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。     

Microassay of heme oxygenase by high-performance liquid chromatography: Application to assay of needle biopsies of human liver

We developed a microassay for heme oxygenase, in which bilirubin (BR) production was measured by HPLC, and compared it to previously reported spectrophotometric methods. The microassay required as little as 5 mg wet human, rat, or chick embryo liver. Using the HPLC assay, values for heme oxygenase activity in extracts (10,000 g supernatant) of normal human liver obtained by needle biopsies were 44 +/- 7 (pmol BR.min-1.mg protein-1). Spectrophotometric assays of homogenates of human liver resulted in low values for heme oxygenase, due to unknown sources of interference. Comparative values of microsomal heme oxygenase activity were 294 +/- 25, 95 +/- 3, and 87 +/- 9 pmol BR.min-1.mg protein-1 for chick, rat, and human livers, respectively.     

Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. Biliverdin is subsequently reduced to bilirubin by the enzyme biliverdin reductase. Increasing evidence has indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. Induction of HO-1 by various chemical inducers that are primarily cell stress inducers or by HO-1 gene transfection confers a protective capacity to cultured cells as well as to cells in several in vivo animal models. In addition, HO-1-deficient mice exhibit a significant increase in susceptibility to tissue injury. The cytoprotective action of HO-1 seems to be mainly a function of the antiapoptotic effects of the enzyme. HO-1 is believed to exert this antiapoptotic action by multiple mechanisms: (a) decreased intracellular pro-oxidant levels, (b) increased bilirubin levels, and (c) elevated CO production. CO may produce an antiapoptotic effect by inhibiting both expression of p53 and release of mitochondrial cytochrome c. HO-1 may also be a target in antitumor therapy because the growth of most tumors depends on HO-1. Our preliminary studies with an HO inhibitor showed a promising antitumor effect. This preliminary work warrants continued investigation for possible novel anticancer chemotherapy.     

Carbon monoxide and bilirubin from heme oxygenase-1 suppresses reactive oxygen species generation and plasminogen activator inhibitor-1 induction

Heme oxygenase-1 (HO-1) responds to a variety of oxidative stresses. We examined whether HO-1 expression influences pro-thrombotic processes, in which the involvement of oxidative stress has been reported. Since HO-1 knockout mice with a C57/BL6J background were not viable, embryonic cells from HO-1 deficient mice (E11.5) were used. Cell viability, the level of plasminogen activator inhibitor-1 (PAI-1) expression and reactive oxygen species (ROS) generation of HO-1 deficient cells in response to the exposures to hydrogen peroxide and oxidized LDL were compared to those with wild-type cells. We also examined the effects of glutathione (GSH), desferrioxamine (DFO) and diphenyleneiodonium (DPI: an NADPH oxidase inhibitor) as well as of the HO reaction products, bilirubin (BR) and carbon monoxide (CO) on PAI-1 expression and ROS generation. PAI-1 expression and ROS generation were markedly elevated in HO-1 deficient cells compared to wild-type cells. Exposure to oxidized LDL significantly elevated PAI-1 expression and ROS production in HO-1 deficient cells. Interestingly, these increases in HO-1 deficient cells were significantly lowered by BR, CO, GSH and DPI while DFO had little effect. Furthermore, BR and CO were effective to improve viabilities of HO-1 deficient cells. These results suggest that HO-1 may be required to suppress ROS generation and the production of pro-thrombotic molecules such as PAI-1.     

Differential heme oxygenase induction by stannous and stannic ions in the heart

Heme oxygenase is the rate-limiting enzyme in heme catabolism, and is induced by oxidative stress, foreign and endogenous chemicals, and many trace elements and heavy metals. This study examined the effect of the oxidative state of the heavy metal tin, on heme oxygenase-1 induction in cardiac tissue. Subcutaneous administration of stannous and stannic chloride failed to induce the enzyme in this tissue. Atomic absorption spectroscopy revealed the absence of tin in the heart cells. Investigation of several metal formulations showed that both stannous and stannic citrate were able to enter the bloodstream from the injection site and into heart tissue. Northern blot analysis revealed that heme oxygenase-1 mRNA was elevated several-fold in rat hearts from animals which received either stannous or stannic citrate, and that mRNA levels corresponded with the increase in enzyme activity. The presence of citrate facilitated the transport of the tin ion into the blood stream and possibly across cardiac cell membrane. The stannous ion was more potent as an inducer of heme oxygenase than was the stannic ion.     

(-)-Hydroxycitric acid attenuates endoplasmic reticulum stress-mediated alterations in 3T3-L1 adipocytes by protecting mitochondria and downregulating inflammatory markers

Abstract

Endoplasmic reticulum (ER) stress is an emerging potential therapeutic target for metabolic syndrome due to its role in synthesis, secretion, and folding of proteins. It leads to an increased production of reactive oxygen species (ROS) which, along with mitochondrial dysfunction and reduced antioxidant defense, causes chronic cell injury. The present investigation aims to observe the alterations in adipocytes due to ER stress and the protective effect of hydroxycitric acid (HCA), a bioactive from Garcinia species, to develop the same as a nutraceutical. ER stress was induced in mature 3T3-L1 adipocytes by treating them with tunicamycin (2μg/ml) for 18 h. Alterations in cell viability, innate antioxidant system (superoxide dismutase, glutathione peroxidase, and glutathione reductase), mitochondria (membrane potential, biogenesis, and transition pore opening), and inflammatory cytokines (tumor necrosis factor, monocyte chemoattractant protein, interferon-γ, interleukin (IL)-10, IL-6, and IL-1β) during ER stress, and co-treatment with HCA were analyzed. Endocrine function of adipocytes was also assessed by measuring adiponectin and leptin secretion levels. HCA protected the cells from ER stress by improving the antioxidant status and mitochondrial functions. The results validate nutraceutical properties of the edible bioactive, commonly used for culinary purpose. A more detailed study on the mechanism of action of HCA is required for developing it as a therapeutic agent for metabolic syndrome.     

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.     

内源性一氧化碳在大鼠高血压发病中的作用

本实验研究内源性血红素氧化酶／一氧化碳系统在大鼠高血压发病听作用。２,４二甘油次卟啉锌是体内ＨＯ活必抑制剂 。     

Carbon Monoxide-Induced Early Thrombolysis Contributes to Heme Oxygenase-1-Mediated Inhibition of Neointimal Growth after Vascular Injury in Hypercholesterolemic Mice

Arterial thrombosis is a critical event in the pathogenesis of lesion development. In this study, we evaluated the effect of heme oxygenase-1 (HO-1), a stress-inducible enzyme with vasoprotective functions, on arterial thrombosis following vascular mechanical injury. The carotid arteries of apoE-deficient mice were subjected to angioplasty with a modified beaded-needle. Arterial thrombosis occurred at 12 h after injury. Treatment of the injured vessels with an adenovirus bearing HO-1 gene (Adv-HO-1) (1× 10⁸ pfu), but not saline or empty adenovirus (Adv), immediately after angioplasty resulted in earlier thrombolysis and restoration of blood flow detected at 24 h. Immunohistochemistry revealed that the arterial plasminogen activator inhibitor-1 (PAI-1) expression was markedly reduced in Adv-HO-1-treated injured arteries as compared to control counterparts. The thrombolytic effect was also observed by exposing animals with existing arterial thrombosis to carbon monoxide (CO) (250 ppm, 2 h), a byproduct derived from heme degradation by HO-1. In parallel with less fibrin(ogen) deposition, the macrophage infiltration, monocyte chemoattractant protein-1 expression and neointimal formation assessed at 2 weeks after angioplasty were substantially reduced in injured arteries treated with Adv-HO-1. These results support a role of early thrombolysis induced by CO in HO-1-mediated protection against intimal hyperplasia after vascular injury.     

内毒素引起的乳鼠心肌细胞血红素加氧酶-1基因的表达

为了探讨在内毒素作用下的乳鼠心肌细胞（neonatal rat cardiomyocytes,NRCMs）血红素加氧酶－1（heme oxygenase-1,HO-1）基因的表达及其在细胞损伤中的作用,分别用10、30及50μg/ml的脂多糖（lipopolysaccharide,LPS）,10μg/ml LPS 10μmol/ml锌原卟啉Ⅸ（Zn-protoporphyrin-Ⅸ,ZnPPⅨ）和单纯10μmol/ml ZnPPⅨ与培养的NRCMs共同孵育6h,以及10μg/ml LPS与NRCMs共同孵育9h和18h。分别观察细胞HO－1 mRNA表达、MDA含量、LDH释放量与台盼蓝摄取率的变化。结果显示,同样与细胞孵育6h,LPS10μg/ml时HO－1 mRNA表达比对照组增加81.2％,30μg/ml时表达量增加126.3％,50μg/ml时表达量增加92.8％;LPS为10μg/ml时,孵育9h后HO－1 mRNA的表达量比对照组增加93.6％,孵育18h后一增加105.8％。LPS30、50μg/ml,10μg/ml LPS＋10μmol/ml ZnPPⅨ与细胞孵育6h及LPS 10μg/ml孵育18h后,细胞MDA含量、LDH释放量与台盼蓝摄取率明显增加（P＜0.01）;单纯10μg/ml LPS与单纯10μmol/ml ZnPPⅨ孵育6h后,上述指标均无明显升高。结果表明,LPS可诱导NRCMs HO－1 mRNA的表达,且在较低LPS剂量范围内具有时间依赖性和浓度依赖性;NRCMs HO-1 mRNA的表达可减低LPS引起的细胞损伤,这可能是细胞产生的一种自身保护性反应。     

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。     

血红素氧合酶-1/一氧化碳通路参与辛伐他汀抗高血压诱发的大鼠心肌肥厚

为了探讨他汀类药物抑制心肌肥厚的作用机制,本研究应用一氧化氮合酶抑制剂左旋硝基精氨酸[N-nitro-L-arginine, L-NNA,15 mg／(kg·d)]制备大鼠高血压心肌肥厚模型,并分别给予不同剂量辛伐他汀[5或30 mg／(kg·d)进行干预。6周后测大鼠左心室功能、左心室重量指数(left ventricular mass index,LVMI)、心肌脑钠素(brain natriuretic peptide,BNP)含量、心肌羟脯氨酸含量和心肌血红素氧合酶(heme oxygenase,HO)活性。在体外培养的新生大鼠心肌细胞中,观察辛伐他汀对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)引起的心肌细胞肥大的抑制作用与细胞血红素氧合酶-1(HO-1)表达、HO活性及CO生成间的关系。结果表明,辛伐他汀干预明显减轻L-NNA处理大鼠的心肌肥厚(LVMI值、心肌BNP和羟脯氨酸含量均显著低于单纯L-NNA处理组),改善左心室舒张功能,而且心肌HO活性显著升高。在离体培养的原代乳鼠心肌细胞,辛伐他汀浓度依赖性地抑制Ang Ⅱ引起的细胞肥大(3H-亮氨酸掺入),并相应增加HO-1 mRNA表达、HO活性和CO生成量。应用HO抑制剂锌卟啉能有效抑制辛伐他汀抗Ang Ⅱ诱导的心肌肥大作用。结果提示:辛伐他汀上调HO-1／CO通路是其抗高血压诱发的心肌肥厚的机制之一。     

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。