硫氧还蛋白还原酶的生理学功能研究进展

硫氧还蛋白还原酶(thioredoxin reductase,TrxR)是二聚体黄素酶,属于吡啶核苷酸二硫化物还原酶家族的一员,广泛表达于从原核生物到人类的各级有机体细胞中[1],因分布区域不同,三种同工酶分别命名为硫氧还蛋白R1(TrxR1)(细胞质型)、TrxR2(线粒体型)和一个主要在睾丸中表达的同工酶TrxR3(又名TGR)[2].TrxR2具有额外的N末端单侧的谷氧还蛋白区域,它是催化氧化型谷胱甘肽(GSH)还原的结构基础,因此TrxR3又被命名为硫氧还蛋白和谷胱甘肽还原酶.胞质型TrxR1发现最早,分布也较广泛,是目前研究得最多的一种同工酶.TrxR以Trx为底物和还原型烟酰胺嘌呤二核苷酸磷酸(NADPH)提供还原反应的还原当量,它们共同构成的氧化还原调控系统,叫做硫氧还蛋白系统.硫氧还蛋白系统在生物体内发挥着广泛的重要生理功能.现对硫氧还蛋白还原酶的生理学功能作一综述.     

硒缺乏和酶诱导剂对大鼠肝硒和非硒谷胱甘肽过氧化物酶的影响

谷胱甘肽过氧化物酶(GSHPx)是体内一种重要的抗氧化剂酶,此酶有含硒和不含硒两种。含硒GSHPx可以过氧化氢和有机氢过氧化物为底物,而非硒GSHPx仅以有机氢过氧化物为底物。本文报告以过氧化羟基异丙苯(CHP)作底物,测定大鼠肝总GSHPx活性的条件及硒缺乏和酶诱导剂对硒和非硒GSHPx活性的影响。     

灯盏花素对大鼠脑缺血再灌注引起抗氧化酶活性改变的影响

本文研究了灯盏花素对大鼠脑缺血再灌注引起抗氧化酶活性改变的影响，结果表明，灯盏花素明显提高脑缺血再灌注引起的脑组织超氧歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH－peroxidase）和过氧化氢酶（Catalase）的活性，减少脑组织丙二醛（MDA）含量，这些作用有利于减轻脑缺再灌注损伤。     

可卡因对性成熟期大鼠睾丸谷胱甘肽过氧化物酶基因表达的影响

目的：研究可卡因对大鼠睾丸谷胱甘肽过氧化物酶（glutathione peroxidase GSH-PX）基因表达的影响。方法：性成熟期健康SD雄性大鼠皮下注射可卡因制造吸毒动物模型。化学比色法检测睾丸氧化应激指标;半定量RT-PCR方法检测睾丸组织的GSH-PXmRNA表达。结果：可卡因处理第7天,实验组GSH-PX活性、MDA含量与对照组比较无显著性差异（P〉0．05）;而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第14天,实验组GSH-PX活性显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）,而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第21天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。可卡因处理第28天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。     

硒对老龄雄性大鼠全血、心、肝、肾谷胱甘肽过氧化物酶的活性及LPO含量的影响

采用老龄(20～21月龄)雄性 VVi—star 大鼠,观察给予老龄雄性大鼠机体补充 Se后对全血、心、肝、肾的硒谷胱甘肽过氧化物酶(SeGSH—Px)活性和脂质过氧化物(LPO)含量的影响。结果表明、机体补充硒后可使老龄大鼠全血和上述脏器组织 SeGSH—Px 活性升高,LPO 含量降低,从而起到延缓衰老的作用。     

硫氧还蛋白还原酶作为新型生物标志物在乳腺癌诊断和治疗中的价值

已有研究证明硫氧还蛋白还原酶在多种人原发肿瘤中过度表达,其活性在肿瘤人群和健康人群中有明显差异。本义研究了健康人群和乳腺癌患者中TrxR活性的不同,并探讨了硫氧还蛋白还原酶作为乳腺癌治疗中的一个新型治疗靶点的可能性。通过研究．我们第一次发现未经治疗的肿瘤患者的TrxR活性值水平明显高于健康人群。存乳腺癌患者中,TrxR活性值在不同的年龄组和癌症分期组没有明显的差异。当TrxR活性值小于10U／mL时,TrxR活性值和肿瘤影像学评价结果有很高的一致性。这些发现证明硫氧还蛋白还原酶可以作为一个有效的新型生物标志物用于对乳腺痛临床治疗效果的监测和评价,并能成为乳腺癌早期检测的一种方法。     

具有谷胱甘肽过氧化物酶活性的硒化环糊精的中试放大研究

将-环糊精的2位羟基进行硒化得到的产物(2-SeCD)作为谷胱甘肽过氧化物酶(GPX)的一种人工模拟酶具有广阔的应用开发前景。为了对其进行进一步的研究及生产,需得到大量的产物。在原有的研究基础上,将中试放大实验的剂量扩大为小试实验的50倍,在7次工艺成熟的中试放大实验中获得了大量的2-SeCD。通过碳氢元素分析、红外光谱分析、核磁共振分析、质谱分析等表征实验验证了放大实验所得产物的结构正确,并测定其活力为5.2 U/mol。在2-SeCD对细胞的生物学效应实验中,发现在2-SeCD浓度较低的条件下可降低细胞死亡率而在浓度较高的条件下却抑制细胞生长。通过形态学观察与DNA ladder检测发现高浓度的2-SeCD可诱导Hela细胞凋亡,并经凋亡因子caspase-3的相对活力检测确定此凋亡是由线粒体caspase激活途径诱发的。     

灯盏花素对CHF大鼠血流动力学的影响

以腹主动脉狭窄法复制心力衰竭动物模型,从血流动力学方面研究讨论灯盏花素对心衰动物的影响,为临床合理用药提供理论基础。实验结果表明,以腹主动脉狭窄法复制的心力衰竭动物模型,左室收缩压、左室内压最大上升及下降速率较假手术组显著降低,而左室舒张末压、动脉压显著增高,产生了心功能不全的血流动力学变化。通过灯盏花素给药6周后,上述指标均得到显著的改善,灯盏花素从而改善血流动力学,而且改善了心衰动物的心室重构,提高了心肌的收缩和舒张功能。     

灯盏花素对急性心肌梗死患者血清超敏C-反应蛋白的影响

目的探讨灯盏花素注射液对急性心肌梗死患者血清超敏C-反应蛋白(hs-CRP)的影响。方法急性心肌梗死患者60例,随机分为对照组和治疗组,各30例。对照组给予尿激酶和低分子肝素,治疗组在对照组基础上加用灯盏花素注射液50 mg溶于0.9%氯化钠注射液250 mL静脉滴注,1次/d,连续14 d。观察治疗前后血清hs-CRP的变化。结果与对照组比较,治疗组患者治疗后血清hs-CRP明显降低(P<0.05);与治疗前比较,对照组无明显变化,而治疗组显著降低(P<0.05)。结论灯盏花素注射液可降低急性心肌梗死患者血清炎症标记物hs-CRP,对心肌有保护作用。     

灯盏花素对离体大鼠肝星状细胞增殖的影响

目的：研究灯盏花素对离体大鼠肝星状细胞增殖与凋亡的影响,探讨灯盏花素在抗肝纤维化方面的作用。方法：用袁桃霞等的肝星状细胞分离方法获得原代培养的肝星状细胞,采用MTT比色法检测肝星状细胞的增殖,TUNEL法检测DNA断裂、凋亡情况。结果：①灯盏花素对肝星状细胞（HSC）增殖有一定的抑制作用,且与其浓度和作用时间有关;②灯盏花素处理组HSC凋亡率与对照组相比差异无显著性。结论：灯盏花素对大鼠肝星状细胞增殖有抑制作用。     

Diphenyl diselenide protects against methylmercury‐induced inhibition of thioredoxin reductase and glutathione peroxidase in human neuroblastoma cells: a comparison with ebselen

Exposure to methylmercury (MeHg), an important environmental toxicant, may lead to serious health risks, damaging various organs and predominantly affecting the brain function. The toxicity of MeHg can be related to the inhibition of important selenoenzymes, such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Experimental studies have shown that selenocompounds play an important role as cellular detoxifiers and protective agents against the harmful effects of mercury. The present study investigated the mechanisms by which diphenyl diselenide [(PhSe)₂] and ebselen interfered with the interaction of mercury (MeHg) and selenoenzymes (TrxR and GPx) in an in vitro experimental model of cultured human neuroblastoma cells (SH‐SY5Y). Our results established that (PhSe)₂ and ebselen increased the activity and expression of TrxR. In contrast, MeHg inhibited TrxR activity even at low doses (0.5 μm ). Coexposure to selenocompounds and MeHg showed a protective effect of (PhSe)₂ on both the activity and expression of TrxR. When selenoenzyme GPx was evaluated, selenocompounds did not alter its activity or expression significantly, whereas MeHg inhibited the activity of GPx (from 1 μm ). Among the selenocompounds only (PhSe)₂ significantly protected against the effects of MeHg on GPx activity. Taken together, these results indicate a potential use for ebselen and (PhSe)₂ against MeHg toxicity. Furthermore, for the first time, we have demonstrated that (PhSe)₂ caused a more pronounced upregulation of TrxR than ebselen in neuroblastoma cells, likely reflecting an important molecular mechanism involved in the antioxidant properties of this compound. Copyright © 2017 John Wiley & Sons, Ltd.     

Drug-resistant human lung cancer cells are more sensitive to selenium cytotoxicity. Effects on thioredoxin reductase and glutathione reductase

The human U-1285 and GLC(4) cell lines, both derived from small cell carcinoma of the lung, are present in doxorubicin-sensitive (U-1285 and GLC(4)) and doxorubicin-resistant MRP-expressing (U-1285dox and GLC(4)/ADR) variants. These sublines were examined here with respect to their susceptibilities to the toxic effects of selenite and compared to the toxic effects of selenite on the promyelocytic leukemia cell line HL-60 and its doxorubicin-resistant P-glycoprotein expressing variant. The drug-resistant U-1285dox and GLC(4)/ADR sublines proved to be 3- and 4-fold, respectively, more sensitive to the cytotoxicity of selenite than the drug-sensitive U-1285 and GLC(4) sublines, whereas no difference was observed between the HL-60 sublines. The presence of doxorubicin at a concentration equal to the IC(10) did not significantly potentiate the toxic effects of selenite. The presence of selenite did not significantly affect the expression of the multi-drug resistant proteins (MRP1, LRP and topoisomerase IIalpha) in the drug-resistant cells. The activities of thioredoxin reductase (TrxR) were higher (50 and 25%, respectively) in the drug resistant cell sublines U-1285dox and GLC(4)/ADR compared to the drug-sensitive parental lines. The activity of glutathione reductase (GR) was essentially the same in the drug-sensitive and -resistant cell lines. Exposure to selenite resulted in a 4-fold increase in both TrxR and GR activities in U-1285 cells, an effect, which was less pronounced in the presence of doxorubicin. Under similar conditions the increase in the TrxR activity in the resistant U-1285dox cell line, was only 30% and the activity of GR was unaltered. Different responses in the activity of the key enzymes in selenium metabolism are one possible mechanism explaining the differential cytotoxicity of selenium in these cells.     

Nuclear and nucleolar glutathione reductase, peroxidase, and transferase activities in livers of male and female Fischer-344 rats.

The present studies were to test the hypotheses that glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities are expressed in nuclei and nucleoli of rat liver cells, and that differences in activities of these enzymes would correlate with the greater resistance of female than of male Fischer-344 rats to hepatic necrosis in vivo, mediated by reactive oxygen species generated by redox-cycling metabolism of diquat. Adult male and female Fischer-344 rats were treated with comparably hepatotoxic doses of diquat (0.1 or 0.2 mmol/kg, respectively), or equal volumes of saline, ip. Six hours later, the livers were harvested, and purified nuclei and nucleoli were isolated by differential centrifugation. Nuclear GR activities in male and female rats were 12 and 15 mU/mg protein, and nucleolar activities were 30 and 51 mU/mg protein, respectively, p < 0.05. Some differences between male and female rats in nuclear and nucleolar activities of GPXs and GSTs were observed, as were some differences in the respective diquat-treated animals, but implications of these differences for susceptibility to diquat-induced oxidant stress effects are not apparent. Nuclear GR, GPX, and GST probably contribute to antioxidant defense mechanisms, but the functions served by localization of GR and GPX in nucleoli are less evident.     

Decreased enzyme activity of hepatic thioredoxin reductase and glutathione reductase in rabbits by prolonged exposure to inorganic arsenate

Chronic exposure of humans to inorganic arsenic, mainly pentavalent arsenate (iAsV), results in drinking water-induced oxidative stress (Pi et al., 2002). Thioredoxin reductase (TR) and glutathione reductase (GR) are the two critical enzymes in the response to oxidative stress in vivo. In the present study we examined alterations in enzyme activities of hepatic TR and GR from prolonged exposure of male New Zealand white rabbits to iAsV. Exposure of rabbits to iAsV in drinking water (5 mg/L) for 18 weeks caused a significant suppression of hepatic TR and GR activities, of approximately 30% and 20%, respectively, below controls. In vitro experiments suggested that trivalent inorganic arsenic (iAsIII) but not pentavalent arsenicals including iAsV, monomethylarsonic acid (MMAsV), and dimethylarsinic acid (DMAsV) affected the hepatic TR activity of rabbit. So it was suggested that in the present study iAsV ingested via drinking water was metabolized to reactive trivalent arsenicals, such as iAsIII, which may play an important role in the decreased TR and GR activities from prolonged exposure to iAsV observed in vivo.     

The role of thioredoxin reductase activity in selenium-induced cytotoxicity

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.     

β-胡萝卜素对阿霉素致大鼠心肌组织的超氧化物歧化酶、谷胱甘肽过氧化物酶mRNA表达改变的影响

目的研究β-胡萝卜素(BC)对阿霉素(ADM)所致大鼠心肌组织的锰超氧化物歧化酶(Mn SOD)mRNA、铜-锌超氧化物歧化酶(Cu-Zn SOD)mRNA、谷胱甘肽过氧化物酶(GPx)mRNA表达改变的影响,探讨BC拮抗ADM导致心肌组织的Mn SOD、Cu-Zn SOD、GPx活性降低的机制。方法大鼠腹腔注射(ip)ADM(10·0mg·kg-1,1次);ADM处理的大鼠灌胃(ig)不同剂量的BC(每天1次,3次;ipADM前igBC,每天1次,11次行预处理)进行干预。分别用硫代巴比妥酸法、硝酸还原酶法、黄嘌呤氧化酶法、二硫代二硝基苯甲酸法、血红蛋白氧化法测定心肌组织的丙二醛(MDA)含量、一氧化氮(NO)含量、Mn SOD及Cu-Zn SOD活性、GPx活性、一氧化氮合酶活性;RT-PCR方法检测心肌组织的Mn SODmRNA、Cu-Zn SOD mRNA、GPx mRNA、诱导型一氧化氮合酶(iNOS)mRNA表达。结果BC(20·0,40·0,80·0mg·kg-1)拮抗ADM所致心肌组织的MDA含量及NO含量增加、iNOS mRNA表达水平增加及其酶活性增加(P<0·01);拮抗ADM所致心肌组织的Mn SOD mRNA、Cu-Zn SOD mR-NA、GPx mRNA表达水平降低及其酶活性降低(P<0·01)。结论BC拮抗ADM导致心肌组织的Mn SOD mRNA、Cu-ZnSOD mRNA、GPx mRNA表达降低而拮抗ADM导致Mn SOD、Cu-Zn SOD、GPx活性降低。其机制可能与BC抑制ADM诱导心肌组织的iNOS mRNA表达而降低iNOS活性使心肌组织产生NO减少,从而减少NO抑制Mn SOD mRNA、Cu-ZnSOD mRNA、GPx mRNA表达有关。     

依布硒的药理作用及反应机制的研究进展

依布硒是一种小分子抗氧化剂 ,易于参加各种氧化还原反应 ,能清除机体内过多的过氧化物 ,阻断产生自由基的链式反应 ,因此可治疗多种疾病 ,维持机体的正常生理功能。长期以来 ,人们普遍认为依布硒在体内主要通过模拟谷胱甘肽过氧化物酶的反应机制发挥作用。然而 ,近来的研究表明依布硒在体内更趋向于通过硫氧还蛋白还原酶和硫氧还蛋白反应系统来发挥作用。本文简要介绍依布硒的药理作用及其反应机制的研究进展     

Effects of lead exposure on the expression of phospholipid hydroperoxidase glutathione peroxidase mRNA in the rat brain.

Oxidative damage associated with lead in the brain has been proposed as a possible mechanism of lead toxicity. Of the many antioxidant enzymes, phospholipid hydroperoxidase glutathione peroxidase (PHGPx) is known to protect cells from lipid peroxide-mediated damage by catalyzing lipid peroxide reduction. In this study, the effects of lead on the activity and expression of PHGPx mRNA were investigated in the brains of rats exposed to lead for 8 weeks. Male Sprague-Dawley rats (3 week old, n = 40) were randomly divided into four groups of 10 and treated with four different concentrations of lead in drinking water: a low dose (0.1% lead acetate), a medium dose (0.3% lead acetate), and a high dose (1.0% lead acetate), and a control group (0% lead acetate). We compared the four groups in terms of body and brain weight, lead concentrations in the brain and blood, and the activities of superoxide dismutase (SOD), gluthatione peroxidase (GPx), and PHGPx mRNA in the brain. Phospholipid hydroperoxidase glutathione peroxidase was found to have a dominant role in lead exposure. We also performed in situ hybridization of PHGPx mRNA in the brain to identity PHGPx mRNA active sites. We found that the level of PHGPx mRNA in brain increased in the medium- and low-dose groups, but decreased in the high-dose group versus the non-lead-treated control group. These results suggest that lead exposure increases the expression of PHGPx mRNA in the low- and medium-dose groups without inducing structural changes, and that the reduced expression of PHGPx mRNA in the high-dose group was associated with structural damage. An In situ hybridization study showed that PHGPx mRNA in the brain is expressed mainly in the white matter of the cerebral hemisphere and in the Purkinje cells of the cerebellar hemispheres; these sites are known to be the vulnerable to lead toxicity.     

Effect of alpha-lipoic acid on lipid peroxidation and anti-oxidant enzyme activities in diabetic rats

1. Oxidative damage has been suggested to be a contributory factor in the development and complications of diabetes. Recently, alpha-lipoic acid (ALA) has gained considerable interest as an anti-oxidant. Various studies have indicated the anti- oxidant effects of ALA and its reduced form dihydrolipoic acid. Therefore, it appears that these compounds have important therapeutic potential in conditions where oxidative stress is involved. The aim of the present study was to investigate the effect of ALA supplementation on lipid peroxidation and anti-oxidant enzyme activities in various tissues in diabetic rats. 2. Male Wistar rats were divided into three groups. Diabetes was induced by streptozotocin (STZ) injection in the two groups of rats to be supplemented and not to be supplemented with ALA. Another group of rats, which received saline injection, formed the control group. After 5 weeks of diabetes, rats were killed. In order to assess the redox status of various organs in the diabetic and control rats, thiobarbituric acid-reactive substances (TBARS) and glutathione (GSH) levels, as well as superoxide dismutase (SOD), glutathione peroxidase (G-Px) and glutathione reductase (G-Red) activities were determined in the liver, pancreas and kidney. 3. In both diabetic groups, TBARS levels and SOD activity were increased in the liver and pancreas, G-Px and G-Red activities were increased in the kidney and GSH levels were decreased in all organs compared with controls. In the ALA- supplemented group, TBARS levels were decreased, GSH levels were increased in the liver and pancreas, SOD activity was decreased in the liver, G-Px activity remained unchanged in all tissues and G-Red activity was increased in the pancreas compared with the diabetic group that did not receive ALA supplementation. 4. In conclusion, ALA supplementation has disparate effects on the redox status of different organs. These data are not sufficient for confirmation the beneficial effects of ALA supplementation on the redox status of various organs in diabetic rats.