Peroxiredoxins家族在H_(2)O_(2)诱导人颗粒细胞凋亡过程中的表达

目的探讨Peroxiredoxins(Prdxs)家族在H_(2)O_(2)诱导人颗粒细胞凋亡过程中的作用。方法选取在华中科技大学同济医学院附属同济医院接受体外受精-胚胎移植技术(in vitro fertilization embryo transfer technology,IVF-ET)的年轻患者的颗粒细胞,经体外培养并给予H_(2)O_(2)诱导人颗粒细胞凋亡,检测Prdx1~6 mRNA表达情况。结果选取患者的激素水平及窦卵泡数(AFC)提示卵巢处于良好状态,体外培养颗粒细胞同时表达卵泡刺激素受体(FSHR)和黄体生成素受体(LHR),符合原代颗粒细胞表型。在H_(2)O_(2)诱导下,细胞核皱缩,发生凋亡。实时定量聚合酶链反应(qPCR)显示,H_(2)O_(2)处理组Prdx1、2、4 mRNA的表达水平显著高于对照组,尤其Prdx4;而Prdx3和Prdx6 mRNA的表达水平低于对照组,差异均具有统计学意义;Prdx5 mRNA的表达水平相对于对照组有升高的趋势,但差异无统计学意义。结论Prdxs可能通过调节颗粒细胞凋亡过程,从而在人卵泡闭锁甚至卵巢衰老过程中发挥重要调节作用。

过氧化物酶2-Cys Peroxiredoxins的研究进展

Peroxiredoxins是最近发现的一类过氧化物酶家族,其中的一个亚类2-Cys Peroxiredoxins具有Peroxiredoxins的典型特征,在生物体内分布广泛且含量较多,可占哺乳动物细胞可溶性蛋白的1%,且具有独特的催化活性;此外2-Cys Peroxiredoxins具有抗氧化(清除活性氧)、信号传导和分子伴侣的功能,对生物体正常的生命活动起重要作用.

Peroxiredoxins在肿瘤中的研究进展

Peroxiredoxins(Prdx)是细胞内存在一类抗氧化酶,它们能催化氧化还原反应而维持细胞内过氧化氢水平的平衡,并作为氧化还原信号通路中的一个重要调节因子,这对于细胞信号转导和代谢十分重要。Prdx家族在肿瘤中发挥着不同的作用。由于肿瘤基因组学的改变,它们的表达发生变化,随之而来是细胞内氧化还原信号通路的改变。这些变化对肿瘤的生物学行为产生重要影响,也为肿瘤的治疗提供了契机。因此,我们对目前Prdx在肿瘤中的研究进展进行简要概括,从而为肿瘤的治疗提供新的方向。

Expression of Peroxiredoxins and Pulmonary Surfactant Protein A Induced by Silica in Rat Lung Tissue

Silicosis is one of the most serious occupational diseases in China and dates back to centuries ago. In this study, we successfully established a rat model of silicosis by intratracheal silica injection for 28 days and determined hydroxyproline levels to evaluate collagen metabolism in lung homogenates. Oxidative stress status was evaluated by detecting catalase and glutathione peroxidase activities.

2-Cys peroxiredoxins的研究进展

Peroxiredoxins是新近发现的抗氧化酶家族成员,广泛存在于各种生物体内。2-Cys Peroxiredoxins(2-Cys Prxs)是其中一个亚类,具有独特的催化特性和可逆性过氧化循环。2-Cys Prxs在细胞内主要起抗氧化和调节过氧化氢(H2O2)介导的信号转导作用,还参与细胞周期进程以及发挥分子伴侣作用。

Relationship between peroxiredoxins protein family levels and osteoporosis in postmenopausal woman

Objective:To observe the expression levels of the main molecules of peroxiredoxins (Prdxs) protein family (Prdx1, Prdx2, Prdx4 and Prdx6) in women with postmenopausal osteoporosis (PMOP), and to analyze their clinical diagnostic values.Methods: Patients diagnosed with PMOP in Hubei Provincial Hospital of Traditional Chinese Medicine and Wuhan Hospital of Traditional Chinese Medicine from May 2016 to March 2018 were included as the study group (72 cases), postmenopausal women with normal bone mineral density (BMD) in the same period were also collected as the control group (51 cases). Levels of Prdx1, Prdx2, Prdx4 and Prdx6 in plasma were determined by ELISA. mRNA levels of Prdx1, Prdx2, Prdx4 and Prdx6 in peripheral blood mononuclear cells (PBMC) were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The correlations between Prdxs and clinical parameters were analyzed. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic values of Prdxs for PMOP.Results: Prdx1, Prdx4 and Prdx6 levels in the study group were significantly lower than those in the control group (P<0.05). The mRNA expression levels of Prdx1 and Prdx6 of PBMC in the study group were significantly lower than those in the control group (P<0.05). Several Prdxs protein levels (plasma) or mRNA levels (PBMC) in the study group were significantly correlated with lipid levels or inflammatory markers levels (P<0.05). The area under the curve (AUC) of plasma Prdx6 for diagnosing PMOP was 0.794 (95% CI =0.714-0.874). And the AUC of mRNA relative expression of Prdx6 in PBMC for diagnosing PMOP was 0.725 (95% CI =0.635-0.814).Conclusion: The decreased expression of Prdxs protein family (especially Prdx1 and Prdx6) is closely related to the incidence of PMOP, and the decreased Prdxs protein family may promote the occurrence of osteoporosis through the abnormal lipid metabolism pathway and the increased systemic inflammation pathway. The detections of Prdx6 levels in plasma and PBMC are of good diagnostic values for PMOP.

Sulfiredoxin-1通过调节peroxiredoxins表达提高大鼠星形胶质细胞抗氧化作用

目的 探讨内源性抗氧化小分子蛋白Sulfiredoxin-1（Srxn1）对H2O2诱导的星形胶质细胞的抗氧化作用以及对内源性氧化应激防御系peroxiredoxins的调节作用.方法 Srxn1干扰慢病毒载体转染体外培养的大鼠原代星形胶质细胞;构建H2O2氧化应激模型,采用LDH方法检测细胞损伤程度,用超氧化物歧化酶（SOD）分析细胞内氧化应激状态;Western blot检测Srxn1的表达与Prdx Ⅰ～Ⅳ和Prdx-SO2/3H（过氧化Prdx）活性的关系.结果 1）ShSrxn1干扰后,Srxn1蛋白水平下调59.2％ （P＜0.01）,基因水平下调63.6％ （P＜0.01）.2）干扰Srxn1增加H2O2后LDH漏出率（P＜0.01）并使氧化应激损伤显著加重、使Prdx Ⅰ ～Ⅳ的表达降低,Prdx-SO2/3H的表达显著增高（P＜0.01或P＜0.05）.结论 Srxn1减轻H2O2诱导的星形胶质细胞的氧化应激损伤,并可能是通过调节Prdxs的活性来完成的.

PRDX6在心血管疾病中的研究进展

在心血管疾病的研究中,Peroxiredoxin 6(PRDX6)是Peroxiredoxin(PRDX)家族中唯一的哺乳动物1-Cys成员而备受关注。PRDX6因其具有过氧化酶活性和磷脂酶A2活性,在氧化应激中发挥着独特的作用,且已被证明参与氧化还原稳态、磷脂代谢。近年来,PRDX6对于心血管疾病发生发展的作用受到极大关注。然而,目前关于PRDX6在心血管系统中的功能尚未形成统一的认识,该文旨在简要概述PRDX6在动脉粥样硬化、肺动脉高压、心肌梗死、心力衰竭及腹主动脉瘤等心血管疾病中的研究进展,系统回顾PRDX6在这些心血管疾病中的表达变化、作用机制以及可能的治疗潜力,期望为心血管疾病的干预提供新的思路和策略。

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species(ROS) and inducing ferroptosis in activated hepatic stellate cells(HSCs). By using activity-based protein profiling(ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay(CETSA), we show that celastrol directly binds to peroxiredoxins(PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6,through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1(HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2,PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.

NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins （2-Cys Prxs） are efficiently reduced by NADPH Thioredoxin reductase C （NTRC）. To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase （GS）, and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wildtype and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.

Selective silencing of 2Cys and type-ⅡB Peroxiredoxins discloses their roles in cell redox state and stress signaling

Peroxiredoxins （Prx） catalyse the reduction of hydrogen peroxide （H2O2） and, in association with catalases and other peroxidases, may participate in signal transduction by regulating intercel ular H2O2 concentration that in turn can control gene transcription and cel signaling. Using virus-induced-gene-silencing （VIGS）, 2-Cys Peroxiredoxin （2CysPrx） family and type-II Peroxiredoxin B （PrxI B） gene were silenced in Nicotiana benthamiana, to study the impact that the loss of function of each Prx would have in the antioxidant system under control （22℃） and severe heat stress conditions （48 ℃）. The results showed that both Prxs, although in different organel es, influence the regeneration of ascorbate to a significant extent, but with different purposes. 2CysPrx affects abscisic acid （ABA） biosynthesis through ascorbate, while PrxIIB does it probably;through the xanthophyl cycle. Moreover, 2CysPrx is key in H2O2 scavenging and in consequence in the regulation of ABA signal-ing downstream of reactive oxygen species and PrxIIB provides an important assistance for H2O2 peroxisome scavenges.

2-Cys Peroxiredoxins Participate in the Oxidation of Chloroplast Enzymes in the Dark

Most redox-regulated chloroplast enzymes are reduced during the day and oxidized during the night.While the reduction mechanism of light-dependent enzymes is well known,the mechanism mediating their oxidation in the dark remains unknown.The thiol-dependent peroxidases,2-Cys peroxiredoxins (Prxs),play a key role in light-dependent reduction of chloroplast enzymes.Prxs transfer reducing equivalents of thiols to hydrogen peroxide,suggesting the participation of these peroxidases in enzyme oxidation in the dark.Here,we have addressed this issue by analyzing the redox state of well-known redox-regulated chloroplast enzymes in response to darkness in Arabidopsis thaliana mutants deficient in chloroplastlocalized Prxs (2-Cys Prxs A and B,Prx ⅡE,and Prx Q).Mutant plants lacking 2-Cys Prxs A and B,and plants overexpressing NADPH-dependent thioredoxin (Trx) reductase C showed delayed oxidation of chloroplast enzymes in the dark.In contrast,the deficiencies of Prx ⅡE or Prx Q exerted no effect.In vitro assays allowed the reconstitution of the pathway of reducing equivalents from reduced fructose 1,6-bisphosphatase to hydrogen peroxide mediated by Trxs and 2-Cys Prxs.Taken together,these results suggest that 2-Cys Prxs participate in the short-term oxidation of chloroplast enzymes in the dark.

刚地弓形虫peroxiredoxin基因的克隆表达与免疫原性分析

目的对刚地弓形虫peroxiredoxin(TgPrx)基因进行克隆、表达和免疫原性分析。方法收集、纯化RH株弓形虫速殖子,提取总RNA;设计合成引物并引入EcoRI和XhoI酶切位点,RT-PCR扩增编码TgPrx的基因片段克隆到原核质粒pET30a(+)中,经双酶切、PCR及测序鉴定阳性克隆;在大肠杆菌BL21/DE3中用IPTG诱导表达,表达产物经SDS-PAGE进行鉴定,重组蛋白用Western blotting分析其免疫原性。结果从弓形虫RH株cDNA中扩增出591bp的TgPrx基因片段,并成功构建重组质粒pET30a(+)/TgPrx;SDS-PAGE结果表明,目的基因在大肠杆菌BL21/DE3中高效表达。重组蛋白的相对分子量约32kDa,Western blotting显示其能被兔抗弓形虫免疫血清识别。结论RH株刚地弓形虫peroxiredoxin可在原核表达系统中高效表达,该重组蛋白具有免疫原性,有望作为弓形虫疫苗的候选抗原。

抗氧化蛋白Peroxiredoxin家族研究进展

Peroxiredoxin是新近发现的抗氧化酶系 ,广泛存在于各种生物体内。根据分子所具有保守半胱氨酸数目的不同 ,哺乳动物的 6个Peroxiredoxin分为 2个亚类。Peroxiredoxin除了具有共同的抗氧化功能外 ,它还具有其它的功能如细胞增殖与分化、细胞信号转导及保护其它蛋白的氧化等。对该类蛋白分子结构的深入研究已初步揭示其抗氧化的作用机制。Peroxiredoxin与肿瘤关系密切 ,它可能成为一个肿瘤标记物 ,可为肿瘤的治疗提供新的思路。

电针预处理通过上调Peroxiredoxin 6减轻大鼠脑缺血再灌注损伤

目的:探讨Peroxiredoxin 6(Prx 6)在电针预处理诱导的SD大鼠脑缺血耐受中的作用。方法:健康雄性SD大鼠80只,随机分为4组:假手术组(Sham组,n=16)、单纯电针组(Electro acupuncture组,EA组,n=16)、局灶性脑缺血再灌注组[通过大脑中动脉栓塞制作脑缺血再灌注(middle cerebral artery occlusion,MCAO)模型,MCAO组,n=32],其中2、6、12、24、48 h各4只和电针预处理组(EA+MCAO组,n=16)。通过梗死容积及神经功能评分(Garcia评分)评价脑损伤程度;通过Western Blot检测Prx 6在脑缺血再灌注后的表达水平;通过Western Blot和免疫组织化学染色法检测电针预处理对Prx 6表达的影响。结果:与MCAO组相比,EA+MCAO组梗死容积减少,神经功能评分显著改善(P<0.05);与Sham组相比,Prx 6在MCAO模型后再灌注24 h时表达最高(P<0.05);与Sham组相比,再灌注后24 h时MCAO组Prx 6表达水平升高(P<0.05);与MCAO组相比,再灌注后24 h时EA+MCAO组Prx 6的表达水平进一步升高(P<0.05)。结论:电针预处理通过促进缺血再灌注后Prx 6的表达升高而发挥脑保护作用。

人源噬菌体抗体对Peroxiredoxin Ⅰ高表达肺腺癌细胞增殖的抑制作用

背景与目的:研究表明过氧化物酶Peroxiredoxin Ⅰ(Prx Ⅰ)与癌症的发展有密切关系。我们已通过噬菌体展示技术构建了肺腺癌相关的人源单链抗体库。本研究对该库进行筛选,得到抗Prx Ⅰ肺腺癌单链抗体,并检测其对肺腺癌细胞A549增殖的抑制作用。方法:PCR法检测TG1中scFv基因插入率,1%琼脂糖凝胶电泳鉴定Sfi Ⅰ和Not Ⅰ双酶切质粒的结果,以A549细胞及在肺癌中高表达的抗氧化蛋白Prx Ⅰ为靶抗原分别对抗体库进行3轮筛选富集。将阳性克隆用IPTG诱导表达并进行检测。放射性核素计数法测定细胞单链抗体内摄水平,MTT法及流式细胞术检测单链抗体对A549细胞的增殖抑制和凋亡情况,免疫印迹法检测抗体作用A549细胞后Prx Ⅰ的表达水平。结果:scFv基因插入率为77%,双酶切鉴定检测到目的条带。在亲和筛选过程中,肺腺癌单链抗体得到富集,收获率逐轮提高,第6轮为第1轮的180倍。ELISA法检测到在随机选取的10个克隆中,有6个与A549细胞呈阳性反应,阳性率60%。SDS-PAGE及ELISA检测证实得到人源抗PrxI肺腺癌单链抗体。被A549细胞内摄的单链抗体介导了细胞的凋亡以及细胞内Prx Ⅰ蛋白表达水平的下降。结论:从噬菌体抗体库中筛选获得具有较高特异性的抗Prx Ⅰ肺腺癌单链抗体。单链抗体与肺腺癌细胞有特异性亲和力,并能有效抑制其增殖。

Peroxiredoxin Ⅰ和Ⅱ在小鼠输卵管和子宫的分布与表达

目的:观察PeroxiredoxinⅠ和Ⅱ(PrxⅠ和Ⅱ)在小鼠输卵管和子宫的分布及其周期性变化。方法:免疫组织化学和蛋白免疫印迹技术。结果:在输卵管中,PrxⅠ和Ⅱ免疫阳性反应见于输卵管上皮;而子宫内,PrxⅠ免疫阳性反应分布于内膜上皮、腺体上皮和内膜基质,而PrxⅡ主要见于内膜基质。蛋白免疫印迹技术也显示这两型蛋白在输卵管和子宫均表达,动情期和动情间期这两型蛋白在输卵管和子宫中的表达水平无显著性差异。结论:提示PrxⅠ与Ⅱ在输卵管和子宫中表达稳定,可能为受精和早期胚胎发育提供抗氧化的良好微环境。

PeroxiredoxinII在小鼠卵巢中卵泡、GV卵、MII卵和植入前胚的表达和定位

目的 观察PeroxiredoxinII在小鼠卵巢中卵泡和植入前胚的表达与定位 ,为探讨PeroxiredoxinII在小鼠卵母细胞发育成熟和早期胚发育的作用提供依据。 方法 根据小鼠PeroxiredoxinIImRNA序列 (AccessionNo :BC0 0 2 0 34)设计引物 ,对小鼠生发泡完整卵细胞 (GV卵 )中的PeroxiredoxinII可能编码区进行扩增。同时 ,取生后 3d和 2月小鼠的卵巢 ,免疫细胞化学染色显示PeroxiredoxinII蛋白在卵泡中的分布。此外 ,运用RT PCR和免疫细胞化学染色方法观察PeroxiredoxinII在植入前胚的表达。 结果 从GV卵中扩增所得 6 84bp的cDNA序列 ,与小鼠PeroxiredoxinII具有 99%同源性 ;其推导的编码氨基酸序列与小鼠PeroxiredoxinII氨基酸序列完全相同。免疫细胞化学染色显示 :生后 3d小鼠卵巢内未见PeroxiredoxinII阳性反应 ;2月小鼠卵巢内阳性反应见于初级卵泡、次级卵泡和近成熟卵泡中卵母细胞的胞质。卵泡细胞未见PeroxiredoxinII阳性反应。RT PCR结果表明 :PeroxiredoxinIImRNA在GV卵中处高水平 ,从MII卵开始略有下降 ,在 4 细胞胚和早期囊胚期间未检到。免疫细胞化学染色显示 :GV卵、MII卵、1 细胞胚、2 细胞胚、4 细胞胚、8 细胞胚、桑椹胚和早期囊胚均呈PeroxiredoxinII阳性反应。

沉默PeroxiredoxinⅠ基因对乳腺癌裸鼠移植瘤放射敏感性的影响

目的探讨PeroxiredoxinⅠ(PrxⅠ)沉默后对乳腺癌裸鼠移植瘤放射敏感性的影响及其作用机制。方法将稳定表达PrxⅠshRNA的pGPU6-PrxⅠ和阴性对照pGPU6-HK细胞接种于裸鼠皮下,建立乳腺癌裸鼠移植瘤模型。实验共分为4组:pGPU6-HK组、pGPU6-PrxⅠ组、pGPU6-HK+照射(ionizing radiation,IR)组、pGPU6-PrxⅠ+IR组。用6MVX线照射裸鼠瘤组织,测量肿瘤体积,称取肿瘤质量,计算抑瘤率;免疫组化检测肿瘤组织中PrxⅠ和Caspase3蛋白表达;电镜观察肿瘤细胞超微结构,Western blot测定肿瘤组织中γ-H2AX和Rad51蛋白表达。结果成功构建了裸鼠移植瘤模型,pGPU6-PrxⅠ+IR组裸鼠移植瘤生长肿瘤明显迟缓,体积较小,抑瘤率为79.76%,与pGPU6-PrxⅠ(34.92%)和pGPU6-HK+IR(56.94%)比较差异具有显著性(P<0.05);给予pGPU6-PrxⅠ及IR处理后,肿瘤细胞凋亡和坏死增多,PrxⅠ和Rad51蛋白表达减少,而Caspase3和γ-H2AX蛋白表达增加,以pGPU6-PrxⅠ+IR组更为显著,其Rad51蛋白下降了84.8%,γ-H2AX蛋白表达升高5.6倍(P<0.05)。结论沉默PrxⅠ表达可提高乳腺癌移植瘤的放射敏感性,其作用机制与DNA双链断裂增加、DNA损伤后修复能力降低有关。PrxⅠ可能是一个理想的乳腺癌放射增敏分子靶点。