促红细胞生成素对压力超负荷大鼠心肌NADPH氧化酶表达的影响

<正>目的:探讨促红细胞生成素(EPO)对压力超负荷大鼠心肌NADPH氧化酶的影响。方法:SD雄性大鼠,腹主动脉结扎复制高血压模型。随机分成3组:模型组(model);假手术组(sham):除不缩窄腹主动脉外,其余操作同腹主动脉缩窄组;重组人促红细胞生成素治疗组(rhE PO,腹腔注射4 000 U/kg,每周2次)。8周后,心动超声和血流动力学评价心功能;Masson染色     

促红细胞生成素对压力超负荷小鼠心肌保护作用的相关分子机制

目的探讨促红细胞生成素(EPO)对压力超负荷小鼠心肌保护作用的分子机制。方法采用主动脉弓缩窄术(TAC)的方法建立小鼠模型,小鼠随机分为假手术组(Sham)、模型组(TAC-PBS)和EPO2000U/Kg治疗组(TAC-EPO2000)。术后8周,测定各组小鼠心肌毛细血管密度及生存率,Western blot方法检测各组小鼠心肌组织p Akt/Akt、p-STAT3/STAT3、p-e NOS/e NOS、p-p38/p38、p-ERK/ERK-1、p-JNK/JNK的表达。结果 EPO治疗能显著提高TAC小鼠生存率(P0.05)。与TAC-PBS组比较,TAC-EPO2000心肌组织p-STAT3、p Akt和p-e NOS的表达水平显著增多(P0.05),p-p38的表达水平显著减少(P0.05),p-ERK/ERK-1,p-JNK/JNK则无明显差异(P0.05)。各组小鼠心肌毛细血管密度没有明显差异。结论 EPO对压力超负荷小鼠心肌保护作用可能与调控p-STAT3、p Akt、p-e NOS和p-p38的表达相关。     

外源性促红细胞生成素对压力超负荷引起的小鼠左心功能障碍的保护作用

目的探讨外源性促红细胞生成素对压力超负荷引起小鼠左心功能障碍的保护作用。方法选取45只C57BL/6J雄性小鼠,主动脉弓缩窄术(TAC)后,将小鼠随机分为假手术组(Sham)、模型组(TAC-PBS)和治疗组(TAC-EPO),每组15只。采用M型超声心动图分别测量小鼠术前和术后2、4、6、8周左室舒张末期直径、左室收缩末期直径、左室短轴缩短率、舒张末期室间隔厚度、左室游离壁厚度和心室率;术后8周进行心导管测定,测量左心室收缩峰压、左室舒张末压和心室率。结果超声心动图示,与假手术组比较,模型组和治疗组小鼠左心室舒张和收缩末期直径均增加(P0.05),左心室短轴缩短率均减少(P0.05);但经EPO治疗后,与模型组相比,治疗组小鼠左心室舒张和收缩末期直径显著降低(P0.05),左心室短轴缩短率显著增加(P0.05)。结论外源性促红细胞生成素对压力超负荷引起的小鼠左心功能障碍的保护作用可能通过降低左心室扩张而实现。     

促红细胞生成素对压力负荷过重小鼠心肌具有保护作用

目的探讨促红细胞生成素(EPO)对压负荷过重引起的左心室重构是否具有保护作用。方法采用主动脉弓缩窄术(TAC)的方法建立小鼠模型,小鼠随机分为假手术组(Sham)、模型组(TAC-PBS)和治疗组(TAC-EPO)。术后8周,测定红细胞压积与左心室(LV)重量,苏木精-伊红染色测定心肌细胞的横截面积,马松染色与狼星红染色测定心肌间质纤维化的面积,TUNEL法检测心肌细胞凋亡,绘制小鼠生存曲线。结果与Sham相比,模型组和治疗组左心室重量均显著升高,给予EPO治疗后,红细胞压积明显增加(P0.01),但是心肌细胞的横截面积无明显变化(P0.05)。与模型组相比,EPO治疗可显著改善TAC小鼠生存率,减少心肌纤维化及TUNEL阳性心肌细胞数量(P0.05)。结论EPO对压负荷过重引起的左心室重构和过早死亡具有保护作用。     

促红细胞生成素对急性心肌梗死大鼠的心肌HIF-1α与Survivin表达的影响

目的探讨促红细胞生成素对急性心肌梗死(AMI)大鼠心肌缺氧诱导因子-1α(HIF-1α)与生存素(Survivin)蛋白表达的影响。方法将36只健康雄性SD大鼠随机分为假手术组、急性心肌梗死组和促红细胞生成素(EPO)治疗组。急性心肌梗死组及EPO治疗组通过冠状动脉左前降支结扎建立急性心梗模型。模型建立4周后,利用心脏超声评价心功能,原位末端凋亡法(TUNEL)检测心肌细胞凋亡情况,采用免疫组织化学方法与Western blot方法检测各组大鼠心梗区域心肌HIF-1α与Survivin蛋白的表达。结果与急性心肌梗死组大鼠相比,EPO治疗组大鼠心功能明显改善,左室射血分数明显增加,左室舒张末期内径(LVEDD)明显缩小(P0.01)。急性心肌梗死组大鼠比假手术组心肌细胞凋亡数明显升高,心肌HIF-1α蛋白表达量显著升高,Survivin蛋白表达量显著降低,而EPO治疗组大鼠比急性心肌梗死组心肌细胞凋亡数明显降低,心肌HIF-1α蛋白表达量显著降低,Survivin蛋白表达量显著升高(P0.01)。结论降低HIF-1α蛋白表达与上调Survivin蛋白表达可能是促红细胞生成素抑制急性心肌梗死大鼠心肌细胞凋亡的机制之一。     

促红细胞生成素对大鼠肾间质纤维化发动蛋白-1表达影响

目的探讨促红细胞生成素（EPO）对大鼠肾间质纤维化发动蛋白．1（Drp-1）表达的影响。方法81只sD大鼠,分为假手术组、对照组和EPO组,每组各27只。EPO组、对照组采用单侧输尿管结扎并剪断建立肾间质纤维化模型,假手术组仅游离输尿管而不结扎和剪断;EPO组予EPO皮下注射;假手术组和对照组给予等量生理盐水皮下注射。各组于术后7、14和21d取动脉血分离血清检测血肌酐和尿素氮水平。取梗阻侧肾组织行苏木精．伊红和Masson染色,观察肾脏病理学变化。用免疫组化方法检测肾组织Drp一1的表达。结果@EPO组各时点血清肌酐和尿素氮水平显著低于对照组（P〈0．01）,但高于假手术组（P〈0．05）。②EPO组各时点肾组织病理改变较对照组明显减轻,肾小管间质损伤评分和肾间质纤维化相对面积均低于对照组（P均〈0．05）,但高于假手术组（P〈0．05）。③EPO组肾组织的Drp．1表达显著低于对照组（P〈0．05）,但高于假手术组（P〈0．05）。结论Drp-1在大鼠肾间质纤维化肾组织中表达增加,参与。肾脏纤维化过程;EPO可以通过抑制Drp一1表达,从而延缓肾间质纤维化的进展。     

重组人促红细胞生成素对人脐静脉内皮细胞PCNA表达的影响

目的研究重组人促红细胞生成素（rHuEPO）对人脐静脉内皮细胞（HUVEC）增殖的影响。方法从脐静脉体外分离培养HUVEC，采用细胞培养及免疫组织化学方法检测不同剂量rHuEP0（10U／ml、20U／ml、40U／m1）对HUVEC增殖细胞核抗原（PCNA）表达的影响。结果对组照及10U／ml、20U／ml、40U／mlrHuEP0实验组内皮细胞PCNA阳性表达百分率分别为（31±3）％、（40±5）％、（68±6）％和（65±5）％，与对照组相比，rHuEPO作用后内皮细胞PCNA表达明显增强（P〈0．05），且随rHuEPO剂量提高作用增强，呈剂量效应关系。结论rHuEP0可促进体外培养内皮细胞增殖。     

促红细胞生成素抑制TGF-β诱导的大鼠心肌成纤维细胞增殖

目的 探讨促红细胞生成素在大鼠心肌成纤维细胞(CFs)增殖中的作用.方法 培养大鼠CFs.实验分为对照组(control)、TGF-β刺激组(TGF-β终浓度为5μg/L)和重组人促红细胞生成素(rhEPO,5 000 U/L)干预组:1h后加入TGF-β.24 h后计数细胞并采用MTT法观察细胞增殖;免疫细胞化学及Western blot法检测α-SMA表达,观察细胞转化;羟脯氨酸定量检测细胞胶原含量;Western blot法检测细胞Ⅰ、Ⅲ型胶原蛋白及MMP-2、MMP-9表达.结果 与对照组比较,TGF-β使细胞明显增殖(P＜0.05),Ⅰ、Ⅲ型胶原蛋白合成(P＜0.05)、α-SMA表达(P＜0.05)、细胞MMP-2、MMP-9表达增多(P＜0.05).使用重组人促红细胞生成素(rhEPO)干预后,CFs增殖、α-SMA表达及Ⅰ、Ⅲ型胶原蛋白合成较TGF-β组均显著降低(P＜0.05),MMP-2、MMP-9表达进一步增多(P＜0.05).结论 生理剂量EPO可抑制TGF-β诱导的大鼠CFs增殖、转化及胶原的合成,促进胶原降解.     

促红细胞生成素对人脐静脉内皮细胞HoxD3 mRNA表达的影响

目的研究促红细胞生成素(EPO)对体外培养内皮细胞同源盒基因Hox D3 mRNA表达的影响。方法体外培养人脐静脉内皮细胞(HUVEC),Ⅷ因子相关抗原(Ⅷ-RAg)鉴定,逆转录-聚合酶链反应(RT-PCR)技术检测细胞Hox D3 mRNA的表达水平。结果与PBS空白对照组比较,EPO可明显上调内皮细胞Hox D3 mRNA表达水平,差异有统计学意义(P<0.05)。结论EPO可通过影响内皮细胞Hox D3 mRNA表达,从而调节EPO促血管生成作用。     

大鼠心肌梗死后心肌NADPH氧化酶亚单位p22phox的表达

目的：探讨心肌梗死大鼠心室重塑与还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶亚单位p22phox和超氧阴离子的关系。 方法： Sprague-Dawley大鼠冠脉左前降支结扎复制心肌梗死模型，8周后，心脏超声、血流动力学、心脏形态学方法检测分析心室重塑，检测血浆和非梗死心肌脂质过氧化物的浓度。用RT-PCR和免疫组化方法检测p22phox mRNA水平和蛋白水平的分布。用激光共聚焦方法检测心肌超氧阴离子分布。 结果： 心肌梗死后大鼠心室重塑过程显著，与正常对照组比较，左室舒张末压、左室舒张末径［(3.09±1.52 vs 18.24±6.58)mmHg，(0.67±0.06 vs 0.90±0.15)mm, P<0.01］和脂质过氧化物水平在血浆和非梗死心肌均显著大于正常对照组(P<0.05)。p22phox mRNA和蛋白表达以及超氧阴离子分布在梗死和非梗死心肌亦均显著增加。 结论： 大鼠心肌梗死后，NADPH氧化酶表达增高，其来源的超氧阴离子可能通过氧化应激参加心室重塑过程。     

NADPH氧化酶家族与高血压血管重构

高血压及其并发症的发生、发展与血管重构密切相关。高血压过程中血管重构的类型、特点以及发生机制已成为高血压研究领域的热点之一。高血压血管重构的机制尚未完全明了,研究表明活性氧诱导的氧化应激参与了高血压血管重构的各个环节。NADPH氧化酶是体内生成活性氧的主要酶类,大量研究证实NADPH氧化酶来源的ROS在高血压血管重构的发生、发展中起重要作用。本文就NADPH氧化酶介导高血压血管重构的作用及机制作一综述。     

吞噬细胞NADPH氧化酶活性的调控

吞噬细胞NADPH氧化酶是一个多组分活化的酶复合体,是吞噬细胞抗菌系统的主要成分。此氧化酶体系包括膜结合的和可溶的胞浆蛋白,其主要特征为依赖刺激而活化,机制为复合体的胞浆因子磷酸化并转位至膜,在这里与细胞色素酶相关联,组装完全的复合体在功能上作为一个电子转运体系,从胞浆NADPH转移电子给分子氧形成超氧阴离子（O2-）,伴随着接下来的活性物质产生,也叫呼吸爆发,发挥抗菌和抗细胞毒素的活性。NADPH氧化酶的这一活化过程受调节亚基、上游调节子等多重因素的调控。     

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.     

Neuregulin-1β对压力超负荷心肌肥大大鼠治疗作用及机制的研究

 目的：研究神经调节蛋白 1β（NRG-1β）对压力超负荷所致大鼠心肌肥大的治疗作用并探讨其机制。方法：Wistar雄性大鼠采用腹主动脉缩窄的方法复制心肌肥大模型。术后8周,将模型动物随机分成模型（model）组、NRG-1β治疗组（尾静脉注射NRG-1β,10 μg·kg-1·d-1）和NRG-1β+赫赛汀(Herceptin, HERCE)治疗组（尾静脉注射NRG-1β的同时给予注射HERCE 10 μg·kg-1·d-1）。假手术（sham）组除不以银夹缩窄腹主动脉外,其余操作同腹主动脉缩窄组。7　d后分别采用心动超声、血流动力学评价心功能;Masson染色观察心肌组织的超微结构;放射免疫法检测心肌组织中血管紧张素II（Ang II）,酶联免疫吸附法测定心肌组织中肿瘤坏死因子 α（TNF-α）的变化;RT-PCR法检测心肌中bcl-2和bax mRMA表达的改变。结果：（1）心动超声显示,和模型组比较,NRG-1β组左室射血分数(LVEF)及短轴缩短率(LVFS)升高,左室收缩末内径(LVESD)及舒张末内径(LVEDD)减小（P<0.01）。（2）血流动力学检测显示,NRG-1β治疗组左室收缩末压(LVESP)和左室内压最大上升和下降速率(±dp/dtmax)均明显高于模型组（P<0.01）;左室舒张末压(LVEDP)低于模型组（P<0.01）。（3）与模型组比较,NRG-1β组心肌胶原容积分数（CVF）下降,心肌中Ang II和TNF-α明显减少,bcl-2 mRNA表达显著升高,而bax mRNA表达下降（P<0.01）。（4）NRG-1β+ HERCE治疗组与模型组相比各项指标无明显改变（P>0.05）。结论：NRG-1可以减少压力超负荷大鼠心肌Ang II和TNF-α的生成,从而减轻Ang II和TNF-α介导的心肌间质重构; NRG-1可通过上调bcl-2 mRNA表达、下调bax mRNA表达,抑制心肌细胞的凋亡,改善压力超负荷大鼠的心功能,进而在心肌肥大的过程中发挥作用。     

NADPH氧化酶与脑缺血/再灌注损伤

氧化应激是发生脑缺血/再灌注(ischemia/reperfusion,I/R)损伤的重要机制.近来研究发现,还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶产生的活性氧对脑I/R后的氧化应激起到重要的作用.一些方法(如常压高氧、缺血后处理)和一些药物(如罗布麻宁、替米沙坦、桦木酸、加兰他敏、雷公藤红素等)可以NADPH氧化酶为靶点治疗脑I/R损伤.     

NADPH oxidase mediated oxidative stress in hepatic fibrogenesis

NADPH oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. ROS is involved as key secondary messengers in numerous signaling pathways, and NADPH oxidases complex has been increasingly recognized as key elements of intracellular signaling of hepatic fibrogenesis. In the liver, NADPH oxidase is functionally expressed both in the phagocytic form and in the non-phagocytic form. The non-phagocytic NADPH oxidase complex is structurally and functionally similar to the phagocytic NADPH, resulting in reduction of molecular oxygen to generate superoxide. There are six homologous NOX proteins in the non-phagocytic forms of NADPH oxidase. An emerging concept is that both phagocytic and nonphagocytic NADPH oxidase components in hepatic stellate cells (HSCs) mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy. The molecular components and signaling pathways of various NADPH oxidase homologues that are critical for the fibrotic activity in HSCs need to be more clearly identified.     

A leading role for NADPH oxidase in an in-vitro study of experimental autoimmune encephalomyelitis

Myelin oligodendrocyte glycoprotein peptide fragment 35–55 (MOG_35–55) is a major autoantigen inducing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis that is characterized by blood–brain barrier (BBB) disruption. Various experimental approaches have employed MOG_35–55 in vivo; however, in vitro BBB models using MOG_35–55 are rarely reported. We investigated MOG_35–55 exposure effects with complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) on brain endothelial cells and elucidated the relationships among NADPH oxidase, MMP-9, ICAM-1, and VCAM-1. These 4 factors significantly increased in MOG_35–55 + CFA + PTX-exposed endothelial cells compared with the control cells. NADPH oxidase inhibition using apocynin reduced MMP-9 activity, ICAM-1, and VCAM-1. MMP-9 inhibitor I decreased expression of ICAM-1 and VCAM-1, and both anti-ICAM-1 and anti-VCAM-1 inhibited MMP-9 activity. Inhibitions of MMP-9, ICAM-1, and VCAM-1 did not change NADPH oxidase activity. Although inhibition of these 4 factors decreased BBB permeability in cells, inhibition of NADPH oxidase exhibited the highest decrease among these. NADPH oxidase directly influenced MMP-9, ICAM-1, and VCAM-1, but not vice versa. MMP-9 and the cell adhesion molecules reversibly affected each other. In conclusion, NADPH oxidase-derived superoxide elevated expression of MMP-9, ICAM-1, and VCAM-1, and these interactions can finally result in increases of BBB permeability in MOG_35–55 + CFA + PTX-exposed endothelial cells.     

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1β and tumour necrosis factor-α

Objectives to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and to study the NADPH oxidase involvement in this production. Material and Methods Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1β and TNF-α, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. Results Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. Conclusions NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4). Received 2 August 2005; returned for revision 12 January 2006; returned for final revision 22 May 2006; accepted by J. Di Battista 9 June 2006