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.     

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。     

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.     

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.     

Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury

Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.     

Cultured astrocytes from heme oxygenase-1 knockout mice are more vulnerable to heme-mediated oxidative injury

Hemin, the oxidized form of heme, is released from hemoglobin after CNS hemorrhage and may contribute to injury to surrounding tissue. The heme oxygenase (HO) enzymes catalyze the breakdown of hemin to biliverdin, carbon monoxide, and ferric iron. Although HO-2, the isoform expressed predominantly in neurons, accelerates heme-mediated neuronal injury, inhibitor studies suggest that HO-1 induction has a protective effect on astrocytes. In the present study, we directly compared the vulnerability of cultured HO-1 knockout and wild-type astrocytes to hemin. Consistent with prior observations, exposure of wild-type cultures to hemin for 24 hr resulted in protein carbonylation and concentration-dependent cell death between 10 and 60 microM, as determined by MTT and lactate dehydrogenase release assays. In cultures prepared from mice lacking the HO-1 gene, oxidative cell injury was approximately doubled. Both protein oxidation and cell death in HO-1 knockout astrocytes were significantly reduced by pretreating cultures with an adenovirus encoding the HO-1 gene prior to hemin exposure. HO-2 expression was observed in both knockout and wild-type cultures and was not altered by HO-1 gene deletion. Cell hemin accumulation after 20 hr hemin exposure was 4.7-fold higher in knockout cells. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Selectively increasing its expression in astrocytes may be beneficial after hemorrhagic CNS injuries.     

Protective effect of heme oxygenase-1 gene transfer against oxyhemoglobin-induced endothelial dysfunction.

The current study was designed to determine the effect of recombinant heme oxygenase-1 (HO-1) gene expression on endothelial function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector (10(10) PFU/mL, total volume 300 microL) encoding either the HO-1 gene (AdCMVHO-1) or the beta-galactosidase (beta-Gal) reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, arterial rings were suspended in organ chamber for isometric force recording. Endothelium-dependent relaxations were obtained in response to bradykinin (10(-10) to 10(-6) mol/L) during contraction to uridine-5'-triphosphate (UTP; 3 x 10(-6) to 3 x 10(-5) mol/L). Certain rings were incubated with oxyhemoglobin (OxyHb; 10(-5) mol/L) overnight (16 to 18 hours of 24 hours). Expression and localization of recombinant protein were shown by Western blot analysis and immunohistochemistry. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to forskolin (10(-9) to 10(-5) mol/L) and DEA-NONOate (10(-10) to 10(-5) mol/L) were identical in beta-Gal- and HO-1-transduced arteries. Exposure to OxyHb caused impairment of endothelium-dependent relaxation to bradykinin (P < 0.01). In contrast, OxyHb did not affect endothelium-dependent relaxation in arteries expressing recombinant HO-1 ( P > 0.05). This protective effect of HO-1 was reversed by coincubation with tin protoporphyrin (SnPP9; 10(-5) mol/L), a selective inhibitor of HO-1 (P < 0.01). Basal levels of 3',5'-cyclic monophosphate (cGMP) in HO-1-transduced vessels were not significantly different from those in beta-Gal-transduced vessels. Pretreatment with OxyHb significantly reduced cGMP level in beta-Gal-transduced rings (P < 0.01), whereas it had no effect in HO-1-transduced rings. These results demonstrate that HO-1 gene transfer does not affect endothelial and smooth muscle function of normal arteries, and that expression of recombinant HO-1 in cerebral arteries protects vasomotor function against OxyHb-induced injury.     

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.     

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood-brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (-60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (-30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.     

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells

Heme oxygenase-1 (HO-1) catalyses the rate-limiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP⁺) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP⁺ and whether HO-1 has a protective role against the MPP⁺-induced cytotoxicity in PC-12 cells. MPP⁺ triggered a relatively rapid induction of HO-1. The MPP⁺-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP⁺-induced cytotoxicity.     

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     

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood–brain barrier breakdown induced by impaired oxidative metabolism

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (−60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (−30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.     

Myelin basic protein induces heme oxygenase-1 in human astroglial cells.

Heme oxygenase-1 (HO-1), also known as heat-shock protein 32 (HSP-32), is induced in many cells by a large variety of stimuli. Its induction in nervous system cells following toxic and oxidative stress was suggested to play a protective role. Its presence was recently detected by immunohistochemical studies at the level of inflammatory lesions of rat experimental autoimmune encephalomyelitis. In the present study, we demonstrate that myelin basic protein (MBP) induces HO-1 in human astroglial cells, as shown by Western blots and RT-PCR. Proteolytic fragments derived from the whole MBP show a different behavior in the HO-1 induction: MBP152-167 was able to produce a light but still significant increase in HO-1 mRNA and protein levels, whereas MBP68-84 was not. The increase in HO-1 production seems to be mediated by a Ca(2+)-dependent mechanism, since MBP addition to astrocytoma cultures induced a strong and immediate increment of [Ca(2+)](i) increase; MBP152-167 elicited a delayed and less pronounced [Ca(2+)](i) increase; no [Ca(2+)](i) changes were induced following cell treatment with MBP68-84. NO pathway involvement in the induction of HO-1 by MBP was ruled out since the expression of the inducible isoform of nitric oxide synthase was not upregulated in treated cells, neither nitrite levels were modified, as demonstrated by Greiss reaction. The possible significance of HO-1 induction following MBP stimulation is discussed.     

Protective effect of hydrogen sulfide on oxidative stress-induced neurodegenerative diseases

Hydrogen sulfide is an antioxidant molecule that has a wide range of biological effects against oxidative stress. Balanced oxidative stress is also vital for maintaining cellular function in biological system, where reactive oxygen species are the main source of oxidative stress. When the normal redox balance is disturbed, deoxyribonucleic acid, lipid, and protein molecules are oxidized under pathological conditions, like diabetes mellitus that leads to diabetic peripheral neuropathy. In diabetes mellitus-induced diabetic peripheral neuropathy, due to hyperglycemia, pancreatic beta cell(β cell) shows resistance to insulin secretion. As a consequence, glucose metabolism is disturbed in neuronal cells which are distracted from providing proper cell signaling pathway. Not only diabetic peripheral neuropathy but also other central damages occur in brain neuropathy. Neurological studies regarding type 1 diabetes mellitus patients with Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have shown changes in the central nervous system because high blood glucose levels(HbA1 c) appeared with poor cognitive function. Oxidative stress plays a role in inhibiting insulin signaling that is necessary for brain function. Hydrogen sulfide exhibits antioxidant effects against oxidative stress, where cystathionine β synthase, cystathionine γ lyase, and 3-mercaptopyruvate sulfurtransferase are the endogenous sources of hydrogen sulfide. This review is to explore the pathogenesis of diabetes mellitus-induced diabetic peripheral neuropathy and other neurological comorbid disorders under the oxidative stress condition and the anti-oxidative effects of hydrogen sulfide.     

Neuroprotective effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis

Ganoderma lucidum polysaccharides have protective effects against apoptosis in neurons exposed to ischemia/reperfusion injury, but the mechanisms are unclear. The goal of this study was to investigate the underlying mechanisms of the effects of ganoderma lucidum poly-saccharides against oxidative stress-induced neuronal apoptosis. Hydrogen peroxide (H2O2) was used to induce apoptosis in cultured cerebellar granule cells. In these cells, ganoderma lucidum polysaccharides remarkably suppressed H2O2-induced apoptosis, decreased ex-pression of caspase-3, Bax and Bim and increased that of Bcl-2. These findings suggested that ganoderma lucidum polysaccharides regulate expression of apoptosis-associated proteins, inhibit oxidative stress-induced neuronal apoptosis and, therefore, have significant neuropro-tective effects.