Curcumin activates defensive genes and protects neurons against oxidative stress

Spices and herbs often contain active phenolic substances endowed with potent antioxidative properties. We had previously shown that curcumin, the yellow pigment in curry, strongly induced HO-1 expression and activity in rat astrocytes. In the CNS, HO-1 has been reported to operate as a fundamental defensive mechanism for neurons exposed to an oxidant challenge. Treatment of astrocytes with curcumin upregulated expression of HO-1 protein at both cytoplasmic and nuclear levels, as shown by immunofluorescence analysis under laser-scanning confocal microscopy. A significant expression of quinone reductase and glutathione S transferase, two members of phase II detoxification enzymes, was found in astrocytes exposed to 5-15 microM curcumin. Moreover, the effects of curcumin on HO-1 activity were explored in cultured hippocampal neurons. Elevated expression of HO-1 mRNA and protein were detected after 6 h incubation with 5-25 microM curcumin. Higher concentrations of curcumin (50-100 microM) caused a substantial cytotoxic effect with no change in HO-1 protein expression. Interestingly, pre-incubation (18 h) with curcumin resulted in an enhanced cellular resistance to glucose oxidase-mediated oxidative damage; this cytoprotective effect was considerably attenuated by zinc protoporphyrin IX, an inhibitor of heme oxygenase activity. This study gives additional support to the possible use of curcumin as a dietary preventive agent against oxidative stress-related diseases.     

Expression of haem oxygenase in cirrhotic rat liver

The haem oxygenase (HO)/carbon monoxide (CO) system has been implicated as a modulator of hepatobiliary function. This study investigated HO expression in the process of cirrhosis development, as well as its relationship to nitric oxide synthase (NOS). Liver cirrhosis was induced in rats by chronic bile duct ligation (BDL). HO mRNA expression was evaluated by competitive RT-PCR, while protein expression was determined by immunoblotting and immunohistochemistry. In liver tissue where cirrhosis had fully developed, the expression levels of HO-1 were greatly enhanced at both mRNA and protein level compared with sham livers. Immunohistochemistry showed that HO-1 was induced in hepatocytes and enhanced in some of the Kupffer-like cells in BDL livers. In contrast, there was no difference between the sham and the BDL livers for the expression levels of HO-2. Interestingly, administration of the NOS inhibitor aminoguanidine (AG) or N(G)-nitro-L-arginine methyl ester inhibited HO-1 expression. To study further the role of HO-1 in the development of liver cirrhosis, hepatocytes were isolated from the rats at different time points after BDL operation. HO-1 was expressed in hepatocytes at high levels during the early onset of cirrhosis but dropped slightly at a later stage of cirrhosis. Zinc protoporphyrin IX (ZnPP), an HO inhibitor, blocked HO-1 expression in hepatocytes from BDL cirrhotic rats, but enhanced the activity of inducible NOS (iNOS) in BDL hepatocytes. In conclusion, HO-1 was induced in the hepatocytes of rats undergoing cirrhosis, suggesting that HO-1 plays a role in the development of liver cirrhosis. Induction of HO-1 may be mediated partially by iNOS. However, once it is induced, HO-1 may be important in modulating iNOS activity, thus playing a protective role in liver cirrhosis.     

Permanent focal and transient global cerebral ischemia increase glial and neuronal expression of heme oxygenase-1, but not heme oxygenase-2, protein in rat brain

Two heme oxygenase (HO) proteins have been identified to date; HO-1, a stress-induced protein, and HO-2, a constitutively expressed isoform. Recently, it was demonstrated that HO-1 mRNA expression is increased following transient global ischemia. The present study examined the effects of global and focal ischemia on HO-1 and HO-2 protein, using immunocytochemistry. Following 20 min of ischemia (rat 4 vessel occlusion model with hypotension) and 6 h of recirculation, increased HO-1 immunoreactivity was evident in hippocampal neurons. After 24 h of recirculation, HO-1 was observed in both hippocampal neurons and astroglial cells. By 72 h, expression was primarily glial and restricted to CA1 and CA3c. In addition to hippocampus, HO-1 was also evident in both neurons and glia in cerebral cortex and thalamus, and in striatal glial cells. Twenty-four hours following permanent focal ischemia, HO-1 immunoreactivity was observed in astroglial cells in the penumbra region surrounding the infarct. In contrast to HO-1, the pattern of HO-2 immunoreactivity was not altered following transient global or permanent focal ischemia. The increased expression of HO-1 following ischemia may confer protection against oxidative stress, but might also contribute to the subsequent neuronal degeneration.     

Highly liver-specific heme oxygenase-1 induction by interleukin-11 prevents carbon tetrachloride-induced hepatotoxicity

Heme oxygenase (HO)-1, the rate-limiting enzyme in heme catabolism, can be induced in response to various oxidative stimuli, and its induction is thought to be critical in the cellular defense against oxidative tissue injuries. Carbon tetrachloride (CCl(4)) treatment of rats causes lipid peroxidation of cell membranes and produces massive hepatic injury. We previously demonstrated that HO-1 induction following CCl(4) treatment is an essential part of the cellular defense against the CCl(4)-inducible toxic changes. As recombinant human interleukin-11 (rhIL-11) has been shown to induce HO-1 in cultured hepatoma cells, we examined the effect of rhIL-11 in vivo in rats on the CCl(4)-induced tissue injury. rhIL-11 treatment of animals by itself markedly induced HO-1 mRNA and its functional protein principally in the liver. rhIL-11 treatment (150 microg/kg) of the CCl(4)-administered (1 ml/kg) animals led to a further increase in HO-1 mRNA, while it markedly suppressed CCl(4)-induced serum alanine transaminase, hepatic malondialdehyde formation, tumor necrosis factor-alpha mRNA, nitric oxide synthase mRNA, nuclear factor-kappaB DNA-binding activity, as well as inflammatory changes of hepatocytes. In contrast, inhibition of HO activity by tin-mesoporphyrin, a competitive specific inhibitor of HO, entirely abolished the cytoprotective effect of rhIL-11. These findings thus demonstrate that rhIL-11 confers significant protection against CCl(4)-induced hepatic injury by virtue of its liver-specific HO-1 induction.     

Expression of heme oxygenase in human airway epithelial cells

Elevated levels of carbon monoxide (CO) are found in the exhaled breath of patients with inflammatory diseases such as asthma and cystic fibrosis. Endogenous CO is derived from heme oxygenase (HO) (EC 1.14.99.3), which catabolizes heme-producing CO and biliverdin. There are three isoforms of HO: HO-1 is inducible by inflammatory cytokines and oxidants, including nitric oxide (NO), whereas HO-2 and HO-3 are expressed constitutively. Primary airway epithelial cells were treated with either 50 ng/ml interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma (cytomix), or the NO donor NOC-18 for up to 24 h. Cytomix-induced HO-1 expression peaked at 4 h, returning to baseline by 24 h, whereas HO-2 expression remained unchanged. This increase in HO-1 expression could not be explained by an increase in NO production as inducible NO synthase expression increased between 12 and 24 h. However, the NO donor NOC-18 (500 microM) increased HO-1 expression twofold and HO activity 25-fold, whereas cytomix treatment increased HO activity eightfold. NO induction of HO-1 was not mediated via guanylyl cyclase and was not attenuated by 1 microM dexamethasone, although dexamethasone increased HO-2 protein. Therefore, airway epithelial cells express HO-2 and can express HO-1; thus, the epithelium may be a source of increased CO in airway diseases.     

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5microM zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress.     

诱导HO-1基因表达的信号通路

血红素氧合酶（HO）的诱导型HO-1与其催化血红素降解生成的产物胆红素和CO一道，组成了机体重要的内源性保护系统，广泛参与抗炎与多种急慢性氧化应激损伤。多种理化因素通过不同的细胞内信号转导通路诱导HO-1的表达，这些信号通路涉及丝裂原活化蛋白激酶（MAPKs）、蛋白激酶C(PKC)、cAMP依赖的蛋白激酶A(PKA)、cGMP依赖蛋白激酶G（PKG）、酪氨酸蛋白激酶（TPK）、蛋白磷酸酶（PPs）、磷脂酰肌醇（-3）激酶（PI3K）/Akt     

脑缺血再灌注后脑内血红素氧合酶-1表达的时相变化

目的 :研究局灶性脑缺血再灌注时血红素氧合酶 1(HO 1)在脑内的表达及其作用。方法 :采用梗塞大鼠大脑中动脉制备脑缺血再灌注模型 ,对 66只大鼠脑缺血及缺血再灌注后不同时间点进行HO 1免疫组化及病理学研究 ,并用计算机图像分析技术计算HO 1表达的强弱。结果 :单纯缺血 1h后皮质及海马即有HO 1阳性神经元胶质细胞的表达 ,梗塞 1h再灌 0 .5hHO 1表达与单纯缺血基本相同。随着再灌注时间的延长 ,HO 1的表达逐渐增强 ,到再灌 12h时达最高 ,以后逐渐下降 ,再灌 7天后仍有HO 1表达。HO 1主要分布在灶周皮质、梗塞皮质区、梨状皮质内的神经元及胶质细胞 ,丘脑、下丘脑、海马齿状回的神经元。结论 :局灶性脑缺血再灌注时脑内HO 1表达变化 ,是脑组织对自身损伤的重要恢复机制之一。HO 1自身具有脑保护作用并通过其下游产物CO起作用     

尼莫地平减轻铝过负荷所致的小鼠神经元退行性变

目的: 探讨尼莫地平对铝过负荷致神经元退行性变的影响。方法: 105只小鼠随机分为对照组、铝过负荷组和尼莫地平治疗组。铝过负荷组和尼莫地平治疗组以0.25%氯化铝脑室内注射2 μL qd×5 d,建立铝过负荷致神经元退行性变小鼠模型;对照组注射等体积人工脑脊液。尼莫地平治疗组以尼莫地平80 mg/kg灌胃bid×30 d,对照组和铝过负荷组以等体积生理盐水灌胃bid×30 d。组织学观察神经元损伤;跳台实验检测学习记忆能力;分光光度计法检测血红素加氧酶(HO)活性;Western blotting和免疫组织化学法检测HO-1蛋白表达水平;电感耦合等离子体原子发射光谱法(ICP-AES)检测铝离子和铁离子水平。结果: 与对照组相比,铝过负荷组学习记忆能力显著降低(P<0.05),神经元损伤严重,HO-1蛋白表达水平、HO活性和铁离子浓度显著升高(P<0.05);与铝过负荷组相比,尼莫地平治疗组学习记忆能力显著提高(P<0.05),神经元损伤减轻,HO-1蛋白表达水平、HO活性和铁离子浓度显著降低(P<0.05)。结论: 尼莫地平可能通过抑制HO-1表达、维持铁离子稳态,减轻铝过负荷所致的神经元退行性变。     

Peritoneal Inflammation in Pigs is Associated with Early Mitochondrial Dysfunction in Liver and Kidney

The objective of this study was to investigate early effects of peritoneal inflammation on the mitochondrial function in the vital organs, liver and kidney, and their relation to inflammatory and oxidative stress mediators. The study was performed on 14 domestic pigs. Peritoneal inflammation was induced in anesthetized pigs after a midline laparotomy by autologous feces. Fluid resuscitation maintained a MAP above 60 mmHg. Animals were sacrificed 12 h later, and tissue samples were obtained to determine mitochondrial function, mRNA levels of relevant genes [inducible NO synthase (iNOS), inducible HO (HO-1), tumor necrosis factor-alpha (TNF-alpha)], generation of reactive oxygen species (ROS), and HO-1 activity. We found impaired mitochondrial function in both liver and kidney, based on decreased state 3 respiration in the liver and increased states 2 and 4 respiration in the kidney at 12 h. This was accompanied by increased TNF-alpha protein in the blood and up-regulation of TNF-alpha mRNA in the liver. Free iron was elevated in the liver but not in the kidney. In the kidney, mitochondrial ROS production was increased. Nitric oxide levels in blood remained unchanged, corresponding to unchanged levels of iNOS mRNA expression in liver and kidney. Similarly, HO-1 mRNA and heme oxygenase (HO)-activity were unchanged. The inflammatory response in the absence of characteristic septic symptoms was not associated with morphological organ damage at this early time point. Peritoneal inflammation in pigs caused mitochondrial dysfunction in liver and kidney, preceding signs of organ damage. We did not find proof that mitochondrial dysfunction was due to increased levels of either nitric oxide (NO) or products of HO, but it was accompanied by increased levels of oxidative stress markers.     

Heme oxygenase expression and activity in immortalized hypothalamic neurons GT1-7

Heme oxygenase (HO), the main enzyme deputed to heme metabolism, has been identified as two main isoforms called HO-1 and HO-2. HO-1 is inducible and plays a main role in the cellular oxidant/antioxidant balance whereas HO-2 is constitutive and involved in the physiological metabolism of heme. However, it is noteworthy to mention that HO contribute to the regulation of the hypothalamic release of neuropeptides such as corticotrophin-releasing hormone and arginine-vasopressin and could modulate the pulsatile release of gonadotropin releasing hormone (GnRH). GT1-7 cells are immortalized hypothalamic neurons and a valuable tool to evaluate hypothalamic neuroendocrine control of reproduction. The aim of this work was to investigate and characterize the presence of HO isoforms in the GT1-7 hypothalamic neurons. Hemin, a well-known inducer of HO-1, significantly increased HO activity, whereas dexamethasone did not modify HO-2 activity. Moreover, hemin and DEX, in combination, did not have any additive effect on HO activity in GT1-7 neurons. Furthermore, basal HO-1 immunoreactivity identified in GT1-7 cells, was significantly up-regulated by hemin. Conversely, no HO-2 immunoreactivity was detected. Taken together, these results suggest the presence of functional HO-1 in GT1-7 immortalized hypothalamic neurons and open new avenues about the use of this cell line for the study of HO modulation of GnRH secretion and reproduction.     

Mechanism of the salutary effects of flutamide on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of estrogen receptor-{beta}-dependent HO-1

Hemeoxygenase (HO)-1 induction following adverse circulatory conditions is known to be protective, and precastrated males have less intestinal damage than sham-operated males following trauma-hemorrhage (T-H). Previous studies have also shown that administration of flutamide up-regulated estrogen receptor (ER) expression in males following T-H. We hypothesized that flutamide administration in males following T-H up-regulates HO-1 via an ER-dependent pathway and protects against intestinal injury. Male Sprague-Dawley rats underwent T-H [mean blood pressure (MBP) 40 mmHg for 90 min and then resuscitation]. A single dose of flutamide (25 mg/kg body weight), with or without an ER antagonist (ICI 182,780), a HO enzyme inhibitor [chromium-mesoporphyrin (CrMP)], or vehicle, was administered subcutaneously during resuscitation. At 2 h after T-H or sham operation, intestinal myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and CINC-3 levels were measured. Intestinal ER-alpha, ER-beta, androgen receptor, and HO-1 mRNA/protein levels were also determined. Results showed that T-H increased intestinal MPO activity, ICAM-1, CINC-1, and CINC-3 levels. These parameters were improved significantly in the flutamide-treated rats subjected to T-H. Flutamide treatment increased intestinal HO-1 and ER-beta mRNA/protein levels as compared with vehicle-treated T-H rats. Administration of the ER antagonist ICI 182,780 or the HO inhibitor CrMP prevented the flutamide-induced attenuation of shock-induced intestinal damage. Thus, the salutary effects of flutamide administration on attenuation of intestinal injury following T-H are mediated via up-regulation of ER-beta-dependent HO-1 expression.     

What is a role of haeme oxygenase‐1 in psoriasis? Current concepts of pathogenesis

The skin is constantly exposed to endogenous and environmental pro-oxidant agents, which lead to harmful generation of reactive oxygen species (ROS). Healthy skin, being a potential target for oxidative stress, is equipped with a large number of defence mechanisms including antioxidant systems. This protection can be corrupted by an imbalance between ROS and antioxidants with pathological level of oxidants prevailing. There is a great body of evidence indicating that some inflammatory skin diseases, such as psoriasis, are mediated by oxidative stress. Keratinocytes of normal skin, the primary target for pro-oxidant agents, show strong expression of ROS-detoxifying enzymes. In addition, normal keratinocytes express haeme oxygenase (HO), an enzyme which might be involved in the protection of cells against oxidative stress. HO (inducible HO-1, constitutive HO-2 and HO-3) is the rate-limiting enzyme in haeme catabolism, which leads to the generation of biliverdin, iron, and carbon monoxide. HO-1 is a stress-responsive protein whose expression is induced by various oxidative agents. HO-1 is known for its cytoprotective, antioxidant and anti-inflammatory properties. Interestingly, a strong overexpression of HO-1 was observed in psoriatic skin. However, the role of HO-1 in psoriasis remains unclear. In this review, we will discuss some current concepts concerning pathogenesis of psoriasis and the contribution of HO-1 in skin inflammation to show the relationships between HO-1, ROS and cytokine network in psoriatic skin. We will try to answer a question whether enhanced HO-1 expression in keratinocytes results in beneficial or detrimental effect on the development and severity of psoriatic lesions.     

大鼠肢体缺血-再灌注后脑HO-1表达的变化及意义

目的:观察肢体缺血-再灌注(I-R)后脑组织内诱导型血红素氧合酶(HO-1)表达的变化及意义。方法:夹闭大鼠双侧股动脉根部4h、开放2-24h复制肢体I-R模型。反转录多聚酶链反应检测脑内HO-1mRNA表达的变化;免疫组化染色法标记HO-1蛋白的组织分布;用锌原卟啉抑制HO-1活性后,光镜观察脑组织的病理学变化。结果:肢体I-R后脑组织HO-1mRNA表达水平明显高于各对照组,再灌12h表达至峰值,再灌至24h仍显著高于各对照组(P＜0.01)。肢体I-R组大脑皮质、海马等区出现大量弥散分布的HO-1免疫阳性胶质细胞,皮质及海马部分锥体细胞呈HO-1免疫阳性;抑制HO-1活性,I-R组脑组织损伤加重。结论:肢体I-R后,脑组织内胶质细胞及神经元HO-1基因表达上调,所诱生的HO-1对神经元具有保护效应。     

Effects of midazolam and phenobarbital on brain oxidative reactions induced by pentylenetetrazole in a convulsion model

Context: Brain oxidative reactions are involved in epilepsy as well as neurodegenerative diseases. In animal convulsion models, some anticonvulsants have been found to suppress oxidative reactions associated with convulsions. However, the effect of anticonvulsants on brain oxidative reactions has not fully been clarified. Objective: Midazolam and phenobarbital are often used as an intravenous anesthetic, and are known to have anticonvulsive effect, but antioxidative effect of these drugs has rarely been studied. Thus, the purpose of this study was to evaluate the effects of these drugs on the degree of convulsions and brain oxidative reactions in an animal convulsion model. Materials and methods: In order to evaluate brain oxidative reactions, we measured malondialdehyde (MDA) level and heme oxygenase (HO)-1 mRNA expression level in the brain of mice in a convulsion model generated by a single injection of pentylenetetrazole (PTZ). We evaluated the effects of midazolam and phenobarbital on the degree of PTZ-induced convulsions and on the changes in brain MDA level and HO-1 mRNA expression level. Results: After PTZ injection, severe convulsions were observed in all mice. MDA level was increased in the whole brain, while HO-1 mRNA expression level was increased only in the hippocampus. Both midazolam and phenobarbital prevented the convulsions and suppressed the increase in both MDA level and HO-1 mRNA expression level in the brain. Conclusion: In this study, both midazolam and phenobarbital suppressed PTZ-induced MDA and HO-1 reactions in the brain, suggesting that these drugs inhibit brain oxidative reactions in a convulsion model.     

血管钙化时血管血红素加氧酶/一氧化碳系统的变化

目的:观察血管钙化时血管血红素加氧酶(HO)-一氧化碳(CO)-环磷酸鸟苷(cGMP)系统的改变, 以探讨血管钙化发病的细胞分子机理。方法:利用维生素D₃(VitD₃)和尼古丁(nicotine)复制大鼠血管钙化模型, 检测HO-1mRNA的相对含量, HO-1免疫组织化学染色, 测定主动脉HO-1活性、碳氧血红蛋白(HbCO)的生成及血管cGMP含量。结果:用竞争性定量RT-PCR的方法发现, 钙化动物血管组织的HO-1mRNA水平较对照组低34.9%(P＜0.05);免疫组织化学方法观察发现钙化血管的HO-1蛋白表达减少, 仅在内膜略有表达, 中膜无明显表达;HO-1活性低60.6%(P＜0.01);CO生成少53.9%(P＜0.01), cGMP的含量低77.1%(P＜0.01)。结论:钙化血管血红素-HO-CO-cGMP途径功能下调。