脂多糖诱导大鼠主动脉血红素氧合酶-1表达及其对血管反应性的影响

目的：探讨血红素－HO－1－CO－cGMP道路对内毒素血症大鼠主动脉血管张力的影响及其分子机制。方法：用离体血管环张力测定技术,观察静脉注射脂多糖（LPS）6h,大鼠胸主动脉环（TARs)对苯肾上腺素（PE）累积收缩反应。分别用一氧化碳（CO）供体正缺血红素（He),血红素氧合酶－1（HO－1）抑制剂锌原卟啉（ZnPP-IX),鸟苷酸环化酶（sGC)抑制剂亚甲兰（MB）预卵育后,测定TARs对PE收缩反应的变化。分别测定主动脉中CO含量,HO－1活性,Western blot测定HO－1蛋白含量,RT－PCR检测HO－1 mRNA表达的改变。结果：LPS组TARs对PE累积收缩反应明显降低,ZnPP-IX可部分逆转低收缩反应,MB可完全逆转低收缩反应,而用He可加重低收缩反应状态;LPS组动脉组织中CO的含量上升,HO－1活性、蛋白表达量和mRNA表达均明显增加。结论：LPS可使主动脉HO－1基因表达上调,蛋白含量及酶活性明显增加,表明启动血红素－HO－1－CO－cGMP通路,是介导ES大鼠主动脉低收缩反应重要机制之一。     

保护基因HO在组织细胞中的作用及其机制研究进展

血红素加氧酶(HO)是血红素降解过程中的限速酶,将血红素降解为胆绿素、CO和游离铁。HO有三种同工酶,HO—1为诱导型,而HO—2和HO—3呈结构性表达。HO—1是一种分布广泛的应激蛋白,具有抗炎、抗凋亡、抗增生效应。各组织细胞中HO—1受不同的应激而诱导,通过上调HO—1基因表达来防御由细胞因子诱导的氧化应激和凋亡。HO—1的细胞保护机制目前尚未明确,可能涉及CO、NO等信号分子,抗凋亡基因的表达,以及NF—κB与p38MAPK信号转导途径的介导。本文就HO在组织细胞中的作用及其可能的机制进行综述。     

斑马鱼HO1基因的表达特征及功能研究

实验对血红素加氧酶1（HO1）在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%86.8%,血红素结合标签相似性为87.5%95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1 mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。       

斑马鱼 HO1 基因的表达特征及功能研究简

实验对血红素加氧酶1(HO1)在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%—86.8%,血红素结合标签相似性为87.5%—95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。     

血红素加氧酶同工酶的结构与功能

血红素加氧酶(Hemeoxygenase,HO)是一种膜结合蛋白,哺乳动物体内共发现3种同工酶,HO 1、HO 2和HO 3.HO能催化血红素降解生成α 胆绿素,铁离子和CO.这些产物都有着重要的生理作用.对HO的结构和功能研究有助于人们正确认识其催化机理和生理意义.因此从HO的催化作用、HO同工酶、HO同工酶的活性部位及HO催化的区域选择性方面作一综述.     

血红素加氧酶与炎症性疾病

血红素加氧酶(HO)是一种降解血红素的限速酶,它能催化血红素降解生成一氧化碳(CO)、胆绿素和游离铁离子。大量证据表明HO-1及其催化产物具有抗炎、抗氧化损伤、抗增生等功能,具有细胞保护作用,能使细胞产生对伤害性刺激的适应和保护性作用。炎症反应是大多数疾病共同的病理特征,研究HO与炎症的关系将为炎症性疾病的治疗开辟新的途径。     

血红素加氧酶-1在缺血/再灌注损伤中的保护作用

血红素加氧酶-1(Heme Oxygenase-1,HO-1)是催化血红素分解的关键酶。近年来,人们对血红素降解产物的抗氧化、抗炎症等功能的认识推动了对HO酶系的研究。缺血／再灌注损伤(IRI)是一个重要的临床问题,而临床上对IRI的防治尚缺乏有效的方法。目前发现HO-1过表达具有抗IRI的作用,其保护作用的可能机制有：抗氧化作用、调节微循环、调节细胞周期和抗炎症作用。     

内源性CO在缺氧性肺动脉高压大鼠肺血重构中的作用

目的和方法：应用逆转录聚合酶链式反应（RT－PCR）、双波长分光光度法,右心导管及维多利亚蓝染色方法,动态观察慢性缺氧不同时间点大鼠肺吕诱导型血红素氧合酶（HO－1）基因表达,内源性CO生成,肺动脉压力及构型的变化,探讨内源性CO大鼠缺氧性肺动脉高压肺血管重构中的作用。结果：（1）正常大鼠肺组织可表达少量HO－1mRNA,缺氧5、10、15d大鼠肺组织HO－1mRNA含量分别增加2．3、3．6、4．0倍（P＜0．01）,动脉血中COHb分别较正常大鼠增加1．2、2．6和2．9倍（P＜0．1或P＜0．05）。同时RVSP升高。光镜下可见IAPA血管壁增厚,管腔变窄。（2）Hemin可使缺氧大鼠肺组织HO－1mRNA和动脉血中COHb保持在高水平（分别高达正常对照组的5．2和3．7倍,P＜0．01或P＜0．05）,能部分地抑制缺氧时大鼠RVSP的升高,减轻IAPA的病理改变。结论：在慢性缺氧性肺动脉高压大鼠肺组织中HO－1基因的表达增加,内源性CO生成增多。Hemin促进HO－1基因表达和内源性CO手成,可抑制肺动脉压升高,阻抑制血管重构,对缺陷氧性肺动脉高压的形成有一定的防治作用。     

植物血红素加氧酶生理功能研究进展(综述)

血红素加氧酶（heme oxygenase, HO）是血红素分解代谢的关键酶类,在植物体内起着重要的作用。本文从HO在植物光敏色素合成中的作用、对植物根形态建成、提高植物抗胁迫反应能力、促进植物种子萌发和调节植物气孔关闭等方面,综述植物HO生理功能的最新研究进展,并对HO未来研究方向进行展望。     

中华按蚊血红素加氧酶基因 AsHO-1的鉴定及其在血餐后虫体中的表达动态

【目的】雌蚊需要吸食血液以完成营养生殖循环,血液蛋白的消化会释放大量的游离血红素。血红素是促氧化剂,必须严格调控血红素的动态平衡。血红素加氧酶 (heme oxygenase, HO) 在血红素的动态平衡调控中起着关键的作用,但不同昆虫HO代谢血红素的途径有差异。本研究旨在鉴定和表达分析中华按蚊 Anopheles sinensis 的 HO-1基因,研究HO-1和血红素在血餐后的动态变化过程,为进一步研究中华按蚊血红素的动态平衡调控机制奠定基础。【方法】通过分析中华按蚊的转录组数据鉴定HO-1基因,采用实时定量PCR技术分析该基因在不同组织和在取食血液、葡萄糖前后的表达谱,并测定取食血液后中肠中不同时间的血红素浓度。【结果】鉴定到中华按蚊血红素加氧酶1基因,命名为 AsHO-1（GenBank登录号为KP994552）。AsHO-1开放阅读框长729 bp,编码242个氨基酸。定量PCR表达分析发现,AsHO-1在幼虫和成虫的中肠和中肠外组织（仅去掉中肠的虫体）中都有表达,中肠外组织中的表达水平显著高于中肠中的表达水平。AsHO-1在吸食血液后的中肠中上调明显,而在中肠外组织中的表达变化较小。吸食葡萄糖后,中肠中AsHO-1在6-12 h表达上升,最高上升了4.2倍,而在吸食血液后AsHO-1在12-24 h表达上升,最高上升了11.67倍。中肠中的血红素含量在吸食血液后的3 h即开始迅速上升,6 h达到最高,18 h开始下降。【结论】研究结果说明AsHO-1在血餐后表达水平显著上调,有可能与血红素的动态调节有关,但有待进一步验证。除中肠外,AsHO-1基因还在幼虫期和在成蚊的中肠外组织中高表达,说明AsHO-1基因还可能参与中华按蚊的其他多种生理过程。     

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.     

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.     

用siRNA沉默人血红素加氧酶-1基因降低胆红素水平

拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。     

Interaction between heme oxygenase-1 and -2 proteins

The three isoforms of heme oxygenase (HO), the rate-limiting enzyme in heme degradation, are the products of different genes that show marked differences in regulation and expression. Why is there redundancy in the heme degradation pathway, and why are there differences in tissue expression of HO isoenzymes are unanswered questions? An interaction between HO-1 and HO-2 is suspected by the co-localization of these enzymes in the lung and regions of the brain. Using multiple models and assays, we demonstrated an interaction between HO-1 and HO-2 at amino acids 0-45 of HO-2 and amino acids 58-80 of HO-1. The latter corresponds to a highly conserved, hydrophilic, and exposed region of the protein. Furthermore, the observed activity of the HO-1.HO-2 complex was lower than that expected from the sum of HO-1- and HO-2-derived activities, suggesting that this interaction serves to limit HO enzymatic activity. We speculate that this HO-1.HO-2 protein interaction may promote non-enzymatic functions of HO.     

Selective induction of heme oxygenase-1 isozyme in rat testis by human chorionic gonadotropin

A radioimmunoassay was developed to assess the response of testicular HO-1 to agents known to increase the microsomal heme oxygenase activity. Treatment of rats with human chorionic gonadotropin (hCG) increased the microsomal heme oxygenase activity in rat testis. The following data suggest that the increase was specific to the HO-1 isozyme: (a) The elution profile of heme oxygenase activity from a DEAE-Sephacel column showed an increase in the HO-1 peak, but not in the HO-2 peak, (b) the Western immunoblot of the testis microsomes showed an increase in HO-1 protein, and (c) the amount of HO-1 protein that was present in the microsomes, when measured by radioimmunoassay, was doubled. Using radioimmunoassay, it was shown that other agents known to increase the testicular heme oxygenase, sodium arsenate and sodium arsenite, also increased the microsomal content of HO-1. An inhibitor of the testicular microsomal heme oxygenase activity, cadmium, also increased the microsomal HO-1 protein. The findings suggest that inducibility of HO-1 extends to tissues other than the liver, in this instance, the testis, and further support the possibility that HO-1 is the only inducible form of heme oxygenase.     

Role of heme oxygenase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to carbon monoxide, bilirubin, and iron. All three products possess biological functions; bilirubin, in particular, is a potent free radical scavenger of which its antioxidant property is enhanced at low oxygen tension. Here, we investigated the effect of severe hypoxia and reoxygenation on HO-1 expression in cardiomyocytes and determined whether HO-1 and its product, bilirubin, have a protective role against reoxygenation damage. Hypoxia caused a time-dependent increase in both HO-1 expression and heme oxygenase activity, which gradually declined during reoxygenation. Reoxygenation of hypoxic cardiomyocytes produced marked injury; however, incubation with hemin or bilirubin during hypoxia considerably reduced the damage at reoxygenation. The protective effect of hemin is attributable to increased availability of substrate for heme oxygenase activity, because hypoxic cardiomyocytes generated very little bilirubin when incubated with medium alone but produced substantial bile pigment in the presence of hemin. Interestingly, incubation with hemin also maintained high heme oxygenase activity levels during the reoxygenation period. Reactive oxygen species generation was enhanced after hypoxia, and hemin and bilirubin were capable once again to attenuate this effect. These results indicate that the HO-1-bilirubin pathway can effectively defend hypoxic cardiomyocytes against reoxygenation injury and highlight the issue of heme availability in the cytoprotective action afforded by HO-1.     

Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.     

Heme oxygenase and the kidney

Heme plays a significant pathogenic role in several diseases involving the kidney. The cellular content of heme, derived either from the delivery of filtered heme proteins such as hemoglobin and myoglobin, or from the breakdown of ubiquitous intracellular heme proteins, is regulated via the heme oxygenase enzyme system. Heme oxygenases catalyze the rate-limiting step in heme degradation, resulting in the formation of iron, carbon monoxide, and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. Recent attention has focused on the biological effects of product(s) of this enzymatic reaction, which have important antioxidant, anti-inflammatory, and cytoprotective functions. Three isoforms of heme oxygenase (HO) enzyme have been described: an inducible isoform, HO-1, and two constitutively expressed isoforms, HO-2 and HO-3. Induction of HO-1 occurs as an adaptive and beneficial response to several injurious stimuli, and has been implicated in many clinically relevant disease states including atherosclerosis, transplant rejection, endotoxic shock, hypertension, acute lung injury, acute renal injury, as well as others. This review will focus predominantly on the role of HO-1 in the kidney.     

Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1)

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The chromatographic elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 (Mr 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.