Differential activation of heme oxygenase-1 by chalcones and rosolic acid in endothelial cells

The induction of heme oxygenase-1 (HO-1) is widely recognized as an effective cellular strategy to counteract a variety of stressful events. We have shown that curcumin and caffeic acid phenethyl ester, two naturally occurring phytochemicals that possess antioxidant, anti-inflammatory, and anticarcinogenic activities, induce HO-1 in many cell types. This suggests that stimulation of HO-1 could partly underlie the beneficial effects exerted by these plant-derived constituents. Here we examined the ability of additional plant constituents to up-regulate heme oxygenase activity and HO-1 in aortic endothelial cells. Incubation of endothelial cells with a series of polyphenolic chalcones (5-50 microM) resulted in increased heme oxygenase activity; interestingly, the chemical structure dictated the pattern of heme oxygenase induction, which was unique to each particular compound employed. We also found that rosolic acid, a constituent isolated from the rhizome of Plantago asiatica L. dramatically increased HO-1 in a concentration- and time-dependent manner. Severe cytotoxicity was observed after prolonged exposure (24 or 48 h) of cells to curcumin and caffeic acid phenethyl ester, whereas 2'-hydroxychalcone and rosolic acid did not affect cell viability. By using different mitogen-activated protein kinase inhibitors, we determined that the extracellular signal-regulated kinase, p38, and c-Jun NH(2)-terminal protein kinase pathways play only a minor role in the induction of HO-1 by rosolic acid and 2'-hydroxychalcone. On the other hand, increased intra- and extracellular thiols markedly reduced the rise in heme oxygenase activity elicited by rosolic acid. Thus, this study identified novel plant constituents that highly induce HO-1 in endothelial cells and investigated some of the mechanisms involved in this effect.     

血红素加氧酶1对模拟移植后犬肺功能的影响

背景:血红素加氧酶1是机体调节和抵抗氧化应激反应的重要生物分子,参与器官移植、缺血再灌注损伤等过程.目的:应用犬离体肺移植再灌注模型模拟肺移植后的循环过程,观察预先诱导犬肺血红素加氧酶1过表达对模拟移植犬肺功能的影响.设计、时间及地点:随机对照动物实验,于2005-12/2006-03在解放军空军总医院实验动物中心完成.材料:健康杂种犬12只用于制备犬离体肺移植再灌注模型.方法:按随机数字表法分3组(n=4):模型组即单纯肺缺血再灌注组;诱导剂组和抑制剂组预先给予血红素加氧酶l诱导剂氯化血红素,抑制剂组于再灌注时向灌注液中加入血红素加氧酶1抑制剂锌原卟啉.主要观察指标:比较分析各组犬肺组织中血红素加氧酶1 mRNA的表达水平.以及模拟移植后血气指标、肺动脉压、肺组织丙二醛含量和超氧化物歧化酶活性.结果:12只犬全部进入结果分析.①诱导剂组血红素加氧酶1 mRNA表达水平高于模型组和抑制剂组(P＜0.01).②模拟移植后各组肺动脉压均呈先增高后降低的趋势.模拟移植后30 min左右肺动脉压达高峰.③模拟移植后120 min,诱导剂组动脉血氧分压高于模型组和抑制剂组(P＜0.05),动脉血二氧化碳分压低于模型组和抑制剂组(P＜0.05):模型组与抑制剂组以上指标差异均无显著性意义(P＞0.05).④模拟移植后120min.各组丙二醛含量均升高,诱导剂组低于模型组和抑制剂组(P＜0.05):各组超氧化物歧化酶活性均下降,诱导剂组高于模型组和抑制剂组(P＜0.05).结论:过表达血红素加氧酶1能明显改善模拟移植的犬肺功能.     

Beneficial effects of heme oxygenase-1 up-regulation in the development of experimental inflammation induced by zymosan

Heme oxygenase-1 (HO-1) is part of the integrated response to oxidative stress. This enzyme may exert anti-inflammatory effects in some animal models, although the precise mechanisms are not fully understood. We have examined the role of HO-1 in the inflammatory response induced by zymosan in the mouse air pouch. Zymosan administration induced HO-1 protein expression in leukocytes migrating to exudates, with maximal levels in the late phase of this response (24-48 h). This was accompanied by ferritin induction and bilirubin accumulation, indicating that this enzyme is active in our model. HO-1 expression by zymosan treatment was partly reduced by aminoguanidine, suggesting the participation of endogenous nitric oxide in the mechanisms leading to HO-1 synthesis in the zymosan-injected mouse air pouch. Up-regulation of HO-1 by hemin administration resulted in inhibition of nitric-oxide synthase-2 activity, cellular infiltration into the air pouch exudate, and plasmatic exudation. Leukotriene B4 levels in exudates were significantly decreased in the early phase of this response (4 h), whereas interleukin-1beta and tumor necrosis factor-alpha were inhibited at all time points. Inhibition of HO-1 activity by zinc protoporphyrin IX prevented most of the effects caused by hemin administration. Our results indicate that HO-1 exerts anti-inflammatory effects on the response to zymosan in the mouse air pouch and support a role for this enzyme in the modulation of inflammatory processes.     

Peroxisome proliferator-activated receptor-gamma agonist rosiglitazone attenuates inflammatory pain through the induction of heme oxygenase-1 in macrophages

Macrophage infiltration to inflammatory sites promotes tissue repair and may be involved in pain hypersensitivity. Peroxisome proliferator-activated receptor (PPAR)γ signaling is known to regulate polarity of macrophages, which are often referred to as proinflammatory (M1) and antiinflammatory (M2) macrophages. We recently showed that the PPARγ agonist rosiglitazone ameliorated the development of postincisional hyperalgesia by increasing the influx of M2 macrophages to inflamed sites. It has been suggested that heme oxygenase (HO)-1, upregulated by PPARγ signaling, promotes differentiation of macrophages to M2 phenotype. In this study, we investigated how rosiglitazone alters pain hypersensitivity by a PPARγHO-1-dependent mechanism during the course of inflammation induced by complete Freund’s adjuvant. Local administration of rosiglitazone alleviated mechanical hyperalgesia, with increased gene induction of HO-1. Phenotype switching of infiltrated macrophages to M2 by rosiglitazone was reversed by an HO-1 inhibitor, tin protoporphyrin, at the inflamed sites. Direct stimulation of peritoneal macrophages with rosiglitazone also increased HO-1 induction in the presence of lipopolysaccharide/interferon-γ. Moreover, rosiglitazone increased gene induction of endogenous opioid proenkephalin, both at inflamed sites and in isolated macrophages. Administration of naloxone blocked the analgesic effects of rosiglitazone. We speculate that rosiglitazone alleviated the development of inflammatory pain, possibly through regulating the M1/M2 balance at the inflamed site by a PPARγ/HO-1-dependent mechanism. PPARγ signaling in macrophages may be a potential therapeutic target for the treatment of acute pain development.     

Clinical restenosis after coronary stent implantation is associated with the heme oxygenase-1 gene promoter polymorphism and the heme oxygenase-1 +99G/C variant

BACKGROUND: Vascular remodeling after percutaneous coronary stent implantation frequently leads to restenosis. Heme oxygenase 1 (HO-1) is involved in the generation of the endogenous antioxidant bilirubin and carbon monoxide, both of which exert antiinflammatory and antiproliferative effects. The aim of the present study was to evaluate the influence of genetic risk factors combined with the conventional risk factors on the development of coronary restenosis after percutaneous coronary intervention (PCI) with stent implantation. METHODS: The HO-1 gene GT dinucleotide repeat promoter polymorphism and HO-1 +99G/C variant were evaluated in 199 patients with coronary artery disease after coronary stent implantation and control angiography at 6 months after the intervention. Coronary restenosis was confirmed by quantitative angiography. RESULTS: Carriers of the long allele of the HO-1 gene promoter (>29 repeats) had a significantly higher risk of developing restenosis after PCI than noncarriers [odds ratio (OR)=1.9; 95% confidence interval (95% CI), 1.0-3.4; P=0.04]. Interestingly, the allele longer than 29 repeats conferred a significantly higher risk of developing restenosis (OR=3.4; 95% CI, 1.2-9.1; P=0.017) in nonsmokers than in smokers (OR=2.0; 95% CI, 0.7-5.2; P=0.18). CONCLUSIONS: The long allele of the HO-1 gene promoter (>29 repeats) polymorphism, which leads to low HO-1 inducibility, may represent an independent prognostic marker for restenosis after PCI and stent implantation. The effect of the >29 repeat allele is attenuated in smokers, who have chronic exogenous CO exposure.     

Dialysis membrane-induced oxidative stress: role of heme oxygenase-1

Dialysis membranes have been reported to induce monocyte apoptosis. We studied the role of oxidative stress in the induction of dialysis membrane-induced monocyte apoptosis. Superoxide dismutase, a superoxide scavenger, prevented dialysis membrane-induced monocyte apoptosis. Similarly, other antioxidants also inhibited dialysis membrane- induced apoptosis. In addition, the interaction of dialysis membranes with monocytes was associated with the generation of molecules leading to oxidative stress such as superoxide and TBARS. Interestingly, pre-induction of heme oxygenase (HO)-1 by hemin prevented dialysis membrane-induced monocyte apoptosis, whereas inhibition of HO-1 activity (treatment with tin protoporphyrin, SN-P) enhanced dialysis membrane-induced monocyte apoptosis. We suggest that oxidative injury associated with dialysis membrane and monocyte interaction plays a role in monocyte injury. Pre-induction of HO-1 may attenuate dialysis membrane-induced monocyte apoptosis.     

血红素加氧酶1分子克隆及表达载体的构建

背景:近年来许多研究发现血红素加氧酶1具有抗炎、抗氧化及抗凋亡等作用,并且在心肌缺血-再灌注损伤等应激反应中有重要的保护作用,从而成为研究热点.目的:克隆大鼠血红素加氧酶1全长基因,并将其于真核表达载体PIRES2-EGFP相连接,构建血红素加氧酶1的真核表达载体.设计、时间及地点:实验于2008-03/06在苏州大学生物技术研究所完成.材料:成年雄件SD大鼠,由苏州大学动物实验中心提供,用于脾脏细胞总RNA的提取;克隆载体PMDl8-T购自Takara公司;表达载体PIRES2-EGFP由苏州大学生物技术研究所保存.方法:分离大鼠脾脏获取脾脏细胞,Trizol法提取脾脏细胞的总RNA,经反转录一聚合酶链反应扩增获得血红素加氧酶1基因,将扩增出来的产物与克隆载体PMDl8.T连接,转化TOP10感受态细菌,挑取阳性克隆经PCR及酶切鉴定后测序确证.测序正确后抽提质粒作为模板进行PCR,Sal-Ⅰ和BamH-Ⅰ同时双酶切PCR产物和载体PIRES2-EGFP,再在T4 DNA连接酶的作用下进行连接,构建其真核表达载体.主要观察指标:血红素加氧酶1基因的克隆和DNA测序结果证实序列是否正确;PIRES2.EGFP/血红素加氧酶1真核表达载体的构建以及测序证实目的基因成功插入载体.结果;①实验完成了大鼠血红素加氧酶1基因的克隆,所获得的序列与GeneBank中收录的大鼠血红素加氧酶1序列完全一致.②构建真核表达载体PIRES2-EGFP/血红素加氧酶1,测序正确说明血红素加氧酶1基因成功插入表达载体PIRES2-EGFP中.结论:实验成功克隆了大鼠血红素加氧酶1基因全长,并构建了其真核表达载体.     

血红素氧化酶-1及氧化应激在老年性痴呆发病中的作用

新近发现血红素氧化酶(hemeoxygenase,HO)与神经变性病有着非常密切的关系。在此就HO-1生物学概况、HO、氧化应激在老年性痴呆发病中的作用、HO-1和阿尔茨海默病病理以及HO-1的检测在阿尔茨海默病中的临床意义作一综述。     

Hepatocyte growth factor modulates H2O2-induced mesangial cell apoptosis through induction of heme oxygenase-1

Oxidative stress plays an important role in the induction of mesangial cell (MC) injury. In the present study, we evaluated the molecular mechanism involved in hydrogen peroxide (H2O2)-induced MC apoptosis. In addition, we examined the role of heme oxygenase-1 (HO-1) in hepatocyte growth factor (HGF)-modulated, H2O2-induced MC injury. H2O2 promoted (p < 0.001) mouse MC (MMC) apoptosis. This effect of H2O2 was associated with translocation of cytochrome c from the mitochondrial to the cytosolic compartment. In addition, a caspase-9 inhibitor partially attenuated this effect of H2O2. These findings suggest that H2O2-induced MMC apoptosis is mediated through the mitochondrial pathway. HGF not only prevented H2O2-induced MMC apoptosis, but also inhibited H2O2-induced translocation of cytochrome c from the mitochondrial to the cytosolic compartment. HGF also promoted the expression of HO-1 by MMCs; interestingly, hemin inhibited (p < 0.001) H2O2-induced MMC apoptosis. On the other hand, zinc protoporphyrin inhibited the protective influence of HGF on H2O2-induced MMC apoptosis. These findings suggest that H2O2-induced apoptosis occurs through the mitochondrial pathway. HGF provides protection against H2O2-induced MMC apoptosis through induction of HO-1.     

Role of heme oxygenase-1 in polymyxin B-induced nephrotoxicity in rats

Polymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 ± 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 μmol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P < 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P < 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P < 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P < 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity.     

血红素氧合酶-1对内毒素休克大鼠肺组织的保护功能

目的　探讨血红素氧合酶是否对内毒素休克大鼠的肺组织有保护作用。方法　将 4 0只健康清洁级Sprague Dawley大鼠随机分为对照组 (C组 ) ,内毒素休克组 (ES组 ) ,内毒素休克 +ZnPP IX组 (EZ组 ) ,ZnPP IX组 (Z组 )。检测肺组织含水率、MDA含量和SOD活性及HO 1mRNA、HO 2mRNA、HO 1和HO 2蛋白水平的变化。结果　①ES组大鼠肺组织含水率及MDA明显低于EZ组 (P值均小于 0 0 5 ) ,但其SOD明显高于EZ组 (P <0 0 5 ) ;②EZ组大鼠其肺组织组织内HO 1mRNA表达水平及HO 1蛋白水平明显低于ES组 (P值均小于 0 0 5 ) ;而各组间肺组织组织内HO 2mRNA表达水平及HO 2蛋白水平均无显著性差异(P值均大于 0 0 5 )。结论　内毒素休克大鼠其肺组织内的HO 1可能对其发挥着保护作用     

Beneficial effects of the heme oxygenase-1/carbon monoxide system in patients with severe sepsis/septic shock

Purpose

We evaluated the relations among the arterial carbon monoxide (CO) concentration, heme oxygenase (HO)-1 expression by monocytes, oxidative stress, plasma levels of cytokines and bilirubin, and the outcome of patients with severe sepsis or septic shock.

Methods

Thirty-six patients who fulfilled the criteria for severe sepsis or septic shock and 21 other patients without sepsis during their stay in the intensive care unit were studied. HO-1 protein expression by monocytes, arterial CO, oxidative stress, bilirubin, and cytokines were measured.

Results

Arterial blood CO, cytokine, and bilirubin levels, and monocyte HO-1 protein expression were higher in patients with severe sepsis/septic shock than in non-septic patients. Increased HO-1 expression was related to the arterial CO concentration and oxidative stress. There was a positive correlation between survival and increased HO-1 protein expression or a higher CO level.

Conclusions

Arterial CO and monocyte HO-1 protein expression were increased in critically ill patients, particularly those with severe sepsis or septic shock, suggesting that oxidative stress is closely related to HO-1 expression. The HO-1/CO system may play an important role in sepsis.     

The interaction of nitric oxide with distinct hemoglobins differentially amplifies endothelial heme uptake and heme oxygenase-1 expression

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.     

血红素氧化酶1基因重组腺病毒的构建及感染效率鉴定

目的：构建含血红索氧化酶1基因的重组腺病毒及鉴定感染效率。方法：实验于2003—05／2004—06在解放军第三军医大学西南医院消化科实验室完成。①用限制性内切酶XhoⅠ＋Hind Ⅲ从克隆载体PRH01中切出血红素氧化酶1基因片段，亚克隆至Puc18中，将之用KpnⅠ＋HindⅢ双酶切，再次亚克隆至质粒pAdTrack-巨细胞病毒中，形成转移质粒pAdTrack-Pucl8-PRH01。②将pAdTrack-Pucl8-PRHO1 Pme Ⅰ酶切线性化后与腺病毒基因组质粒pAdEasy—1共转化大肠杆菌BJ5183，得到含目的基因的重组体质粒。③重组腺病毒PacⅠ酶切后用脂质体转染293细胞，包装成重组体腺病毒AdH01。④对重组体腺病毒进行鉴定采用聚合酶链反应方法。结果：①利用氯化钙法由pAdTrack-Puc18-PRHO1和pAdEasy-1共转化BJ5183感受态菌，可获得阳性重组体细菌克隆。②重组腺病毒基因组DNA，用目的基因血红素氧化酶1引物进行聚合酶链反应检测，可特异性扩增出356bp的预期条带，而空载体组未能扩增出此片段，表明血红素氧化酶1基因已克隆入重组体腺病毒基因组中，根据绿色荧光蛋白记数法测得腺病毒滴度1．4&;#215;10^13空斑形成单位／L。⑧重组腺病毒感染效率的测定结果：用浓缩后的重组腺病毒以感染复数为10感染肾小球内皮细胞，约85％的内皮细胞出现荧光，表明腺病毒AdH01在体外能有效表达相应的基因产物。结论：细菌内同源重组法获得AdH01在体外能有效表达。     

血红素氧化酶1基因重组腺病毒的构建及感染效率鉴定

目的:构建含血红素氧化酶1基因的重组腺病毒及鉴定感染效率。方法:实验于2003-05/2004-06在解放军第三军医大学西南医院消化科实验室完成。①用限制性内切酶XhoⅠ+HindⅢ从克隆载体PRH01中切出血红素氧化酶1基因片段,亚克隆至Puc18中,将之用KpnⅠ+HindⅢ双酶切,再次亚克隆至质粒pAdTrack-巨细胞病毒中,形成转移质粒pAdTrack-Puc18-PRHO1。②将pAdTrack-Puc18-PRHO1PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,得到含目的基因的重组体质粒。③重组腺病毒PacⅠ酶切后用脂质体转染293细胞,包装成重组体腺病毒AdH01。④对重组体腺病毒进行鉴定采用聚合酶链反应方法。结果:①利用氯化钙法由pAdTrack-Puc18-PRHO1和pAdEasy-1共转化BJ5183感受态菌,可获得阳性重组体细菌克隆。②重组腺病毒基因组DNA,用目的基因血红素氧化酶1引物进行聚合酶链反应检测,可特异性扩增出356bp的预期条带,而空载体组未能扩增出此片段,表明血红素氧化酶1基因已克隆入重组体腺病毒基因组中,根据绿色荧光蛋白记数法测得腺病毒滴度1.4×1013空斑形成单位/L。③重组腺病毒感染效率的测定结果:用浓缩后的重组腺病毒以感染复数为10感染肾小球内皮细胞,约85%的内皮细胞出现荧光,表明腺病毒AdH01在体外能有效表达相应的基因产物。结论:细菌内同源重组法获得AdH01在体外能有效表达。     

血红素加氧酶-1对内毒素血症大鼠胸主动脉和肺动脉反应性作用的比较

目的:比较改变血红素加氧酶-1(HO-1)水平对内毒素血症大鼠胸主动脉和肺动脉血管舒缩功能的影响.方法:将SD大鼠随机分为对照组和内毒素模型组(LPS 4 mg/kg,腹腔注射).应用RT-PCR技术检测血管HO-1 mRNA表达;应用血管张力检测技术检测各组主动脉和肺动脉血管环在HO-1抑制剂和诱导剂孵育前后反应性变化.结果:内毒素组胸主动脉与肺动脉的HO-1 mRNA均明显增高.HO-1抑制剂预孵育后,内毒素组胸主动脉对苯肾上腺素(PE)的收缩反应较孵育前提高了31.42%(P＜0.01),但对乙酰胆碱的舒张反应无显著变化;内毒素组肺动脉对PE的收缩值亦增加(P＜0.01).HO-1诱导剂预孵育后,内毒素组胸主动脉收缩性较孵育前显著下降,肺动脉收缩性较孵育前虽有下降趋势,但差异无统计学意义.结论:HO-1对内毒素血症胸主动脉和肺动脉的舒缩功能的影响存在差异.