少弱精子症患者精子候选差异蛋白Trx 2、TrxR 1的验证及在少精、弱精子症中的表达研究

目的:根据TMT技术筛选少弱精子症患者精子差异蛋白的结果,选取硫氧还蛋白2(thioredoxin 2,Trx 2)、硫氧还蛋白还原酶1(thioredoxin reductase 1,TrxR 1)进行验证,探讨二者在少精、弱精和少弱精子症中的表达变化及其意义。方法:收集105例少精子症组(O组)、150例弱精子症组(A组)、50例少弱精子症组(OA组)和106例正常精液男性(N组)精液,分离出精子,对少弱精子症进行串联质谱标签(Tandem Mass Tag,TMT)技术蛋白质组学分析,根据少弱精子症组的精子差异蛋白结果选取Trx 2、TrxR 1,通过免疫荧光和免疫印迹方法检测其在O组、A组、OA组的表达情况。结果:TMT技术蛋白质组学结果显示Trx 2为上调差异蛋白(为N组的1.31倍),TrxR 1为下调差异蛋白(为N组的0.82倍)。免疫荧光和免疫印迹结果显示O组、A组、OA组Trx 2表达显著高于N组(P0.05),O组、OA组TrxR 1的表达显著低于N组(P0.05)。二者在OA组的结果与蛋白质组学结果一致。结论:Trx 2、TrxR 1可能在少精、弱精及少弱精子症的发生中起着重要的作用,并有望成为少弱精子症患者精子的候选标志物及治疗靶点。     

一种新的硒酶——硫氧还蛋白还原酶

必需微量元素硒的生物功能主要是通过各种硒酶和硒蛋白来实现的,如谷胱甘肽过氧化物酶（ＧＰｘ）类具有抗氧化功能,碘甲腺原氨酸脱碘酶（ＤＩ）类在甲状腺激素的生物合成和代谢中具有重要作用。已有研究表明,在哺乳动物各种组织中可能大约存在２５种左右硒蛋白,但是迄...     

硫氧还蛋白还原酶1(TrxR1)在胃癌中的表达及对胃癌细胞生长的影响

目的:探讨胃癌组织硫氧还蛋白还原酶1(TrxR1)表达与生存时间的关系及其对胃癌细胞生长的影响。方法:用Real-time PCR法检测76例胃癌组织及癌旁TrxR1 mRNA表达,并分析其与胃癌患者临床病理特征及预后的关系;随机选取3例胃癌组织及癌旁组织,采用免疫组化法、Western blot法检测TrxR1蛋白表达。采用Western blot法和Real-time PCR法检测胃癌细胞系及人胃粘膜上皮细胞中TrxR1的表达。采用小RNA干扰序列(siRNA)处理AGS细胞,根据处理方法不同将AGS细胞分为3组:阴性对照组:转染NC-siRNA、TRXR1 siRNA干扰1组:转染TRXR1-siRNA1、TRXR1 siRNA干扰2组:转染TRXR1-siRNA2。使用Real-time PCR法检测各组AGS细胞中TrxR1 mRNA的表达,克隆形成试验和MTT法检测AGS细胞生长情况。结果:胃癌组织中TrxR1 mRNA和蛋白表达量均显著性上调,TrxR1主要定位于细胞质中。TrxR1高表达与患者TNM分期及淋巴结转移有关,且TrxR1高表达组患者的中位生存时间短于低表达组...     

地钱,肾蕨和中山柏的NADP硫氧还蛋白系统

硫氧还蛋白(Td)是一类低分子量酸性蛋白,具有二硫键(-s-s-),通过氧化还原互变来参与很多反应(周志民等1986)。Td可被NADP-硫氧还蛋白还原酶(NTR)还原:     

猪链球菌2型硫氧还蛋白还原酶介导细菌应激与致病作用

【背景】硫氧还蛋白还原酶（thioredoxin reductase,TRR）是硫氧还蛋白系统关键组成部分,对病原菌应对体内外氧化应激、调节细菌稳态和介导致病过程具有重要作用。【目的】探究硫氧还蛋白还原酶TRR在人畜共患猪链球菌2型感染过程中参与的生物学效应。【方法】同源重组法构建猪链球菌2型硫氧还蛋白还原酶trr基因缺失株（Δtrr）及回补株（cΔtrr）,通过细菌染色、点板计数、体外细胞和动物感染模型等试验比较分析trr基因对细菌形态、抗应激反应及致病过程的影响。【结果】缺失trr对猪链球菌2型形态与生长特性的影响不大,但可增强细菌抗热应激、氧化应激和酸应激能力,缺失株对上皮细胞黏附力下降,侵袭进入脑血管内皮细胞作用显著降低,易于被吞噬细胞吞噬清除,对小鼠模型致病效应显著减弱。【结论】猪链球菌2型TRR因子参与细菌应激反应,介导细菌黏附、侵袭等致病过程,是猪链球菌2型新的潜在毒力因子。     

玉米过氧化物还原蛋白BAS1的原核表达及其功能研究

植物过氧化物还原蛋白BAS1是巯基依赖的过氧化物酶,通过催化的Cys残基还原过氧化氢,依赖NADPH的叶绿体硫氧还蛋白还原酶保持BAS1的还原态。玉米含有两种BAS1:2-Cys PrxA和2-Cys PrxB。利用RT-PCR方法从玉米幼叶中克隆了编码成熟2-Cys PrxA的基因,并将蛋白Cys34残基突变成Ser34。SDS-PAGE显示纯化的野生型和突变体蛋白为一条主带,分子量约为23kDa;体外蛋白结合实验表明纯化的叶绿体硫氧还蛋白还原酶通过分子间二硫键结合纯化的2Cys PrxA的C34S突变体,非还原SDS-PAGE显示纯化的野生型2Cys PrxA含有分子间二硫键组成的二体,而纯化的C34S突变体呈现单体,巯基专一性标记化合物AMS修饰及活性分析表明纯化的BAS1还原态是催化还原过氧化氢所所必须的,它由硫氧还蛋白还原酶及其辅酶NADPH所催化。     

硫氧还蛋白-1(Trx1)氧化还原状态的检测

硫氧还蛋白-1(thioredoxin-1,Trx1)是一种广泛存在于生物体内的氧化还原调节蛋白,其氧化还原状态的变化是细胞内发挥氧化还原调控作用的重要过程.本文建立了Trx1氧化还原状态的检测方法—氧化还原蛋白免疫印迹法(redox Western blot),即通过碘乙酸(IAA)标记Trx1,根据蛋白所带负电荷的不同,达到分离蛋白氧化与还原状态的目的,并根据能斯特方程计算出相应的氧化还原电势.本方法是在蛋白免疫印迹(Western blot)的基础上建立的,具有低成本、易操作的特点.实验中分别采用H2O2和DTT处理样本,利用此方法检测了细胞裂解液中、细胞内及过表达Trx1氧化还原电势的变化;并检测了HEK293细胞不同生长时期Trx1的氧化还原状态.     

硫氧还蛋白结合蛋白-2在糖和脂代谢中的作用

硫氧还蛋白结合蛋白-2(thioredoxin binding protein-2,TBP-2)属于硫氧还蛋白结合蛋白家族成员,与还原型硫氧还蛋白结合,抑制其还原活性。在生物体内,TBP-2不但参与细胞内的氧化还原调节,还具有调节细胞生长繁殖和促进细胞凋亡的作用。TBP-2在葡萄糖和脂肪代谢中的作用被广泛研究。TBP-2高表达时,胰岛细胞凋亡,与糖尿病发生相关;而TBP-2缺失,与高脂血症发病有关。本文综述了TBP-2在糖脂代谢中的作用。     

阿纳其根脂肪酸成分分析及其对蛋白酪氨酸磷酸酯酶1B抑制作用研究

利用三种不同溶剂(正己烷、乙醚、氯仿)经索氏提取法提取阿纳其根中的油脂成分,经甲酯化后,用气相色谱质谱法(GC-MS)对脂肪酸组成进行了分析和鉴定。试验结果显示,正己烷提取油脂中共鉴定出了8种脂肪酸,其中亚油酸为36.341%、棕榈酸为34.268%、油酸为19.656%;乙醚提取油脂中共鉴定出了12种脂肪酸。其中亚油酸为34.102%、棕榈酸为32.916%、油酸为20.000%、亚麻酸为5.329%;氯仿提取油脂中共鉴定出了5种脂肪酸。其中亚油酸为42.427%、棕榈酸为40.952%、油酸为11.250%。对阿纳其根脂肪酸进行蛋白酪氨酸磷酸酯酶1B(PTP1B)的抑制作用研究,结果表明脂肪酸对PTP1B有较强的抑制作用,其IC50分别为5.27±0.40、4.71±0.08、7.01±1.98μg/m L。该研究为深入研究阿纳其根的药理作用提供了科学依据。     

硫氧还蛋白还原酶：一种新型有效的胃癌化疗疗效监测标志物

胃癌是国际上发病率较高的肿瘤,寻找新型有效的肿瘤标志物用于胃癌的诊断和疗效评估,对于胃癌的临床治疗有较大的帮助。本研究选取经临床确诊的155例胃癌患者为研究对象,探究血浆硫氧还蛋白还原酶（thioredoxin reductase, TR）在胃癌化疗后临床获益人群与未获益人群中检测水平的差异,以及其与治疗效果的关系。结果显示,未获益组TR阳性率为82.67%,显著高于获益组TR阳性率（48.94%）。受试者操作特征曲线（receiver operating characteristic curve,ROC）分析显示,未获益组人群与获益组人群的TR活性诊断阈值为8.95 U/mL,ROC曲线下面积（area under the curve,AUC）为0.8423,明显优于常规胃癌标志物癌胚抗原（carcinoembryonic antigen,CEA）,肿瘤特异性抗原（cancer antigen,CA）19-9和CA72-4。同时,TR与常规胃癌标志物的联合检测提高阳性检出率至98.49%。TR被认为是一种新型的胃癌临床化疗疗效监测标志物,与临床疗效评价具有较高的一致性。     

Stress defense mechanisms of NADPH-dependent thioredoxin reductases (NTRs) in plants

Plants establish highly and systemically organized stress defense mechanisms against unfavorable living conditions. To interpret these environmental stimuli, plants possess communication tools, referred as secondary messengers, such as Ca²⁺ signature and reactive oxygen species (ROS) wave. Maintenance of ROS is an important event for whole lifespan of plants, however, in special cases, toxic ROS molecules are largely accumulated under excess stresses and diverse enzymes played as ROS scavengers. Arabidopsis and rice contain 3 NADPH-dependent thioredoxin reductases (NTRs) which transfer reducing power to Thioredoxin/Peroxiredoxin (Trx/Prx) system for scavenging ROS. However, due to functional redundancy between cytosolic and mitochondrial NTRs (NTRA and NTRB, respectively), their functional involvements under stress conditions have not been well characterized. Recently, we reported that cytosolic NTRA confers the stress tolerance against oxidative and drought stresses via regulation of ROS amounts using NTRA-overexpressing plants. With these findings, mitochondrial NTRB needs to be further elucidated.     

Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2

It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1^−/−) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H₂O₂ production. This death and excessive H₂O₂ levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H₂O₂. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells.     

Down-regulation of aldose reductase renders J774A.1 cells more susceptible to acrolein- or hydrogen peroxide-induced cell death

Aldose reductase (AR) is abundantly expressed in a variety of cell lineages and has been implicated in the cellular response against oxidative stress. However, the exact functional role of AR against oxidative stress remains relatively unclear. This study investigated the role of AR in acrolein- or hydrogen peroxide-induced apoptosis using the J774.A.1 macrophage cell line. Ablation of AR with a small interference RNA or inhibition of AR activity significantly enhanced the acrolein- or hydrogen peroxide-induced generation of reactive oxygen species and aldehydes, leading to increased apoptotic cell death. Blockade of AR activity in J774A.1 cells markedly augmented the acrolein- or hydrogen peroxide-induced translocation of Bax to mitochondria along with reduced Bcl-2 and increased release of cytochrome c from the mitochodria. Taken together, these findings indicate that AR plays an important role in the cellular response against oxidative stress, by sequestering the reactive molecules generated in cells exposed to toxic substances.     

Insights about resveratrol analogs against trypanothione reductase of Leishmania braziliensis: Molecular modeling,computational docking and in vitro antileishmanial studies

In this work, we combined molecular modeling, computational docking and in vitro analysis to explore the antileishmanial effect of some resveratrol analogs (ResAn), focusing on their pro-oxidant effect. The molecular target was the trypanothione reductase of Leishmania braziliensis (LbTryR), an essential component of the antioxidant defenses in trypanosomatid parasites. Three-dimensional structures of LbTryR were modeled and molecular docking studies of ResAn1-5 compounds showed the following affinity: ResAn1?>?ResAn2?>?ResAn4?>?ResAn5?>?ResAn3. Positive correlation was observed between these compounds’ affinity to the LbTryR and the IC₅₀ values against Leishmania sp (ResAn1?<?ResAn2?<?ResAn4), which allows for TryR being considered an important target for them. As the compound ResAn1 showed the best antileishmanial activity, and docking studies showed its high affinity for NADP binding site (NS) of TryR, plus having been able to induce ROS production in L. braziliensis promastigotes treated, ResAn1 probably occupies NS interfering in the electron transfer processes responsible for the catalytic reaction. The in silico prediction of ADMET properties suggests that ResAn1 may be a promising drug candidate with properties to cross biological membranes and high gastrointestinal absorption, not violating Lipinski’s rules. Ultimately, the antileishmanial effect of ResAn can be associated with a pro-oxidant effect which, in turn, can be exploited as an antimicrobial agent.

Communicated by Ramaswamy H. Sarma     

Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase

The mammalian thioredoxin reductases (TrxR) are selenoproteins with a catalytic selenocysteine residue which in the oxidized enzyme forms a selenenylsulfide and in the reduced enzyme is present as a selenolthiol. Selenium compounds such as selenite, selenodiglutathione and selenocystine are substrates for the enzyme with low K_m-values and the enzyme is implicated in reductive assimilation of selenium by generating selenide for selenoprotein synthesis. Redox cycling of reduced metabolites of these selenium compounds including selenide with oxygen via TrxR and reduced thioredoxin (Trx) will oxidize NADPH and produce reactive oxygen species inducing cell death at high concentrations explaining selenite toxicity. There is no free pool of selenocysteine since this would be toxic in an oxygen environment by redox cycling via thioredoxin systems. The importance of selenium compounds and TrxR in cancer and cardiovascular diseases both for prevention and treatment is discussed. A selenazol drug like ebselen is a direct substrate for mammalian TrxR and dithiol Trx and ebselen selenol is readily reoxidized by hydrogen peroxide and lipid hydroperoxides, acting as an anti-oxidant and anti-inflammatory drug.     

综述: 细胞氧化还原调控与衰老

利用酵母、线虫、果蝇、小鼠等模式生物进行的研究表明,细胞的衰老过程与氧化还原紧密相关.伴随衰老,细胞内GSSG水平升高,GSH、NADPH等水平降低,而氧化还原状态变化将直接影响蛋白质的功能,特别是氧化还原敏感的含巯基蛋白质的功能,从而影响细胞信号转导和细胞命运.氧化还原失衡可能是衰老发生的重要因素.本综述将从氧化还原平衡与衰老、氧化还原调控与信号转导及衰老、氧化损伤与衰老等方面阐述细胞氧化还原调控与衰老研究的最新进展,提出并探讨氧化还原平衡的维持、氧化还原平衡的系统调控及氧化还原调控的个体化等延缓衰老及健康衰老的新策略.     

Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer

In recent years, a number of studies have implicated the potent antioxidant property of astaxanthin in various experimental systems; however, these studies employed only the all-trans isomer. On the other hand, it has been reported that all-trans natural astaxanthin is readily isomerized to cis-trans, especially 9-cis and 13-cis isomers, under certain conditions by chemical analysis; however, the biological activities of the cis isomers of astaxanthin are little known. In the present study, we investigated the antioxidant activity of 9-cis and 13-cis astaxanthin compared to the all-trans isomer in vitro. In a stable radical DPPH scavenging activity test and in rat microsome and rabbit erythrocyte ghost membrane lipid peroxidation systems induced by AAPH and t-BuOOH, respectively, the results apparently showed that cis-astaxanthin, especially 9-cis astaxanthin, exhibited a higher antioxidant effect than the all-trans isomer. In addition, during polyunsaturated fatty acid (PUFA) oxidation, both DHA and linoleic acid hydroperoxides formation were markedly inhibited by astaxanthin isomers addition in the order 9-cis >13-cis >all-trans. Furthermore, 9-cis also exhibited the most effective inhibition of the generation of ROS induced by 6-hydroxydopamine (6-OHDA) in human neuroblastoma SH-SY5Y cells among the astaxanthin isomers, as well as on the degradation of collagen type II induced by DHA and linoleic acid hydroperoxides. The above-mentioned results suggest, for the first time, that cis isomer astaxanthin, especially 9-cis astaxanthin, has a much higher antioxidant potency than that of the all-trans isomer.     

The therapeutic effects of edaravone on collagen-induced arthritis in rats

Current research suggests that synovial phagocytic cells remove excessive amounts of free oxygen radicals (reactive oxygen species [ROS]), thereby preventing damage to synovial tissues. Moreover, ROS may affect the expression of growth arrest and DNA damage inducible α (GADD45A), thus further promoting the activation of synovial fibroblasts. Male adult rats were assessed for progression of collagen-induced arthritis (CIA) using a macroscopic arthritis scoring system of the hind paws and by measuring the changes in the rat's body weight, and activity level before and after diagnosis of CIA. Rats were intraperitoneally injected twice daily with edaravone at doses of 3, 6, and 9 mL/kg. Samples were taken at 2, 4, and 6 weeks, respectively. Edaravone was found to significantly reduce macroscopic arthritis and microscopic pathology scores in CIA rats. The concentration of endothelial nitric oxide synthase-6, glutathione, and heme oxygenase-1 in the serum of rats decreased, as was the production of ROS around the synovium and inflammatory factors. Moreover, ROS-1 increased the expression of the nuclear factor-κB (NF-κB) p65 protein by altering the expression level of GADD45A, causing aggravation of tissue damage. Edaravone also significantly improved the physiological condition of CIA rats, including appetite, weight changes, and loss of fur, as well as limb mobility. We believe that edaravone acts to reduce the expression of NF-ĸB p65 by clearing ROS, which causes reduced expression of GADD45A, and subsequently reduces the level of apoptosis and inflammatory response proteins, thereby reducing the symptoms of CIA. We, therefore, propose that edaravone is an effective option for clinical treatment of rheumatic arthritis.     

Schistosoma mansoni: ferredoxin-NADP(H) oxidoreductase and the metabolism of reactive oxygen species

Mitochondrial-type ferredoxin-NADP(H) oxidoreductases (FNR) catalyze the electron transport between NADPH and substrates such as ferredoxins. Even though enzymes belonging to this family are present in several organisms, including prokaryotes, their biological function is not clearly understood. In a previous work, we reported the existence of a mitochondrial-type FNR in the trematode Schistosoma mansoni (SmFNR). This enzyme conferred tolerance to oxidative stress conditions when tested in an heterologous system. In this work, we demonstrate that the SmFNR can be imported to mitochondria in mammal cells and show that its expression is induced in parasite cultures by reactive oxygen species (ROS). The results reported herein give further support to the involvement of SmFNR in ROS metabolism.