分离培养小鼠骨髓来源EPC,不同浓度t-AUCB及过氧化体增殖物激活型受体γ(PPARγ)阻断剂GW9662预干预EPC,检测上述EPC体外增殖、黏附、迁移、血管生成情况。结果在1~100μmol/L范围内,t-AUCB呈浓度依赖性增强EPC体外增殖、黏附、迁移、血管生成能力,而5μmol/L PPARγ阻断剂GW9662可抑制EPC上述功能。结论可溶性环氧化物水解酶抑制剂t-AUCB参与EPC增殖、黏附、迁移、血管生成等功能的调控,其机制可能与激活PPARγ通路有关。     

EETs的心血管效应及心肌保护

研究表明,环氧-二十碳三烯酸(EETs)是花生四烯酸经细胞色素P450氧化代谢途径的产物之一,可作用于多种心血管细胞的离子通道,具有强大的心血管效应,本文就其心血管效应及心肌保护的意义作一综述.     

微粒体环氧化物水解酶与肺疾病

微粒体环氧化物水解酶(mEH)是一种生物转化酶,催化范围广泛的各种外源性环氧化物水解,形成更易溶于水的反式二氢二醇,是参与化学去毒和/或活化过程的重要控制点。近来发现该酶与慢性阻塞性肺病和肺气肿关系密切,是可能的易感因素。本文就mEH的分子生物学研究进展及其在肺部疾病中的作用与机制作一介绍。     

细胞色素P450基因对高脂饮食诱导不同周龄小鼠动脉粥样硬化的保护作用

目的研究血管内皮高表达CYP2C8基因能否改善小鼠动脉粥样硬化。方法以20和40周龄APOEKO+/-CYP2C8Tg+/-和CYP2C8Tg+/-基因(血管内皮特异性CYP2C8+/-转基因)的小鼠(n=10/组)为研究对象,相同基因背景和周龄的同窝APOEKO+/-和C57BL/6小鼠设为对照。采用PCR技术鉴定小鼠CYP2C8+/-基因;油红O染色检测APOEKO+/-CYP2C8Tg+/-、CYP2C8Tg+/-、APOEKO+/-和C57BL/6小鼠主动脉斑块形成面积;酶比色检测APOEKO+/-CYP2C8Tg+/-、CYP2C8Tg+/-、APOEKO+/-和C57BL/6小鼠血清TG、TCH、HDL。结果 20和40周龄APOEKO+/-CYP2C8Tg+/-和CYP2C8Tg+/-小鼠主动脉斑块形成面积与同周龄野生型小鼠相比明显减少,并且明显改善了血脂代谢状态。结论高表达CYP2C8基因能够改善西方饮食诱导的不同周龄小鼠的动脉粥样硬化。     

Soluble Epoxide Hydrolase Inhibitors and Heart Failure

Cardiovascular disease remains one of the leading causes of death in the Western societies. Heart failure (HF) is due primarily to progressive myocardial dysfunction accompanied by myocardial remodeling. Once HF develops, the condition is, in most cases, irreversible and is associated with a very high mortality rate. Soluble epoxide hydrolase (sEH) is an enzyme that catalyzes the hydrolysis of epoxyeicosatrienoic acids (EETs), which are lipid mediators derived from arachidonic acid through the cytochrome P450 epoxygenase pathway. EETs have been shown to have vasodilatory, antiinflammatory, and cardioprotective effects. When EETs are hydrolyzed by sEH to corresponding dihydroxyeicosatrienoic acids, their cardioprotective activities become less pronounced. In line with the recent genetic study that has identified sEH as a susceptibility gene for HF, the sEH enzyme has received considerable attention as an attractive therapeutic target for cardiovascular diseases. Indeed, sEH inhibition has been demonstrated to have antihypertensive and antiinflammatory actions, presumably due to the increased bioavailability of endogenous EETs and other epoxylipids, and several potent sEH inhibitors have been developed and tested in animal models of cardiovascular disease including hypertension, cardiac hypertrophy, and ischemia/reperfusion injury. sEH inhibitor treatment has been shown to effectively prevent pressure overload‐ and angiotensin II‐induced cardiac hypertrophy and reverse the pre‐established cardiac hypertrophy caused by chronic pressure overload. Application of sEH inhibitors in several cardiac ischemia/reperfusion injury models reduced infarct size and prevented the progressive cardiac remodeling. Moreover, the use of sEH inhibitors prevented the development of electrical remodeling and ventricular arrhythmias associated with cardiac hypertrophy and ischemia/reperfusion injury. The data published to date support the notion that sEH inhibitors may represent a promising therapeutic approach for combating detrimental cardiac remodeling and HF.     

Cardiovascular Therapeutic Aspects of Soluble Epoxide Hydrolase Inhibitors

Soluble epoxide hydrolase (sEH) is an enzyme responsible for the conversion of lipid epoxides to diols by the addition of water. Biological actions on the cardiovascular system that are attributed to epoxides include vasodilation, antiinflammatory actions and vascular smooth muscle cell antimigratory actions. Conversion of arachidonic acid epoxides to diols by sEH diminishes the beneficial cardiovascular properties of these epoxyeicosano‐ids. Cardiovascular diseases in animal models and humans have been associated with decreased epoxygenase activity or increased sEH activity and these changes are responsible for the progression of the disease state. More recently, sEH gene polymorphisms in the human population have been associated with increased risk for cardiovascular diseases. Thus the biological actions of epoxyeicosanoids and the sEH enzyme are ideal therapeutic targets for cardiovascular diseases. The rapid development of 1,3‐disubstituted urea based sEH inhibitors over the past five years has resulted in a number of studies demonstrating cardiovascular protection. sEH inhibitors have antihypertensive and antiinflammatory actions and have been demonstrated to decrease cerebral ischemic and renal injury in rat models of hypertension. These findings of beneficial actions in animal models of disease position the sEH enzyme as a promising therapeutic target for cardiovascular diseases.     

Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression.Depression is the most severe and debilitating of the psychiatric illnesses. The World Health Organization estimates that more than 350 million individuals of all ages suffer from depression (1). Almost one million lives are lost annually because of suicide, which translates to 3,000 deaths daily (1). Although antidepressants are generally effective in the treatment of depression, it can still take weeks before patients feel the full antidepressant effects. However, approximately two-thirds of depressed patients fail to respond fully to pharmacotherapy. Furthermore, there is a high rate of relapse, and depressed patients have a high risk of committing suicide (2–4).Accumulating evidence suggests that inflammation plays a central role in the pathophysiology of depression (5–9). Meta-analyses showed higher blood levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), in drug-free depressed patients compared with healthy controls (10–13). Studies using postmortem brain samples showed elevated gene expression of proinflammatory cytokines in the frontal cortex of people with a history of depression (14, 15). Taking these data together, we find that it is likely that both peripheral and central inflammations are associated with depression and that antiinflammatory drugs, such as cyclooxygenase inhibitors, could ameliorate depressive symptoms in depressed patients (16, 17).Epoxyeicosatrienoic acids (EETs), which are produced from arachidonic acid by the action of cytochrome P450s, have potent antiinflammatory actions. These mediators are broken down into the corresponding diols by soluble epoxide hydrolase (sEH), and inhibition of sEH enhances the beneficial effects of EETs (18–21). It is also reported that sEH inhibitors have potent antiinflammatory effects in a number of animal models (18–20, 22, 23). Although sEH has been associated with the onset of anorexia nervosa (24), the role of sEH in the pathophysiology of depression has not been studied to date.The purpose of this study was to examine the role of sEH in the pathophysiology of depression using a potent sEH inhibitor and sEH knockout (KO) mice. Furthermore, we examined the role of brain-derived neurotrophic factor (BDNF) and its receptor TrkB signaling in selected brain regions, because BDNF-TrkB signaling plays a key role in the pathophysiology of depression (25–30).     

基于心血管系统疾病的量表研制进展

患者报告的临床结局直接来源于患者,可以体现患者最关心的症状和问题,捕捉细微的心理变化和功能改变,作为临床疗效的补充。心血管疾病患者,尤其慢性病程的患者,往往客观指标改善以后,自我症状不缓解,借助量表可以有效补充疗效评价,因此,关于心血管系统疾病的评价量表种类繁多,兹归类综述,供临床参考。     

结缔组织生长因子在心血管疾病的作用及研究进展

结缔组织生长因子是一种新发现的可刺激成纤维细胞增殖和胶原沉积的生长因子,结缔组织生长因子具有广泛的生物学功能如促纤维化、促有丝分裂、趋化作用、促细胞增殖,诱导凋亡以及促血管新生等。结缔组织生长因子在多种细胞均可表达,在高血压、心力衰竭、动脉粥样硬化病变,心肌梗死以及心肌炎等各种心血管疾病中结缔组织生长因子表达均明显增高,并可能通过其多种生物学行为参与心血管重塑、心脏纤维化和动脉粥样硬化病变的发生发展。     

The Relationship between Age of Asthma Onset and Cardiovascular Disease in Canadians

Purpose: To quantify the association between cardiovascular disease (CVD) and asthma in Canadian adults and to determine whether age of asthma onset is a moderator of this association. Methods: We used a sample of 74 342 participants with a mean age of 56.4 ± 12.5 from cycle 1.1 of the Canadian Community Health Survey. Asthma age of onset was categorized into early-onset (0–20 years) and adult-onset (21–54 years). Three major outcomes were used to estimate the relationship between asthma and CVD, namely: high blood pressure, heart disease, and stroke. Results: Multiple logistic regression models revealed that asthmatics were 43% (OR = 1.43, CI = 1.19–1.72) more likely to have heart disease, and 36% (OR = 1.36, CI = 1.21–1.53) more likely to have high blood pressure than non-asthmatics. There were no consistent results for age of onset with high blood pressure, heart disease, or stroke. Conclusion: Using a population-based dataset we confirmed that asthmatics are at increased odds of cardiovascular disease compared to non-asthmatics; furthermore, age of asthma onset did not appear to moderate this relationship. Future research should focus on determining whether asthma severity or allergic/non-allergic phenotypes have a differential effect on the asthma-CVD relationship.