血红素氧合酶-1与脑出血的继发性损害

自发性脑出血是指非外伤性脑实质出血,发病率高,死亡率高[1],且脑出血后患者多遗留不同程度的神经功能障碍。因此,探讨脑出血后脑组织损伤的病理生理机制对于改善脑出血病情及预后是十分必要的。大量研究表明自发性脑出血后造成的脑损伤存在多种机制[2]:早期血肿机械占位效应、     

血红素氧合酶-1在大鼠蛛网膜下腔出血后脑血管中的表达

目的探讨蛛网膜下腔出血(SAH)后脑血管中血红素氧合酶-1(HO-1)的表达变化及其与迟发性脑血管痉挛(DCV)之间的关系。方法采用大鼠枕大池二次注血法建立SAH后DCV模型,用逆转录聚合酶链反应(RT-PCR)检测基底动脉HO-1mRNA表达的变化。结果注水对照组3、5、7d基底动脉内径周长分别为(996.20±43.25)μm、(1019.05±58.16)μm和(965.25±49.98)μm,血管壁厚分别为(9.82±0.57)μm、(9.65±0.65)μm和(10.11±0.48)μm;SAH组3、5、7d基底动脉内径周长分别为(705.65±66.57)μm、(738.70±42.19)μm和(665.31±49.85)μm,血管壁厚分别为(14.41±0.51)μm、(13.25±0.63)μm和(17.43±0.55)μm。注水对照组不同时相基底动脉中无HO-1mRNA表达;SAH组注血后3、5、7d,HO-1mRNA的相对表达量分别为0.61±0.042、0.55±0.039和0.48±0.052,以注血后3d表达水平最高。结论SAH后大鼠基底动脉有HO-1mRNA的低表达,但其不能拮抗SAH后DCV的发生。     

Ammonia-induced heme oxygenase-1 expression in cultured rat astrocytes and rat brain in vivo

Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE), which is a major complication in acute and chronic liver failure and other hyperammonemic states. The molecular mechanisms underlying ammonia neurotoxicity and the functional consequences of ammonia on gene expression in astrocytes are incompletely understood. Using cDNA array hybridization technique we identified ammonia as a trigger of heme oxygenase-1 (HO-1) mRNA levels in cultured rat astrocytes. As shown by Northern and Western blot analysis, HO-1 mRNA levels were upregulated by ammonia (0.1-5 mmol/L) after 24 h and protein expression after 72 h in astrocytes. These ammonia effects on HO-1 are probably triggered to a minor extent by ammonia-induced glutamine synthesis or by astrocyte swelling, because HO-1 expression was not inhibited by the glutamine synthetase inhibitor methionine sulfoximine (which abrogated ammonia-induced cell swelling in cultured astrocytes), and ammonia-induced HO-1 expression could only partly be mimicked by hypoosmotic astrocyte swelling. Hypoosmotic (205 mOsm/L) exposure of astrocytes led even to a decrease in HO-1 mRNA levels within 4 h, whereas hyperosmotic (405 mOsm/L) exposure increased HO-1 mRNA expression. After 24 h, hypoosmolarity slightly raised HO-1 mRNA expression. Taurine and melatonin diminished ammonia-induced HO-1 mRNA or protein expression, whereas other antioxidants (dimethylthiourea, butylated hydroxytoluene, N-acetylcysteine, and reduced glutathione) increased HO-1 mRNA levels under ammonia-free conditions. An in vivo relevance is suggested by the finding that increased HO-1 expression occurs in the brain cortex from acutely ammonia-intoxicated rats. It is concluded that ammonia-induced HO-1 expression may contribute to cerebral hyperemia in hyperammonic states.     

N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury

Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI.     

血红素氧合酶-1及血红素氧合酶-2在大鼠局灶性脑缺血中的意义

目的　研究血红素氧合酶 1(HO 1)及血红素氧合酶 2 (HO 2 )在局灶性脑缺血中的作用。方法　采用大鼠大脑中动脉栓塞脑缺血模型 ,对 6 6只大鼠脑缺血后不同时间点进行HO 1、HO 2免疫组化染色及病理学研究 ,并用计算机图像分析技术计算两者表达水平。结果　栓塞后 30min大鼠皮质及海马即有HO 1阳性神经元及胶质细胞的表达 ,且随着时间推移HO 1的表达逐渐增强 ,到栓塞后 12h达峰值 (P <0 0 1) ,以后逐渐下降 ,栓塞后 1周仍有HO 1表达。HO 2在正常大鼠及梗死大鼠脑组织内均有表达。栓塞后不同时间段 ,HO 2阳性神经元的数量无明显变化 (P >0 0 5 ) ,但HO 2表达呈动态变化 ,2 4h时最高 (P <0 0 1) ,以后逐渐下降。结论　脑缺血时脑内HO 1、HO 2表达的不同变化 ,是脑组织对损伤恢复重要的机制之一。HO 1修复受损的神经元和胶质细胞 ,而HO 2在于维护正常细胞的稳定     

血红素氧合酶-1/一氧化碳系统在新生大鼠缺氧缺血性脑损伤中的作用

目的 观察新生大鼠缺氧缺血性脑损伤(HIBD)后血红素氧合酶-1/一氧化碳系统(HO-1/CO)变化,探讨其在HIBD中的作用.方法 7日龄Wistar新生大鼠按随机数字表法分为假手术组(Sham组)、缺氧缺血组(HIBD组)及HO抑制剂锌原卟啉组(Znpp组),每组6只.采用实时荧光定量PCR法、硫代巴比妥酸法(TBA法)、流式细胞术(FCM)和双波长定量测定法分别检测脑组织中HO-1 mRNA的表达、丙二醛(MDA)含量、脑组织细胞凋亡率及血中CO浓度.结果 与Sham组比较,HIBD组、Znpp组HO-1 mRNA表达均增强(分别为0.166±0.042、2.289±0.333、1.839±0.322),CO浓度升高[分别为(0.460±0.009)%、(1.026±0.145)%、(0.735±0.079)%],差异有统计学意义(P＜0.05);但与HIBD组比较,Znpp组HO-1 mRNA表达明显减少,CO浓度降低,差异有统计学意义(P＜0.05).与Sham组比较,HIBD组、Znpp组MDA含量及细胞凋亡率均明显升高[MDA含量分别为(1.016±0.210)nmol/mg prot、(1.945±0.312)nmol/mg prot、(3.202±0.693)nmol/mg prot,凋亡率分别为(0.108±0.009)%、(1.412±0.307)%、(2.458±0.565)%],差异有统计学意义(P＜0.05);与HIBD组比较,Znpp组MDA含量及细胞凋亡率明显增高,差异有统计学意义(P＜0.05).结论 缺氧缺血性脑损伤后HO-1/CO系统可能在脑损伤的恢复中起到一定的保护作用.     

Neuroinflammation associates with antioxidant heme oxygenase-1 response throughout the brain in persons living with HIV

Journal of NeuroVirology - Previous studies showed that persons living with HIV (PLWH) demonstrate higher brain prefrontal cortex neuroinflammation and immunoproteasome expression compared to...     

Sustained induction of heme oxygenase-1 in the traumatized spinal cord

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.     

血红素氧合酶-2基因缺失对氧化应激性脑损伤的保护作用

目的 探讨血红素氧合酶-2基因缺失对血红素诱导氧化应激性脑损伤的保护作用.方法 分别将6 μl (8 μmol/L)灭菌氯高铁血红素定向注入野生型小鼠和基因(HO-2)敲除小鼠的纹状体内,72 h后分别检测纹状体细胞生存率,蛋白和脂类的氧化作用.用蛋白质印迹法检测血红素氧合酶-1,2(HO-1)的表达.结果 与野生型相比,基因(HO-2)敲除小鼠纹状体内蛋白和脂类的氧化作用显著降低,而纹状体细胞的存活率显著增加;HO-1的表达在两种小鼠注射前后没有明显差异.结论 结果提示,血红素氧合酶-2基因缺失对血红素诱导的氧化应激性脑损伤具有保护作用;选择性抑制神经元血红素氧合酶-2基因的表达可减轻氧化应激性脑损伤.     

Astrocyte-specific heme oxygenase-1 hyperexpression attenuates heme-mediated oxidative injury

In prior studies, we have observed that HO activity protects astrocytes from heme-mediated injury, but paradoxically increases neuronal injury. In this study, we tested the hypothesis that an adenovirus encoding the human HO-1 gene driven by an enhanced glial fibrillary acidic protein promoter (Ad-GFAP-HO-1) would increase HO-1 expression selectively in astrocytes, and provide cytoprotection. Treatment with 100 MOI Ad-GFAP-HO-1 for 24 h resulted in HO-1 expression that was 6.4-fold higher in cultured primary astrocytes than in neurons. Astrocyte HO activity was increased by approximately fourfold over baseline, which was sufficient to reduce cell death after 24-h hemin exposure by 60%, as assessed by both MTT and LDH release assays. A similar reduction in cell protein oxidation, quantified by carbonyl assay, was also observed. These results suggest that HO-1 transgene expression regulated by an enhanced GFAP promoter selectively increases HO-1 expression in astrocytes, and is cytoprotective. Further investigation of this strategy in vivo is warranted.     

Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier

The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood–brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood–brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.     

Impaired spatial navigation learning in transgenic mice over-expressing heme oxygenase-1

Transgenic mice expressing heme oxygenase-1 (HO-1) using the neuron-specific enolase promoter were impaired in learning the Morris water maze compared to nontransgenic littermates. The memory of the HO-1 mice for the location of the platform was similarly impaired when tested using a probe trial after 7 training blocks, but performance on visible platform trials was similar for both groups of mice. Importantly, both HO-1 and nontransgenic mice had normal sensorimotor function, and performed the same on a Y-maze alternation task, highlighting the specificity of memory deficit in the spatial navigation task. These results suggest that carbon monoxide, one product of HO-1 activity, interferes in the development of spatial navigation memory, and may play a role in normal memory function.     

Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury.

Heme oxygenase-1 (HO-1) is a stress protein induced in response to a variety of oxidative challenges. After treatment of the hybrid septal cells SN 56 with beta-amyloid peptide (beta-AP1-40) or hydrogen peroxide (H2O2), we detected high levels of reactive oxygen species, accompanied by a significant elevation in HO-1 expression. Levels of HO-1 increased and then decreased following cell loss. Pretreatment of SN 56 cells with HO-1 antisense oligonucleotides dramatically decreased the immunoreactivity of HO-1 and significantly enhanced the cytotoxicity of beta-AP1-40 and H2O2. In contrast, pretreatment with hemin, an HO-1 inducer, increased the expression of HO-1 and decreased the beta-AP1-40- and H2O2-induced cytotoxicity. These findings support the importance of HO-1 in protecting neurons against oxidative stress-induced injury.     

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：成年雄性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PMD18-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-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PMD18-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。     

丹参对大鼠移植肝血红素氧合酶1表达的影响*

背景：器官移植前使用丹参预处理能够保护组织缺血-再灌注损伤,改善移植器官存活率。 目的：观察含丹参的冷灌注液对同种异体大鼠移植肝脏中血红素氧合酶1表达的影响,以及对供体肝脏缺血-再灌注损伤的保护作用。 方法：将SD雄性大鼠随机分成UW液组(术中使用UW液灌注保存)、丹参+UW液组(术中使用丹参+UW液灌注保存)、ZnPP预处理组(移植前24 h腹腔内注射ZnPP,术中使用丹参+UW液灌注保存),建立稳定的大鼠同种异体肝移植模型。同时取10只正常大鼠作为正常对照。 结果与结论：丹参+UW液组和UW液组血清总胆红素、谷丙转氨酶、谷草转氨酶水平明显低于ZnPP预处理组(P < 0.01)。血红素氧合酶1mRNA及其蛋白在丹参+UW液组中较UW组表达更明显,在ZnPP预处理组中表达明显受到抑制(P < 0.05)。丹参+UW液组肝脏Suzuki标准评分明显低于ZnPP预处理组及UW液组(P < 0.05)。表明丹参能上调同种异体的大鼠移植肝脏中血红素氧合酶1 mRNA及其蛋白的表达,减轻供肝缺血-再灌注损伤,保护移植大鼠肝脏。     

Characterization of alpha1-antitrypsin as a heme oxygenase-1 suppressor in Alzheimer plasma

Heme oxygenase-1 (HO-1) mRNA and protein levels are diminished in Alzheimer disease (AD) blood, cerebrospinal fluid (CSF) and choroid plexus. Herein, the presence of a heme oxygenase-1 suppressor (HOS) factor was ascertained by astroglial bioassay, biochemical techniques and immunofluorescence confocal microscopy. We report significantly augmented plasma HOS activity in AD patients relative to healthy elderly and neurological controls. The HOS factor was determined to be a 50-100 kDa heat-labile, heparin-binding glycoprotein that is unrelated to antioxidant ingestion, plasma total antioxidant capacity, circulating cortisol levels or apolipoprotein E epsilon4 carrier status. HOS bioactivity was recapitulated by exogenous alpha(1)-antitrypsin. alpha(1)-antitrypsin levels were significantly increased in AD plasma and correlated with HOS activity and MMSE scores. alpha(1)-antitrypsin immunodepletion attenuated HOS activity of AD plasma. In AD brain, alpha(1)-antitrypsin immunoreactivity was augmented and co-distributed with HO-1. HOS activity of alpha(1)-antitrypsin may curtail HO-1-dependent derangement of cerebral iron homeostasis and account for diminished HO-1 expression in AD peripheral tissues.     

Long-term expression of heme oxygenase-1 (HO-1, HSP-32) following focal cerebral infarctions and traumatic brain injury in humans

Extracellular heme derived from hemoglobin following hemorrhage or released from dying cells induces the expression of heme oxygenase-1 (HO-1, HSP-32) which metabolizes heme to the gaseous mediator carbon monoxide (CO), iron (Fe) and biliverdin. Biliverdin and its product bilirubin are powerful antioxidants. Thus, expression of HO-1 is considered to be a protective mechanism against oxidative stress and has been described in microglia, astrocytes and neurons following distinct experimental models of pathological alterations to the brain such as subarachnoidal hemorrhage, ischemia and traumatic brain injury (TBI) and in human neurodegenerative diseases. We have now analyzed the expression of HO-1 in human brains following TBI (n = 28; survival times: few minutes up to 6 months) and focal cerebral infarctions (FCI; n = 17; survival time: < 1 day up to months) by immunohistochemistry. Follwing TBI, accumulation of HO-1+ microglia/macrophages at the hemorrhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months. In contrast, after FCI HO-1+ microglia/macrophages accumulated within focal hemorrhages only and were absent in non-hemorrhagic regions. Further, HO-1 was weakly expressed in astrocytes in the perifocal penumbra. In contrast to experimental data derived from rat focal ischemia, these results indicate a prolonged HO-1 expression in humans after brain injury.     

血红素加氧酶-1基因转染对神经元损伤的保护作用

目的观察腺病毒介导的血红素加氧酶-1（HO—1）基因转染大鼠后对脑缺血再灌注损伤的保护作用。方法雄性SD大鼠随机分为4组：假手术对照组（SH）、生理盐水组（V）、空载体组（Ad）和Ad—HO-1转染组（HO）。后三组在缺血前3d于右侧脑室部分别注射20μl生理盐水、含1μl空载体腺病毒（1．0×10^10plaque-forming unit／ml,PFU／ml）的生理盐水或含1μl重组HO-1腺病毒（1．0×10^10PFU／ml）的生理盐水,连续注射3d后,采用右侧大脑中动脉栓塞法（MCAO）建立脑缺血再灌注模型。每组大鼠测定神经功能后,处死大鼠并取全脑标本,测定右脑梗死体积及细胞凋亡指标,荧光显微镜下观察脑组织荧光蛋白的表达情况,Western blot检测脑组织HO-1的表达。结果HO组中HO-1表达量明显高于Ad组和V组,Ad组和HO组可见有荧光蛋白表达,转染率为34．5％±3．4％。HO组神经功能显著优于Ad组和V组（P〈0．001）。与SH组比较,V组、Ad组脑梗死体积、神经细胞凋亡明显升高。与V组及Ad组比较,HO组脑梗死体积显著减小（P〈0．01）、神经元凋亡显著减少（P〈0．01）。结论腺病毒携带的HO-1基因能有效的转染脑组织,并在脑内稳定表达;HO-1基因转染显著减轻脑缺血再灌注后神经细胞损伤。