脱氢抗坏血酸对高糖诱导系膜细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导系膜细胞产生氧自由基(ROS)的影响。 方法 (1)原代培养大鼠系膜细胞；(2)以Fe3+还原法检测细胞内抗坏血酸(AA)和DHA浓度，观察系膜细胞摄取AA和DHA的情况及葡萄糖、葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B对其的影响；(3)采用激光扫描共聚焦显微镜检测细胞内ROS，观察高糖诱导系膜细胞ROS产生的情况及不同浓度DHA对其的影响；(4)采用凝胶电泳迁移率法(EMSA)检测活性蛋白1(AP-1)和DNA的结合活性，观察DHA对高糖诱导的系膜细胞内AP-1 活性的影响。结果 (1)AA不能由细胞外进入系膜细胞，而DHA可以进入，并且随着细胞外葡萄糖浓度的增加，其进入速度减慢；细胞松弛素B则完全抑制了DHA进入到系膜细胞。(2)高糖快速诱导系膜细胞ROS产生增多；DHA抑制了高糖的这种作用，并且该抑制作用在≤4 mmol/L的浓度范围内呈浓度依赖性。(3)DHA抑制了高糖诱导的系膜细胞内AP-1 的激活。 结论 (1)系膜细胞是依赖DHA利用Vit C的细胞型；DHA进入该细胞依赖GLUT介导，高糖可抑制其进入细胞。(2)DHA可有效抑制高糖诱导的系膜细胞ROS产生增多，并在一定范围内呈浓度依赖性。(3)DHA在抑制ROS产生的同时，也显著抑制了高糖诱导的AP-1 的激活。     

脱氢抗坏血酸对高糖诱导肾小管上皮细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导肾小管上皮细胞产生氧自由基的影响｡方法 (1)传代培养肾小管上皮细胞;(2)以Fe3+还原法检测细胞内抗坏血酸(AA)浓度,观察肾小管上皮细胞摄取AA和DHA的情况及葡萄糖和葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B(cytochalasin B)对其影响;(3)激光扫描共聚焦显微镜检测细胞内氧自由基,观察高糖诱导肾小管上皮细胞氧自由基产生的情况及不同浓度DHA对其影响｡结果 (1)AA不能由细胞外进入肾小管上皮细胞,而DHA可以进入,并且随着细胞外葡萄糖浓度的增加,其进入速度减慢｡细胞松弛素B则完全抑制了DHA进入到肾小管上皮细胞, 而固定葡萄糖浓度(25 mmol/L)时,随着DHA浓度的增加,DHA进入细胞内的速度逐渐增快｡(2)高糖快速诱导了肾小管上皮细胞氧自由基产生增多,DHA抑制了高糖的这种作用,并且该抑制作用在浓度小于､等于4 mmol/L的范围内呈浓度依赖性｡结论 (1)肾小管上皮细胞是依赖DHA利用VitC的细胞型,DHA进入该细胞依赖GLUT介导,高糖可抑制其进入细胞;(2)DHA可有效抑制高糖诱导的肾小管上皮细胞氧自由基产生增多,并在一定范围内呈浓度依赖性｡     

表没食子儿茶素没食子酸酯对高糖环境下体外小鼠足细胞活性氧的影响

目的　观察不同浓度表没食子儿茶素没食子酸酯（EGCG）对高糖造成氧化应激状态下体外小鼠足细胞损害的作用并探讨其机制。 方法　以高糖（25 mmol/L）培养的小鼠足细胞为研究对象,维生素E培养为对照。首先以MTT法检测细胞活力,再在激光共聚焦显微镜下以CM-H2DCFDA荧光探针观察不同浓度EGCG（0.2、10、100 μmol/L）刺激足细胞6、12、24 h后活性氧（ROS）生成,并以流式细胞仪定量分析ROS平均荧光强度。RT-PCR法检测足细胞内ROS产生的主要通路NADPH氧化酶各亚基mRNA（ph22phox、p47phox、p67phox）的表达。 结果　高糖刺激下6 h,足细胞ROS生成显著增加（P < 0.01）。正常糖组和甘露醇组培养12 h ROS生成无显著增加（P > 0.05）。EGCG 0.2 μmol/L作用6 h可显著降低高糖环境下体外小鼠足细胞ROS水平（P < 0.01）。与高糖组比较,EGCG（100 μmol/L）显著减少NADPH氧化酶亚基p22phox和p67phox mRNA表达（均P < 0.05）。与维生素Ｅ组比较,EGCG（0.2 μmol/L）和维生素E（0.2 mmol/L）协同作用组显著减少p47phox mRNA表达（P < 0.05）。 结论　EGCG能缓解高糖环境下体外足细胞氧化应激状态,对高糖培养下足细胞有保护作用。     

脂联素对高糖诱导系膜细胞活性氧和内皮一氧化氮合成酶产生的影响

氧化应激在糖尿病肾病(DN)发病中起重要作用[1].脂联素是脂肪细胞分泌的一种特异性蛋白质,具有抗炎、改善胰岛素抵抗、抗动脉粥样硬化、降血糖、降血脂及抗氧化作用.研究发现脂联素与DN关系密切,其血浆浓度随.肾功能不全进展而增加,原因可能是为减轻内皮和血管损伤的正反馈调节[2,3].因此,我们推测脂联素可能通过抑制氧化应激而对DN发挥保护作用,并通过本研究进一步明确脂联素抗糖尿病微血管病变的机制.     

Mitochondrial reactive oxygen species are required for hypothalamic glucose sensing

The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucose-sensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling.     

In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species

BACKGROUND: We postulated that in mesangial cells exposed to high glucose, protein kinase C-zeta (PKC-zeta) is necessary for the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and that the requirement of PKC-zeta for filamentous (F)-actin disassembly may involve ROS. To identify signaling mechanisms relevant to PKC-zeta activation and ROS generation, including phosphoinositide 3 kinase (PI3 kinase), we examined mesangial cell stimulation with platelet-derived growth factor (PDGF). METHODS: In primary rat mesangial cells cultured in 5.6 mmol/L or 30 mmol/L d-glucose, PKC-zeta expression was identified with immunoblotting and activity was analyzed in cell membrane immunoprecipitates and by confocal immunofluorescence imaging. ROS generation was measured by dichlorofluorescein fluorescence using confocal microscopy and was inhibited by transfection of antisense against NADPH subunits p22(phox) or p47(phox) or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt. RESULTS: In high glucose, generation of NADPH oxidase-dependent ROS was dependent on PKC-zeta. Conversely, sustained PKC-zeta activity was dependent on ROS generation, suggesting a positive feedback. PKC-zeta-dependent F-actin disassembly in high glucose required ROS generation. PDGF stimulated NADPH oxidase generation of ROS through a PKC-zeta mechanism that was independent of Akt phosphorylation and remained unchanged in high glucose. CONCLUSION: In high glucose, mesangial cell PKC-zeta is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-zeta-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-zeta in high glucose.     

Effect of reactive oxygen species in semen on the pregnancy of infertile couples

Objective:   We intended retrospectively to investigate whether reactive oxygen species (ROS) levels, detected in whole semen, were correlated with the actual pregnancy rate.
Methods:   A total of 89 patients with data of ROS in semen, attending our male infertility clinics from April 1994 to June 2000, were evaluated. Semen parameters were determined with computer assisted semen analyzer (CASA) and ROS production levels were measured using a computer-driven LKB Wallac 1251 Luminometer after the addition of 40 µL of 4 mM luminol at the patients' first visits. All of the participants were inquired about their partners' pregnancies after the mean follow-up of 24.0 months (range 1.4 to 74.1). They were divided into two groups (pregnant group: n  = 41, non-pregnant group: n  = 48) and their characteristics, semen profiles and integrated ROS levels were analyzed.
Results:   There was no difference between the pregnancy rate of ROS detectable cases and negative cases. However, the mean integrated ROS level in detectable cases of the non-pregnant group was significantly higher than that in detectable cases of the pregnant group (115.61 ± 74.32 mV/30 min/10⁶ sperm versus 7.22 ± 4.69 mV/30 min/10⁶ sperm, P  = 0.0033). Then, by calculating the receiver operating characteristics curve with 95% confidence intervals, 4.35 mV/30 min/10⁶ sperm was considered as a cut-off value of ROS in semen for pregnancy.
Conclusion:   These results indicate that (i) highly detectable ROS in whole semen of infertile patients may have implications in their partners' pregnancies and that (ii) detection of ROS in whole semen has a prognostic value for idiopathic male infertility.     

Effect of rotation on the generation of reactive oxygen species in human semen

Summary. Rotation of semen after liquefaction is a standard procedure in an Andrology laboratory. During rotation under aerobic conditions the semen is exposed to oxygen. Oxygen toxicity induces lipid peroxidation which is responsible for the loss of sperm function. The generation of reactive oxygen species was significantly increased after rotation. This may have a detrimental effect on sperm function during assisted reproduction.     

活性氧影响骨重建在骨质疏松发病中的作用

骨重建是成年骨组织改建的主要形式,同时受生物力学因素和非生物力学因素调控。机体内活性氧产生和清除失衡会造成氧化应激,在此状态下活性氧作为非生物力学因素参与调控的骨重建与骨质疏松的发生有密切关系。活性氧损伤骨细胞转导力学信号的能力,上调骨重建阈值,导致骨组织已经习惯的力学刺激不足以维持骨量,而发生失用型骨重建。同时,在更新骨质和修复微损伤时,活性氧抑制成骨细胞分化和骨形成,促进破骨细胞形成和骨吸收,导致负性骨重建。总的效应是骨量逐年丢失,伴随骨结构逐渐退行性变,骨质疏松随年龄的增加而加重。     

Targeting reactive oxygen species in hypertension

PURPOSE OF REVIEW: Hypertension is a major risk factor for vascular diseases such as stroke, myocardial infarction, and renal microvascular disease. The mechanism by which vascular disease develops is complex, and growing evidence suggests that an increase in reactive oxygen species during hypertension is a major contributing factor. NADPH oxidase, the primary source of reactive oxygen species in the cardiovascular system, is a strong candidate for the development of therapeutic agents to ameliorate hypertension and end-organ damage. RECENT FINDINGS: Various scavengers and inhibitors of reactive oxygen species have been proposed for use in animal as well as human studies. While many of these agents are effective at lowering tissue reactive oxygen species levels, their specificity is a serious concern. Our laboratory has developed cell-permeant peptidic inhibitors targeting key interactions among the different NAD(P)H oxidase homologues. One of these inhibitors targeting nox2 and p47-phox interaction has proven useful in attenuating target neoplasia and hypertrophy. SUMMARY: Strategies aimed at specifically inhibiting NAD(P)H oxidase have proven effective in attenuating cardiovascular oxidative stress. The development of new inhibitors targeting novel oxidase homologues appears to hold significant promise for clarifying the physiologic role of these homologues as well as for the development of new antioxidant therapies.     

Infection and reactive oxygen species

Summary. In previous years the physiologic and pathophysiologic significance of reactive oxygen species (ROS) on sperm function has been recognized. The impact of ROS during the invasion, adhesion and multiplication of microorganisms in the male genital tract are largely unknown. However, it is known that the resulting activation of leukocytes leads to an increased generation of ROS. There is growing evidence that spermatozoa are protected from detrimental ROS effects by the powerful antioxidants in seminal plasma since disturbances of sperm function by ROS were demonstrated in the absence of seminal plasma, i.e., during epididymitis or after semen preparation. If seminal plasma is present, ROS generated by physiologic numbers of granulocytes (< 1 times 10⁶ ml⁻¹) apparently do not damage spermatozoa. Interestingly, ROS generated by leukocytes during male genital tract infections are critical for the techniques of semen preparation for assisted reproduction. These ROS impair sperm function if the protective effects of seminal plasma are not present. The relevance of ROS production by higher leukocyte numbers in human semen is presently unknown as is the relevance of ROS generated in the female reproductive tract.
In previous years the ambiguous role of reactive oxygen species (ROS) generation in human semen has been recognized. On one hand, ROS appear to be involved in physiologic reactions such as capacitation (De Lamirande & Gagnon, 1995; Griveau et al. , 1995a), while on the other hand, there is ample evidence that an increased ROS production impairs sperm functions and the fertilizing capacity of spermatozoa (Ochsendorf & Fuchs, 1993; Aitken, 1994; Griveau et al. , 1995b). The objective of this review is to summarize the relevance of reactive oxygen species during infections of the male genital tract.     

糖皮质激素对骨髓微血管内皮细胞活性氧代谢影响的实验研究

目的糖皮质激素的应用是引起非创伤性股骨头缺血性坏死的主要原因之一。通过研究经糖皮质激素处理后的骨髓微血管内皮细胞活性氧(reactive oxygen species,ROS)的代谢变化,进一步探讨激素性股骨头缺血性坏死的发病机制。方法取行人工全髋关节置换术患者自愿捐献的股骨头内松质骨,利用酶消化法分离培养骨髓微血管内皮细胞,倒置相差显微镜下观察细胞形态。取第3代细胞,分别与不同浓度(0、0.03、0.10、0.30、1.00 mg/mL)氢化可的松培养后,采用MTT法测定细胞增殖抑制率;流式细胞仪观测细胞凋亡率;采用荧光探针检测细胞中ROS含量变化,以及与ROS代谢相关的黄嘌呤氧化酶(xanthine oxidase,XOD)含量变化。结果原代培养2～3 d后,镜下见单个细胞为梭形,呈铺路石样排列;1周后细胞趋于融合状态。0.03、0.10、0.30、1.00 mg/mL组细胞增殖抑制率分别为20.22%±2.97%、22.94%±4.52%、43.98%±3.35%、78.29%±3.85%,0.30、1.00 mg/mL组的细胞增殖抑制率明显高于0.03、0.10 mg/mL组(P<0.05)。0、0.03、0.10、0.30、1.00 mg/mL组细胞凋亡率分别为0.10%±0.01%、0.23%±0.02%、1.83%±0.04%、6.34%±0.11%、15.33%±0.53%,0.30、1.00 mg/mL组细胞凋亡率明显高于0 mg/mL组(P<0.05)。0、0.30、1.00 mg/mL组ROS含量分别为57.35±7.11、120.47±15.68、166.15±11.57,XOD含量分别为0.017 9±0.000 9、0.028 3±0.001 7、0.067 7±0.004 1;以上两指标3组间两两比较差异均有统计学意义(P<0.05)。结论糖皮质激素通过使血管内皮细胞内ROS生成增加,引起氧化应激反应,进而导致细胞功能受损。     

Effect of lipid and propofol on oxidation of haemoglobin by reactive oxygen species

The clinical formulation of the i.v. anaesthetic propofol contains both the substrate for and inhibitor of lipid peroxidation. In these in vitro experiments we have investigated the importance of this potential conflict in a system where haemoglobin was oxidized by reactive oxygen species generated by hypoxanthine and xanthine oxidase. The presence of lipid in the system accelerated the rate of haemoglobin oxidation. Propofol inhibited lipid-induced acceleration but not the underlying rate of reactive oxygen species-induced oxidation. The rate of conjugated diene production, measured semi-quantitatively by ultraviolet absorption at 234 nm, was not reduced by propofol. Propofol may act by preventing haemoglobin oxidation by lipid hydroperoxides.      

Reactive oxygen species in the presence of high glucose alter ureteric bud morphogenesis

Renal malformations are a major cause of childhood renal failure. During the development of the kidney, ureteric bud (UB) branching morphogenesis is critical for normal nephrogenesis. These studies investigated whether renal UB branching morphogenesis is altered by a high ambient glucose environment and studied underlying mechanism(s). Kidney explants that were isolated from different periods of gestation (embryonic days 12 to 18) from Hoxb7-green fluorescence protein mice were cultured for 24 h in either normal d-glucose (5 mM) or high d-glucose (25 mM) medium with or without various inhibitors. Alterations in renal morphogenesis were assessed by fluorescence microscopy. Paired-homeobox 2 (Pax-2) gene expression was determined by real-time quantitative PCR, Western blotting, and immunohistology. The results revealed that high d-glucose (25 mM) specifically stimulates UB branching morphogenesis via Pax-2 gene expression, whereas other glucose analogs, such as d-mannitol, l-glucose, and 2-deoxy-d-glucose, had no effect. The stimulatory effect of high glucose on UB branching was blocked in the presence of catalase and inhibitors of NADPH oxidase, mitochondrial electron transport chain complex I, and Akt signaling. Moreover, in in vivo studies, it seems that high glucose induces, via Pax-2 (mainly localized in UB), acceleration of UB branching but not nephron formation. Taken together, these data demonstrate that high glucose alters UB branching morphogenesis. This occurs, at least in part, via reactive oxygen species generation, activation of Akt signaling, and upregulation of Pax-2 gene expression.     

Effects of inosine monophosphate dehydrogenase inhibition on high glucose-induced cellular reactive oxygen species in mesangial cells

Mesangial cell extracellular matrix (ECM) synthesis plays an important role in chronic renal diseases including chronic renal allograft dysfunction and diabetic nephropathy. Although inosine monophosphate dehydrogenase 2 (IMPDH2), as a target of mycophenolic acid (MPA), is important for de novo guanosine synthesis in lymphocytes, mesenchymal cells are not wholly dependent on it. To explore the importance of IMPDH2 on the inhibitory effects of MPA in mesangial cells (MC), we compared the effects of MPA and IMPDH2 siRNA on high glucose (HG)-induced fibronectin secretion and cellular reactive oxygen species (ROS). Mouse mesangial cells (MMC) were stimulated with HG (30 mmol/L D-glucose) in the presence or absence of MPA pretreatment or IMPDH2 siRNA transfection. Fibronectin secretion was measured by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS assessed by flow cytometry. HG increased fibronectin secretion by 1.8-fold at 24 hours and DCF-sensitive cellular ROS by 1.5-fold at 1 hour. MPA at 10 μmol/L totally inhibited HG-induced fibronectin secretion and cellular ROS in MMC. However, IMPDH2 siRNA only partially suppressed HG-induced fibronectin secretion and cellular ROS. These results suggested that MPA may inhibit HG-induced fibronectin secretion partially through inhibiting cellular ROS and the inhibition of IMPDH2 may be partially involved in the mechanism of MPA.     

精子功能与氧自由基

本文综述了精液内氧自由基的产生系统和拮抗系统 ,以及氧自由基对精子功能和形态的影响。精液中一定水平的氧自由基是精子获能和发生顶体反应所必需的 ,但氧自由基生成量增加 ,超过精子所需的生理剂量 ,则会导致精子受损 ,影响人类受精和家畜繁殖