Postischemic cardiac recovery in heme oxygenase‐1 transgenic ischemic/reperfused mouse myocardium

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na⁺ and Ca²⁺ gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K⁺ loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO^−/− hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.     

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified β-actin mRNA.

The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.     

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.     

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。     

Role of rice heme oxygenase in lateral root formation

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. In rice, exposure to auxin, methyl jasmonate (MJ), apocynin, and CoCl₂ has been shown to increase LR formation. This review provides evidence showing a close link between rice heme oxygenase (HO) and LR formation. The effect of auxin and MJ is nitric oxide (NO) dependent, whereas that of apocynin requires H₂O₂. The effect of CoCl₂ on the LR formation could be by some other pathway unrelated to NO and H₂O₂. This review also highlights future lines of research that should increase our knowledge of HO-involved LR formation in rice.     

Biliverdin-promoted lateral root formation is mediated through heme oxygenase in rice

In this study, we examined the effect of biliverdin (BV), a product of heme oxygenase (HO) catalyzed reaction, on lateral root (LR) formation in rice. Treatment with BV induced LR formation and HO activity. As well, BV, could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) reduced LR number, HO activity and OsHO1 mRNA level induced by BV. Our data suggest that HO is required for BV-induced LR formation in rice.     

性激素对血红素氧化酶在大鼠前列腺腹侧叶表达的影响

血红素氧化酶(heme oxygenase,HO)是产生内源性一氧化碳(carbon monoxide,CO)的限速酶,最近发现内源性CO在调节平滑肌张力方面起重要作用。而人的良性前列腺增生(benign prostates hyperplasia,BPH)所致的膀胱出口梗阻与前列腺平滑肌张力有密切关系,但还不清楚内源性HO/CO系统是否介导了前列腺平滑肌的活动。为了观察性激素对大鼠前列腺腹侧叶中血红素氧化酶-1(heme oxygenase-1,HO-1)和血红素氧化酶-2(heme oxygenase-2,HO-2)基因表达的影响,我们采用睾丸切除术建立雄性SD大鼠去势模型,用RT-PCR方法观察HO-1和HO-2的转录水平,应用免疫组织化学结合图像分析技术,观察去势、外源性雄激素和雌激素对前列腺腹侧叶中HO—1和HO-2蛋白水平的影响。结果表明,HO-1和HO-2在正常大鼠前列腺腹侧叶中都有表达,腺上皮细胞和纤维平滑肌间质呈现HO-1的免疫活性,HO-2的免疫染色仅在腺上皮细胞内检测到;去势组HO-1的mRNA和蛋白表达水平显著低于正常对照组(P<0.01):外源性给予雄激素组和雌激素组的HO-1表达水平明显增高(P<0.01),且雌激素主要增加前列腺纤维平滑肌间质的HO-1表达:HO-2在各组间的表达无明显差异(P>0.05)。这些结果提示,性激素对HO-1有诱导作用,但对HO-2无明显的影响,因此推测一氧化碳-血红素氧化酶(CO—HO)     

川芎嗪对兔肺缺血/再灌注损伤时血红素氧合酶-1表达与活性的影响

目的:探讨川芎嗪注射液对兔肺缺血/再灌注损伤时血红素氧合酶-1(HO-1)表达与活性的影响.方法:采用在体兔单肺原位缺血/再灌注损伤模型.实验兔20只,随机均分为肺缺血-再灌注损伤组(模型组)和川芎嗪注射液治疗组(川芎嗪组).用免疫组化、原位杂交方法观察HO-1在兔肺组织中的表达变化;测定肺组织湿干重比(W/D)及肺泡损伤数(IAR);电镜观察肺组织超微结构的改变.结果:HO-1在模型组、川芎嗪组肺血管内皮、部分血管平滑肌、外膜层及部分气道上皮均有阳性表达,免疫组化(原位杂交)的平均吸光度值分别为0.168±0.016(0.148±0.013)、0.186±0.014(0.158±0.012).川芎嗪组HO-1的表达水平明显高于模型组(P＜0.01),W/D、IAR值显著低于模型组(P＜0.01),肺组织形态学异常改变轻于模型组.结论:川芎嗪注射液可通过提高肺组织HO-1的表达水平,对肺缺血/再灌注损伤发挥积极的保护作用.     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

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.     

上调血红素氧合酶1对糖尿病心肌梗死大鼠心功能的远期影响

目的：研究上调血红素氧合酶1（HO-1）的表达对糖尿病心肌梗死大鼠心功能的影响及其机制。方法：将60只成年Wistar雄性大鼠随机分为5组（n=12）,分别为假手术组、糖尿病组、模型组、诱导剂组、抑制剂组。心梗造模后次日开始给药,1次／周,持续6周,术后28周,应用心脏超声、经颈动脉左心室内插管术等方法观察HO-1诱导剂钴原卟啉（CoPP）及HO活性抑制剂锡中卟啉（SnMP）干预后对心室重构及心功能各项指标的远期影响;测定血糖（GLU）、总胆红素（TB）、C反应蛋白（CRP）、血清肌酐（cr）、转氨酶（ALT）等指标;采用ELISA测定白介素-6（IL-6）、肿瘤坏死因子（TNE）、一氧化氮（NO）、前列环素（PG12）、脂联素、超敏CRP（HsCRP）等水平。结果：应用CoPP上调HO-1水平,能够改善糖尿病心梗大鼠左心室压力最大变化速率、左室射血分数、左室短轴缩短率,缩小左室舒张末期内径,升高血清胆红素、一氧化氮和前列环素水平,提高心肌组织磷酸化的内皮型一氧化氮合酶（peNOS）、磷酸化的活化蛋白激酶（pAkt）、磷酸化的腺苷活化的蛋白激酶（pAMPK）的表达,降低血清TNF-α、hs-CRP水平。使用SnMP后,能够阻断CoPP的上述作用。结论：上调HO-1通过peNOS、pAMPK途径能够长期地改善血管内皮功能,抑制炎症反应,提升血清胆红素等,有效抑制心室重塑,改善梗死后糖尿病大鼠远期的心功能。     

Induction of heme oxygenase-1 improves cold preservation effect of liver graft

We have examined the protective effect and mechanisms of heme oxygenase-1 (HO-1) induction in rat liver model of ex vivo cold ischemia preservation using cobalt protoporphyrin (CoPP) as HO-1 inducer and zinc protoporphyrin (ZnPP) as HO-1 inhibitor. There was a decrease in both aspartate transaminase and lactate dehydrogenase activities and in malondialdehyde level in liver of the CoPP-treated group compared with controls (p < 0.05). In the CoPP-treated rats, the histological signs of reperfusion injury were much lower than in control. Up-regulation of HO-1 expression was also associated with reduced levels of tumor necrosis factor α and interleukin-6. Markedly fewer apoptotic liver cells (determined by TUNEL assay) could be detected in CoPP-treated group compared with the control group. These protective effects were prevented by administration of ZnPP. In conclusion, induction of HO-1 provides protection against liver injury during cold ischemia preservation and improves the preservation of liver graft. The mechanisms underlying these beneficial effects include reduction of oxidative injury and of inflammatory response and prevention of apoptosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 674–681.     

Photic regulation of heme oxygenase activity in the golden hamster retina: involvement of dopamine

The photic regulation of heme oxygenase (HO) activity was examined in the golden hamster retina. This enzymatic activity was significantly higher at midday than at midnight. When the hamsters were placed under constant darkness for 48 h and killed at subjective day or at subjective night, the differences in HO activity disappeared. Western blot analysis showed no differences in HO levels among these time points. Dopamine significantly increased this activity in retinas excised at noon or at midnight, with a higher sensitivity at night. The effect of dopamine was reversed by SCH 23390 but not by spiperone and clozapine and it was not reproduced by quinpirole. In vitro, the increase in HO activity found in retinas incubated under light for 1 h was significantly reduced by SCH 23390. Two cAMP analogs increased HO activity and their effect, as well as the effect of dopamine was blocked by H-89, a protein kinase A (PKA) inhibitor. Tin protoporphyrin IX, an HO inhibitor, significantly decreased cGMP accumulation with maximal effects during the day. Low concentrations of bilirubin decreased retinal thiobarbituric acid substances levels (an index of lipid peroxidation) in basal conditions and after exposing retinal cells to H2O2. These results suggest that hamster retinal HO activity is regulated by the photic stimulus, probably through a dopamine/cAMP/PKA dependent pathway.     

Gene transfer of human heme oxygenase into coronary endothelial cells potentially promotes angiogenesis

Heme oxygenase (HO-1) is a stress protein that has been suggested to participate in defense mechanisms against agents that induce oxidative injury such as hemoglobin/heme, hypoxia-ischemia and cytokines. Overexpression of HO-1 in endothelial cells (EC) might, therefore, protect against oxidative stress produced under these pathological conditions, by generation of CO, a vasodilator, and bilirubin, which has antioxidant properties that enhance blood vessel formation to counteract hypoxia-induced injury. A plasmid containing the cytomegalovirus promoter (pCMV) neomycin human HO-1 gene complexed to cationic liposomes, lipofectin, was used to transfect rabbit coronary microvessel EC. Cells transfected with human HO-1 gene demonstrated a twofold increase in HO activity and maintained a similar phenotype as in the nontransfected cells. Cell number in transfected cells with human HO-1 gene increased by about 45%, as compared to nontransfected or those transfected with control pCMV. Transfected and nontransfected EC revealed a similar response to basic fibroblast growth factor (bFGF) in capillary formation. However, transfected cells with the human HO-1 gene exhibited a twofold increase in blood vessel formation. The angiogenic response of EC to overexpression of HO-1 gene provides direct evidence that the inductive form of HO-1 following injury represents an important tissue adaptive mechanism for moderating the severity of cell damage produced in inflammatory reaction sites of hemorrhage, thrombosis and hypoxic-ischemia. Thus, HO-1 may participate in the regulation of EC activation, proliferation and angiogenesis. J. Cell. Biochem. 68:121–127, 1998. © 1998 Wiley-Liss, Inc.     

Guanosine protects C6 astroglial cells against azide‐induced oxidative damage: a putative role of heme oxygenase 1

Guanosine, a guanine‐based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide‐induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor‐κB activation and the release of proinflammatory cytokines tumor necrosis factor α and IL‐1β. Guanosine strongly prevented these effects, protecting glial cells against azide‐induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage.

     

CO-trapping site in heme oxygenase revealed by photolysis of its co-bound heme complex: mechanism of escaping from product inhibition

Heme oxygenase (HO) catalyzes physiological heme degradation using O(2) and reducing equivalents to produce biliverdin, iron, and CO. Notably, the HO reaction proceeds without product inhibition by CO, which is generated in the conversion reaction of alpha-hydroxyheme to verdoheme, although CO is known to be a potent inhibitor of HO and other heme proteins. In order to probe how endogenous CO is released from the reaction site, we collected X-ray diffraction data from a crystal of the CO-bound form of the ferrous heme-HO complex in the dark and under illumination by a red laser at approximately 35 K. The difference Fourier map indicates that the CO ligand is partially photodissociated from the heme and that the photolyzed CO is trapped in a hydrophobic cavity adjacent to the heme pocket. This hydrophobic cavity was occupied also by xenon, which is similar to CO in terms of size and properties. Taking account of the affinity of CO for the ferrous verdoheme-HO complex being much weaker than that for the ferrous heme complex, the CO derived from alpha-hydroxyheme would be trapped preferentially in the hydrophobic cavity but not coordinated to the iron of verdoheme. This structural device would ensure the smooth progression of the subsequent reaction, from verdoheme to biliverdin, which requires O(2) binding to verdoheme.     

Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium

Myocardial adaptation to ischemia involves up-regulated expression of a number of genes implicated in conferring cytoprotection. We have previously shown that myocardial ischemia followed by reperfusion leads to a co-ordinated expression of mRNAs encoding heme oxygenase-1 (HO-1) and ubiquitin in pigs. HO-1 participates in biological reaction leading to the formation of the antioxidant, bilirubin and the putative cellular messenger, carbon monoxide. In the present study, we examined the expression and cellular localization of HO-1 in the heart during myocardial stunning in anesthetized pigs. After thoracotomy, the LAD was occluded for 10 min and reperfused for 30 min (group I, n = 4), again occluded for 10 min and reperfused for 30 min (group II, n = 6), 90 min (group III, n = 4), 210 min (group IV, n = 5) and for 390 min (group V, n = 4). Myocardial tissue specimens were collected in 10% formalin as well as in liquid nitrogen and processed for immunohistochemistry and mRNA expression analysis, respectively. In the distribution territory of the LAD (experimental, E), systolic wall thickening was significantly decreased (39 ± 6%) as compared to that of the area perfused by left circumflex coronary artery (LCx, control) in group I and remained depressed in all subsequent groups. Northern blot analysis revealed that the expression of a single mRNA species of 1.8 kb encoding HO-1 was significantly induced in E as compared to control in groups II and III with maximum mRNA levels in group II (1.9 ± 0.4 fold vs. control). Immunoreactive HO-1 was localized in the cytoplasm of cardiomyocytes as well as in the perivascular regions in all groups. Semiquantitative analysis of HO-1 staining showed significantly enhanced levels of HO-1 in perivascular region in E as compared to respective controls derived from groups III and IV. These results suggest that myocardial adaptive response to ischemia involves up-regulation of HO-1 in cells of perivascular region indicating that this enzyme may participate in regulating vascular tone via CO and thereby, contributing in pathophysiologically important defense mechanism(s) in the heart.     

细胞自噬在大鼠缺血/再灌注肺损伤中的调控作用

目的: 探讨细胞自噬在大鼠缺血/再灌注肺损伤中的作用。方法: 随机将40只SD大鼠分为5组(n=8),分别为 ① 假手术组(Sham组):只开胸3.5 h;② 缺血/再灌注组(I/R组):开胸夹闭肺门缺血0.5 h后再灌注3 h;③ 溶剂组(DMSO组):术前1 h腹腔注射DMSO溶液;④自噬激动剂组(Rap组):术前腹腔注射雷帕霉素溶液;⑤自噬抑制剂组(3-MA组):术前1 h腹腔注射3-MA溶液;后三组的其余操作同I/R组。实验结束后处死大鼠,取肺组织,记录并计算肺组织湿/干重比(W/D)、总肺含水量变化(TLW) ,光镜和电镜观察肺组织及细胞形态,计算肺泡损伤率(IAR),Western blot检测自噬相关蛋白的表达情况。结果: 相对于sham组,其余四组肺W/D、TLW、IAR均明显升高,自噬相关蛋白表达明显上升,p-AMPK、Beclin 1、LC3 II 蛋白明显增多,p-mTOR、p62蛋白明显减少(P＜0.05或P＜0.01),光镜下其余各组肺组织有不同程度的水肿渗出,肺泡结构紊乱,电镜下细胞超微结构损伤加重,部分可见自噬小体;与DMSO组相比,3-MA组肺W/D、TLW、IAR明显下降(P＜0.05或P＜0.01),自噬相关蛋白表达明显下降,肺间质水肿较轻,细胞渗出较少,细胞超微结构损伤减轻,未见自噬小体。而I/R、DMSO、Rap组的各项指标变化无统计学差异(P＞0.05)。结论: 肺缺血/再灌注可诱发细胞自噬增强,从而引起大鼠肺损伤。