Nicotinamide adenine dinucleotide phosphate oxidase activation and neuronal death after ischemic stroke

Nicotinamide adenine dinucleotide phosphate oxidase(NOX) is a multisubunit enzyme complex that utilizes nicotinamide adenine dinucleotide phosphate to produce superoxide anions and other reactive oxygen species. Under normal circumstances, reactive oxygen species mediate a number of important cellular functions, including the facilitation of adaptive immunity. In pathogenic circumstances, however,excess reactive oxygen species generated by NOX promotes apoptotic cell death. In ischemic stroke, in particular, it has been shown that both NOX activation and derangements in glucose metabolism result in increased apoptosis. Moreover, recent studies have established that glucose, as a NOX substrate, plays a vital role in the pathogenesis of reperfusion injury. Thus, NOX inhibition has the potential to mitigate the deleterious impact of hyperglycemia on stroke. In this paper, we provide an overview of this research,coupled with a discussion of its implications for the development of NOX inhibition as a strategy for the treatment of ischemic stroke. Both inhibition using apocynin, as well as the prospect of developing more specific inhibitors based on what is now understood of the biology of NOX assembly and activation, will be highlighted in the course of our discussion.

Excessive Copper Induces the Production of Reactive Oxygen Species,which is Mediated by Phospholipase D, Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Antioxidant Systems

Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu^2＋. Treatment with 100 μmol/L Cu^2＋ generated a large amount of HzOz and thiobarbituric acid-reactive substances （TBARS） in cells. Using phospholipase D （PLD） specific inhibitor （1-butanol） or phosphatidic acid （PA）, we demonstrated that PLD plays an important role in the generation of H2O2 and TBARS. Semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme activity assays with wild type and nicotinamide adenine dinucleotide phosphate （NADPH） oxidaseoverexpressing BY-2 cells revealed that PLD and PA are the key factors leading to NADPH oxidase activation, which is responsible for H2O2 and TBARS production induced by Cu^2＋. Moreover, the content of ascorbic acid （AsA）, an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu^2＋. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu^2＋-elevated reactive oxygen species （ROS） production is caused by upregulated PLD that elevates the activity of NADPH oxidase and its collapsed antioxidant systems that scavenges ROS.

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.

Potential role of NADPH oxidase in pathogenesis of pancreatitis

Studies have demonstrated that reactive oxygen species(ROS) are closely related to inflammatory disorders. Nicotinamide adenine dinucleotide phosphate oxidase(NOX), originally found in phagocytes, is the main source of ROS in nonphagocytic cells. Besides directly producing the detrimental highly reactive ROS to act on biomolecules(lipids, proteins, and nucleic acids), NOX can also activate multiple signal transduction pathways, which regulate cell growth, proliferation, differentiation and apoptosis by producing ROS. Recently, research on pancreatic NOX is no longer limited to inflammatory cells, but extends to the aspect of pancreatic acinar cells and pancreatic stellate cells, which are considered to be potentially associated with pancreatitis. In this review, we summarize the literature on NOX protein structure, activation, function and its role in the pathogenesis of pancreatitis.

Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

AIM:To investigate the role of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium(DSS)-induced colitis.METHODS:Balb/c mice were divided into three groups:8 mice with acute DSS-induced colitis(3.5% DSS solution;7 d),8 mice with chronic DSS-induced colitis(3.5% DSS solution for 5 d + water for 6 d;4 cycles;total:44 d) and 12 mice without DSS supplementation as a control group.Primary colonic epithelial cells were isolated using chelation method.The cells were cultivated in the presence of mediators(lipopolysaccharide(LPS),apocynin or diphenyleneiodonium).Viability of cells was assessed by fluorescent microscopy.Production of reactive oxygen species(ROS) by the cells was measured fluorometrically using Amplex Red.Production of tumour necrosis factor-alpha(TNF-α) by the colonic epithelial cells was analysed by ELISA.Nox1 gene expression was assessed by real-time PCR.RESULTS:Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups,whereas decreased viability,increased ROS production,and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls.The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis.Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.CONCLUSION:Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.

The Notch pathway attenuates burn-induced acute lung injury in rats by repressing reactive oxygen species

Background:Acute lung injury(ALI)is a common complication following severe burns.The underlying mechanisms of ALI are incompletely understood;thus,available treatments are not sufficient to repair the lung tissue after ALI.Methods:To investigate the relationship between the Notch pathway and burn-induced lung injury,we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations,including hematoxylin and eosin(H&E)staining,lung injury scoring,bronchoalveolar lavage fluid and wet/dry ratio analyses,myeloperoxidase immunohistochemical staining and reac-tive oxygen species(ROS)accumulation analysis.To explore whether burn injury affects Notch1 expression,we detected the expression of Notch1 and Hes1 after burn injury.Then,we extracted pulmonary microvascular endothelial cells(PMVECs)and conducted Notch pathway inhibition and activation experiments,via aγ-secretase inhibitor(GSI)and OP9-DLL1 coculture,respectively,to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs.To investigate the regulatory effect of the Notch pathway on ROS accumulation,we detected the expression of oxidative-stress-related molecules such as superoxide dismutase,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)2,NOX4 and cleaved caspase-3.NOX4-specific small interfering RNA(siRNA)and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4.Results:We successfully established a burn model and revealed that lung injury,excessive ROS accumulation and an inflammatory response occurred.Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury.In PMVECs challenged with burn serum,ROS and cell death were elevated.Moreover,when the Notch pathway was suppressed by GSI,ROS and cell apoptosis levels were significantly increased.Conversely,these parameters were reduced when the Notch pathway was activated by OP9-DLL1.Mechanistically,the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs.Conclusions:The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs.The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.

Role of reactive oxygen species in the renal fibrosis

Renal fibrosis is a common pathway of progressive renal diseases leading to end-stage renal disease regardless of the etiology. Accumulating evidence indicates that oxidative stress, resulting in generation of reactive oxygen species （ROS）, plays a critical role in the initiation and progression of fibrotic diseases. Nicotinamide adenine dinucleotide phosphate （NADPH） oxidase is the predominant enzyme source for ROS generation and is now recognized as a key mediator of cell proliferation and matrix accumulation in renal disease. Multiple stimuli and agonists, such as transforming growth factor , tumor necrosis factor, platelet derived growth factor, angiotensin II, hyperglycemia, oxidized low-density lipoprotein and albumin have been shown to alter the activity or expression of the NADPH oxidase and ultimately increase ROS production. ROS directly incites damage to biologically important macromolecules and leads to generation of the so-called advanced oxidation protein products （AOPPs） and advanced glycation end products, which are not only markers of oxidative stress but also cause renal injury. Targeting NADPH oxidase and/or reducing AOPPs production miaht be a novel strateav for the theraoeutic intervention of varietv of fibrotic kidney disorders.

NADPH氧化酶4在心血管损伤中的作用机制

心血管结构和功能损伤是许多心血管疾病的重要病理基础,许多研究表明氧化应激在缺血性心脏病、动脉粥样硬化、高血压等诸多病理性心血管损伤中发挥重要作用。NADPH氧化酶(Nox)是调控氧化还原信号的关键酶,而血管内的活性氧主要来源于Nox4。随着研究的不断深入,发现Nox4在不同阶段或不同刺激下会发挥不同甚至截然相反的作用,如双向调节动脉粥样硬化的进展、双向作用影响血压等。现总结Nox4在不同心血管损伤中的不同影响及作用机制,为后续的研究提供一定的理论基础。

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2） was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco＇s modified Eagle＇s medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-＇reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine （8-OHdG）, 4-hydroxy-2-nonenal （4-HNE） and 3-nitrotyrosin （3-NT） in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

子痫前期孕妇外周血NOX2和胎盘组织中NOX2、ROS的表达及其临床意义

目的探讨子痫前期孕妇外周血、胎盘组织中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)及胎盘组织中活性氧(ROS)的表达及其临床意义。方法选取2021年3月—2022年10月在徐州医科大学附属徐州妇幼保健院就诊的子痫前期孕妇60例,其中早发型子痫前期孕妇30例(早发组),晚发型子痫前期孕妇30例(晚发组),另取同期在该院就诊的健康孕妇30例作为对照(正常组)。检测各组外周血NOX2、胎盘组织中NOX2 mRNA相对表达量和ROS表达。结果早发组外周血NOX2、胎盘组织NOX2阳性表达率、NOX2mRNA相对表达量、ROS阳性表达率均高于对照组和晚发组(P<0.0125);晚发组与对照组外周血NOX2、胎盘组织NOX2阳性表达率、NOX2 mRNA相对表达量、ROS阳性表达率比较,差异均无统计学意义(P>0.05)。早发组≥30岁孕妇外周血NOX2高于<30岁孕妇(P<0.05)。早发组不同体质量指数、孕妇类型、严重程度孕妇外周血NOX2、胎盘组织NOX2阳性表达率、NOX2 mRNA相对表达量、ROS阳性表达率比较,差异均无统计学意义(P>0.05)。晚发组不同年龄、体质量指数、孕妇类型、严重程度孕妇外周血NOX2、胎盘组织NOX2阳性表达率、NOX2 mRNA相对表达量、ROS阳性表达率比较,差异均无统计学意义(P>0.05)。子痫前期外周血NOX2与胎盘组织NOX2蛋白、NOX2 mRNA相对表达量呈正相关(r=0.411和0.455,P=0.017和0.012);胎盘组织NOX2蛋白与胎盘组织NOX2 mRNA相对表达量呈正相关(r=0.425,P=0.013)。早发组胎盘组织NOX2和ROS阳性表达率高于晚发组和对照组(P<0.05)。结论外周血NOX2和胎盘组织中NOX2、ROS表达水平升高可能与早发型子痫前期的发病有关。子痫前期早发型与晚发型的发病机制可能有所不同。

高葡萄糖刺激血管内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调、活性氧增加及细胞凋亡

目的研究在高葡萄糖刺激的条件下,人脐静脉内皮细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的表达、细胞内活性氧以及细胞凋亡的变化。方法倒置显微镜下观察人脐静脉内皮细胞形态;免疫荧光法检测人脐静脉内皮细胞Ⅷ因子相关抗原的表达;高葡萄糖刺激人脐静脉内皮细胞,用逆转录聚合酶链反应检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA水平,Western blotting检测尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4蛋白表达的变化,DCFH-DA检测细胞内活性氧生成量,流式细胞仪和Hoechst染色检测细胞凋亡。结果高葡萄糖刺激人脐静脉内皮细胞时,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4mRNA及蛋白表达上调,细胞内活性氧生成增加,细胞凋亡增加。结论高葡萄糖处理促进人脐静脉内皮细胞尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4表达上调并引起细胞内活性氧生成和凋亡增加。

尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达对人脐静脉内皮细胞凋亡和活性氧水平的影响

目的研究尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4表达水平的改变对内皮细胞活性氧生成和凋亡的影响。方法转染尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4表达质粒或GFP质粒到人脐静脉内皮细胞和ECV304中,用逆转录聚合酶链反应检测转染后尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4mRNA的水平,用流式细胞仪检测细胞内活性氧水平和细胞凋亡率,用Hoechst染色和TUNEL法观察细胞凋亡。结果转染后的人脐静脉内皮细胞中尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4mRNA水平明显高于GFP质粒组和对照组,不表达尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶的ECV304细胞系经转染后亦表达尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4mRNA;流式细胞仪检测发现,与GFP质粒组(ECV304和人脐静脉内皮细胞的凋亡率分别为1.56%±0.33%和4.56%±0.62%)和对照组(ECV304和人脐静脉内皮细胞的凋亡率分别为1.05%±0.25%和2.28±0.37%)相比,转染尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4质粒组内皮细胞的活性氧生成和凋亡率(ECV304和人脐静脉内皮细胞的凋亡率分别为9.60%±0.92%和12.41%±1.12%)明显增加(P<0.05,n=3);Hoechst和TUNEL染色发现,转染尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4质粒后有部分内皮细胞胞核出现凋亡特征性改变。结论尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4表达质粒可有效地转染人脐静脉内皮细胞,引起尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4mRNA水平升高;尼克酰胺腺嘌呤二核苷酸磷酸盐氧化酶4过表达可诱导人脐静脉内皮细胞凋亡。

NADPH氧化酶抑制剂对遗传性视网膜色素变性感光细胞凋亡的抑制作用

背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性（RP）过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇（ROS）的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d（P9）腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg（0.01 ml/kg）,每日1次,连续5d（至P13）;PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d（P14）处死并制备视网膜冰冻切片,采用二氢乙锭（DHE）荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR（real-time PCR）法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义（t=2.360,P=0.000）;香荚兰乙酮注射组小鼠视网膜外核层厚度为（35.95±1.63） μm,明显厚于PBS对照组的（23.17±1.38） μm,差异有统计学意义（t=3.850,P=0.016）.结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.

NADPH氧化酶NOX家族与疾病的关系

还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶的非吞噬细胞氧化酶(non-phagocytic cell oxidase,NOX)家族是许多非吞噬细胞中活性氧(reactive oxygen species,ROS)的主要来源。正常状态下,通过该途径产生的ROS作为信号分子参与了细胞分化、增殖、凋亡等的调节,但在环境胁迫下,NOX蛋白家族在感受细胞外信息刺激时,能够迅速活化产生过量的ROS,引起的氧化压力会诱导机体多种疾病的发生、发展。本文主要从NADPH氧化酶NOX家族蛋白的结构、活化、功能及与疾病发生、发展的关系等方面进行简述。

β淀粉样蛋白通过升高ROS水平上调晚期糖基化终末产物受体的表达

目的探讨β淀粉样蛋白（Aβ）调控海马晚期糖基化终产物受体（RAGE）表达及其机制。方法通过脑立体定向仪及微量注射器于大鼠海马内注射Aβ1~40,注射3周后,利用Western blot检测海马组织RAGE、还原型烟酰胺腺嘌呤二核苷酸（NADPH）氧化酶（gp9lphox及p47phox亚基）、核因子-κb（NF-κB）、NF-κB抑制蛋白（IκB）的表达变化,利用流式细胞仪检测大鼠海马组织活性氧（ROS）的变化。结果海马注射Aβ1~403周后,海马组织的RAGE的表达显著升高（P〈0.01）,同时检测到注射Aβ1~40引起海马组织内ROS水平明显增高（P〈0.01）,gp9lphox、p47phox的表达及p47phox的磷酸化水平均显著升高（P〈0.01）,IκBα的表达显著降低（P〈0.01）,IκBα的磷酸化水平显著增加（P〈0.01）,NF-κB的表达及NF-κB的磷酸化水平均显著升高（P〈0.01）。结论 Aβ可引起海马组织RAGE的表达升高,NADPH氧化酶/ROS/NF-κB信号通路可能在Aβ诱导海马神经元RAGE的表达过程中发挥重要作用。

活性氧与肾纤维化的研究进展

肾纤维化是各种慢性肾疾病进行性发展的主要病理基础,活性氧在肾纤维化的发生和发展中扮演着重要的角色。有关抗氧化治疗肾纤维化的研究较多,N-乙酰半胱氨酸、血管紧张素转换酶抑制剂、氟非尼酮、维生素E等被认为有一定的肾保护作用,这也为肾纤维化的临床防治提供新的研究方向和治疗靶点。

夹竹桃麻素对高肺血流肺动脉高压大鼠的治疗作用

目的观察夹竹桃麻素(apocynin)对肺高血流肺动脉高压大鼠肺动脉内皮细胞活性氧(ROS)及NADPH氧化酶4(NOX4)蛋白水平的影响,研究Apocynin对肺高血流肺动脉高压形成的拮抗作用。方法将30只雄性SD大鼠随机均分为对照组、分流给药组、分流组,通过腹主动脉-下腔静脉分流法建立左向右分流肺高血流肺高压大鼠模型,测定大鼠平均肺动脉压、肺小动脉中膜厚度百分比(WT%)、肺小动脉管壁面积百分比(WA%);体外培养模型大鼠肺动脉内皮细胞,流式细胞仪检测2,7-二氢二氯荧光黄双乙酸钠(DCFH-DA)荧光探针标记的肺动脉内皮细胞活性氧水平,Western Blot法检测大鼠肺动脉内皮中NOX4蛋白的表达情况。结果与分流组对比,分流给药组大鼠肺动脉压升高和肺血管重构情况明显改善,肺动脉内皮细胞中NOX4蛋白的表达较分流组虽无明显减少,但细胞内ROS的水平显著下降。结论持续使用夹竹桃麻素干预,可使左向右分流肺动脉高压大鼠肺动脉内皮细胞中ROS水平明显下降,从而拮抗大鼠肺动脉高压的形成。

NADPH氧化酶家族成员促肝纤维化的研究进展

肝纤维化是由肝脏在各种慢性肝损伤条件下的损伤修复反应造成,以细胞外基质过度沉积为主要特征.许多证据表明NADPH氧化酶(nicotinamide adenine dinucleotide phosphate oxidase,NOX)及其产生的活性氧簇(reactive oxygen species,ROS)引起的氧化应激在肝纤维化中起关键作用.NOX是一个多亚基复合体,在肝脏中,吞噬细胞型和非吞噬细胞型的NOX均有功能性表达,并对引起肝纤维化的主要细胞-肝星状细胞(hepatic stellate cells,HSCs)均具有明显的促纤维化作用.本文就NOX家族在肝纤维化的发生、发展中的作用的近几年研究进展做一综述.

NOX家族在肝纤维化中的作用

还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH oxidase,NOX)是一种多蛋白质亚基组成的复合物,存在于吞噬细胞和非吞噬细胞中,能生成消除病原微生物的活性氧簇(reactive oxygen species,ROS).在肝脏,NOX参与肝纤维化过程,并发挥重要作用.激活后具有功能的NOX不仅在Kupffer细胞(KC)中(吞噬细胞型)存在,在肝星状细胞(hepatic stellate cells,HSCs)中(非吞噬细胞型)同样存在,非吞噬细胞型NOX在HSCs的活化中发挥作用.本文就NOX在肝纤维化形成过程中所起的作用进行综述.

高糖对人脐静脉内皮细胞NADPH氧化酶表达和活性氧生成的影响

目的:探讨高糖对内皮细胞活性氧生成和NADPH氧化酶(nicotinamide adenine dinucleotide phosphate oxidase,NOX)表达的影响。方法:本实验以人脐静脉内皮细胞为研究对象,分为正常对照组、20 mmol/L甘露醇处理4 h高渗对照组、20 mmol/L葡萄糖处理2 h组、20 mmol/L葡萄糖处理4 h组、20 mmol/L葡萄糖处理6 h组和NOX特异性抑制剂二苯基碘(diphenylene iodonium,DPI)5 mol/L预处理0.5 h再加入20 mmol/L葡萄糖处理组(DPI预处理组)。用流式细胞仪检测各组细胞内活性氧的浓度;反转录聚合酶链反应及Western印迹法分别检测各组细胞内NOX各亚基m RNA和蛋白的表达变化。结果:1)与正常对照组相比,20 mmol/L葡萄糖处理2,4和6 h组活性氧增高(均P<0.05),而DPI组活性氧生成与正常对照组比较差异无统计学意义(P>0.05);2)与正常对照组相比,20 mmol/L葡萄糖处理4 h组NOX4 m RNA和蛋白的表达明显增加(均P<0.05),NADPH氧化酶亚基NOX2,P67phox,P22phox和Rac表达差异均无统计学意义(均P>0.05);3)与正常对照组相比,DPI预处理组NOX4,NOX2,P67phox,P22phox和Rac表达差异均无统计学意义(均P>0.05)。结论:高糖可能通过上调NOX4表达以增加活性氧生成而发挥氧化应激作用。