Contribution of superoxide to reduced antioxidant activity of glycoxidative serum albumin

Hyperglycemia increases oxidative stress in various tissues and leads to diabetic cardiovascular complication. Dyslipidemia, such as an increase in oxidized low-density lipoprotein (LDL), is well recognized in diabetic patients with hyperglycemia. However, the mechanism by which hyperglycemia causes the increased LDL oxidation remains unclear. Albumin is the most abundant protein in the circulation, and can function as an antioxidant. Therefore, we examined whether glycoxidative modification inhibits the antioxidant activity of albumin to LDL oxidation and clarified the mechanism by which this modification may suppress its antioxidant activity. Human serum albumin (HSA) was incubated in phosphate-buffered saline with and without glucose at 37°C for up to 8 weeks under aerobic conditions (referred to as glycoxidation (goHSA) and oxidation (oHSA), respectively). Metal chelator-treated, nonoxidative HSA (chHSA) and freshly prepared HSA (fHSA) were used as controls. N ^ε-(carboxymethyl)lysine (CML), a glycoxidative product, was determined by enzyme-linked immunosorbent assay. Oxidation was estimated by measuring the thiols of the HSA molecule. Copper-mediated oxidation of LDL was conducted in the presence or absence of modified HSAs at 37°C for 6 days. Malondialdehyde and negative charge of LDL were measured. To clarify the mechanism of reduced antioxidant activity of HSA, we examined firstly the binding activity of modified HSAs to copper, and secondly the effects of free radical scavengers on the formation of malondialdehyde. CML was formed in goHSA in a time- and concentration-dependent manner. Both goHSA and oHSA significantly decreased the contents of free thiol groups compared to ch- and fHSAs. The antioxidant activity of goHSA to LDL oxidation was the lowest among various modified HSAs. The oHSA showed a moderate decrease in antioxidant activity. The binding activity of go- and oHSAs to copper was lower than that of ch- and fHSAs. The formation of MDA from LDL oxidation in the presence of goHSA was completely inhibited by Tiron (1,2-dihydroxy-3,5-benzenedisulfonic acid) and superoxide dismutase. In contrast, catalase and mannitol had no effect. Our results indicate that in vitro glycoxidation of HSA induced a marked loss of antioxidant activity of this molecule to copper-mediated oxidation of LDL, which may be caused by the generation of superoxide. Received: December 17, 2001 / Accepted: June 28, 2002 Acknowledgments The authors thank Drs. Ryoji Nagai and Seikoh Horiuchi (Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto, Japan) and Drs. Hiroyuki Itabe and Tatsuya Takano (Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan) for kindly supplying antibodies. We also thank Associate Professor Takeo Yamaguchi (Department of Chemistry, Faculty of Science, Fukuoka University) for the ESR experiment and Miss Satoko Nagano for her excellent technical assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 14570171) and in part by funds from the Central Research Institute of Fukuoka University (No. 016004). Correspondence to N. Sakata     

The Complement—Fixing Activity of Immune Complexes Containing IgG Antibodies of Different Functional Affinities: Effects on Superoxide Production by Rabbit Neutrophils

When neutrophil phagocytes are stimulated by IgG containing immune complexes (IgG‐IC), with or without the participation of the complement system, they show a sharp increase in oxygen uptake and begin to release large quantities of superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂) into the surrounding medium. The aim of the present investigation was to provide insights into the production and release of O₂^? by rabbit neutrophils activated with immune complexes (IC) containing IgG antibodies of different functional affinity, opsonized and not opsonized by complement system components. For this purpose, two populations of polyclonal anti‐ovalbumin (OVA) IgG antibodies with different functional affinity, 5 × 10⁸ M^{? 1} and 2 × 10⁷ M^{? 1}, were prepared. The production of O₂^? was measured spectrophotometrically by a method using the superoxide dismutase‐inhibited reduction of ferricytochrome C to the ferrous form. The activation of complement by different IgG‐IC was determined by estimating the total residual haemolytic activity of the alternative and classical pathways in sera treated with different concentrations of anti‐OVA IgG/OVA immune complexes formed at equivalence. The results showed that: 1) antibody functional affinity influenced O₂^? production and the complement‐fixing activity induced by the IC. In general, the higher functional affinity antibodies were more efficient in stimulating the respiratory burst of neutrophils and in activating complement by the classical and alternative pathways than the lower functional affinity antibodies at all IC concentrations tested; 2) complement components incorporated into the immune complex lattice caused an increase in the stimulatory activity of both IgG antibodies to produce O₂^? (? 15% for the IC of IgG with K_a = 5 × 10⁸ M^{? 1} and ? 7% for the IC of IgG with K_a = 2 × 10⁷ M^{? 1}). This effect was dependent on antibody affinity and concentration; 3) there was a direct relationship between the overall level of complement activation, antibody affinity and superoxide production by neutrophils. Thus, we conclude that antibody affinity influences immune complex lattice formation, modulating its three‐dimensional structure and the disposition of Fc fragments interfering with the antibody's biological properties. These results can help understand the precise role of antibody functional affinity in antigen‐antibody complex diseases and define the immunochemical characteristics of pathogenic complexes.     

Experimental periodontitis promotes transient vascular inflammation and endothelial dysfunction

Objectives

This study aimed to evaluate the systemic inflammatory response and cardiovascular changes induced by experimental periodontitis in rats.

Design

Experimental periodontitis was induced by placing a cotton ligature around the cervix of both sides of mandibular first molars and maxillary second molars in each male rat. Sham-operated rats had the ligature removed immediately after the procedure. Seven, 14 or 28 days after procedure, the effects of acetylcholine, sodium nitroprusside and phenylephrine were evaluated on blood pressure, aortic rings and isolated and perfused mesenteric bed. The blood was obtained for plasma Interleukin-6 (IL-6), C-reactive protein (CRP) and lipid evaluation. The mesenteric vessels were obtained to evaluate superoxide production and nitric oxide synthase 3 (NOS-3) expression.

Results

Ligature induced periodontitis reduced endothelium-dependent vasodilatation, a hallmark of endothelial dysfunction. This effect was associated with an increase in systemic inflammatory markers (IL-6 and CRP), worsens on lipid profile, increased vascular superoxide production and reduced NOS-3 expression. It is interesting to note that many of these effects were transitory.

Conclusion

Periodontitis induced a transient systemic and vascular inflammation which leads to endothelial dysfunction, an initial step for cardiovascular diseases. Moreover, the animal model of periodontitis used here may represent a valuable tool for studying the relationship between periodontitis and endothelial dysfunction.     

外源性胆绿素对中波紫外线诱导的角质形成细胞光损伤的保护作用和机制

【摘要】 目的 探讨外源性胆绿素对中波紫外线（UVB）照射的HaCaT细胞光损伤的保护作用。方法 将HaCaT细胞分为加入0、0.1、1、10 μmol/L胆绿素并照射UVB的UVB组、0.1 μmol/L UVB组、1 μmol/L UVB组、10 μmol/L UVB组及不做处理的对照组。UVB照射剂量为30 mJ/cm2,照射后继续培养24 h,分别检测细胞活性氧（ROS）水平、超氧化物歧化酶（SOD）活力、丙二醛（MDA）含量,ELISA法检测各组细胞的炎症因子白细胞介素6（IL-6）、IL-8水平。多组间均数比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果 UVB组、0.1 μmol/L UVB 组、1 μmol/L UVB组、10 μmol/L UVB组、对照组细胞ROS水平（3 613.33 ± 206.61、2 958.67 ± 193.87、2 678.33 ± 178.24、2 274.67 ± 118.81、1 905.67 ± 250.25）、SOD活力（24.41 ± 1.78、28.96 ± 2.21、29.75 ± 1.75、30.19 ± 2.29、37.52 ± 2.31）、MDA含量（5.61 ± 0.32、5.46 ± 0.55、4.65 ± 0.22、2.55 ± 0.93、1.31 ± 0.05）、IL-6水平、IL-8水平差异均有统计学意义（F值分别为 34.02、57.36、214.09、29.73、11.40,均P < 0.05）,UVB组ROS水平、MDA含量及IL-6、IL-8水平均高于另4组（均P < 0.05）,SOD活力均低于另4组（均P < 0.05）。结论 外源性胆绿素减轻UVB引起的HaCaT细胞的氧化损伤、减轻炎症反应和抑制脂质过氧化作用,对细胞光损伤有一定的保护作用。     

真菌抗氧化酶的研究进展

真菌对抗宿主的氧化损伤作用是真菌能顺利入侵宿主的一个重要因素。抗氧化酶的调节是真菌发挥抗氧化作用的重要途径之一,也是目前研究的热点。其中,抗氧化酶包括过氧化氢酶、超氧化物歧化酶、谷胱甘肽还原酶与谷胱甘肽过氧化物酶、硫氧还蛋白系统以及海藻糖酶系统。几种常见真菌的抗氧化酶及编码抗氧化酶的相关基因的研究取得了进展,包括编码过氧化氢酶的相关基因如CTT1、CATA;编码超氧化物歧化酶的SOD基因;谷胱甘肽系统相关的GRX、GST基因;编码硫氧还蛋白系统的TRX、TRR基因以及海藻糖系统相关的TPS、ATH1基因等。     

溶血磷脂酸与饮食所致高血脂幼兔主动脉壁病变发展的关系

目的探讨血浆溶血磷脂酸(LPA)水平变化与高脂喂养幼兔主动脉粥样硬化(AS)病变发展的关系。方法2月龄家兔随机分成普通饮食组(A组,n=6)与高脂喂养组(B组,n=11)。分别饲喂高脂饲料与普通饲料。动态测定血脂水平、血浆LPA水平、血清一氧化氮(NO)和丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性。另于各个时间点选取B组中兔一只,处死,进行胸主动脉病理学检查。结果高脂喂养1~2周动脉内膜显示微观结构改变。脂质条纹(FS)形成于高脂喂养第4周并进行性加重。血脂水平自高脂喂养1周后即显著升高,以后进行性升高。血浆LPA水平于FS形成前期即显著升高并达到峰值。血清NO,MDA水平及SOD活性均在FS形成中后期发生显著改变。结论测定高脂饮食哺乳动物血浆LPA水平,同时结合血清NO,MDA水平及SOD活性可预警并判定主动脉脂质条纹性病变的严重程度。     

高脂血症,脂质过氧化,抗氧化酶活性与动脉粥样硬化的关系

用大剂量胆固醇(1.5g/日)喂家兔60天后停胆固醇30天塑造动脉粥样硬化(AS)模型。观察血胆固醇、过氧化脂(LPO)含量和抗氧化酶活性与AS病变发生发展的关系。发现:血清胆固醇水平随喂胆固醇时间延长而升高,至60天时达高峰,停饲胆固醇,血清胆固醇水平迅速下降,而同样升高的LPO水平不但未降,反而继续升高,明显高于对照的水平。抗氧化酶SOD和GSH-Px活性在LPO升高的早期显示代偿性增高,以后即降低并保持在低于对照的水平。主动脉、肺动脉和冠状动脉均发生程度不等的AS病变,即使在停饲胆固醇一个月后亦可见进行性病变,如大量平滑肌细胞增生和炎细胞浸润,且其病变较前更重。以上结果提示,血中过量的LPO抑制了抗氧化酶活性,可能在AS的发生发展中起重要作用。     

急性脑卒中患者血清一氧化氮、一氧化氮合酶、丙二醛、超氧化期歧化酶及过氧化脂质水平动态变化与病情预后分析

目的通过对216例急性脑卒中患者血清一氧化氮（NO）、一氧化氮合酶（NOS）、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化脂质（LPO）水平变化的动态观察,探讨这5项指标与急性脑卒中病情变化及预后之间的关系。方法采用生物化学方法检测急性脑卒中患者血清第1～4周NO、NOS、MDA、SOD、LPO含量水平,并与正常对照组比较。结果①急性脑卒中组NO、NOS、MDA、LPO水平均明显高于正常对照组（P均（0.01）,发病后第1周显著升高,随着病情的好转,第2～4周逐渐下降;SOD水平明显低于正常对照组（P（0.01）,发病后第1周显著降低,第2～4周逐渐升高。②出血性脑卒中NO、NOS、MDA、LPO水平明显高于缺血性脑卒中组（P（0.01）,而SOD水平则明显降低（P（0.05）。结论急性脑卒中患者存在NO、NOS、MDA、SOD、LPO含量失衡的变化,并随病情的严重与演变而变化。观察这5项指标的水平动态变化,可作为判定病情和评价预后的参考指标。     

实验性Ⅱ型糖尿病大鼠主动脉早期超微结构改变及影响因素

本实验测定了实验性Ⅱ型糖尿病大鼠血脂、血浆过氧化脂质及超氧化物歧化酶，观察了１０周时大鼠主动脉超微结构的改变。结果表明：糖尿病组及单纯肥胖组大鼠均有主动脉内膜病变，前者较明显。与对照组大鼠比较，前二者过氧化脂质、甘油三脂及低密度脂蛋白胆固醇均增高，而高、低密度脂蛋白胆固醇比值下降，差异非常显著（Ｐ＜０．０１），糖尿病组大鼠超氧化物歧化酶下降而单纯肥胖组大鼠则升高。以上结果提示，实验性Ⅱ型糖尿病大鼠主动脉内膜有早期超微病理改变，这种病变与过氧化脂质升高及超氧化物歧化酶活性下降有关。与脂代谢紊乱也有一定的关系。     

太子参醇提物对大鼠听性脑干反应阈及其相关因素的影响

目的:观察太子参醇提物(EPHPH)对自然衰老模型大鼠听性脑干反应(ABR),耳蜗组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)及分型一氧化氮合酶(iNOS)的影响,探讨其对老年性聋的作用及其机制。方法:自然衰老模型大鼠分别灌胃给予太子参2.16、4.32及8.64g·kg-1·d-1的醇提物,每日1次,共6个月,分别测定自然衰老模型大鼠ABR,耳蜗组织中MDA、SOD、NOS及iNOS。结果:自然衰老模型大鼠ABR阈值明显升高,并伴有耳蜗组织中MDA明显升高,SOD、NOS及iNOS活力明显下降。相关性分析表明,ABR阈值升高与耳蜗组织中MDA升高呈正相关;与SOD、NOS及iNOS活力明显下降均呈负相关。自然衰老模型大鼠灌胃太子参2.16、4.32及8.64g·kg-1·d-1的醇提物,能不同程度对抗ABR阈值升高及耳蜗组织中MDA升高,其量效关系均呈负相关;能不同程度抑制SOD、NOS和iNOS活力降低,其量效关系均呈正相关。结论:EPHPH可减轻老年性聋程度,其机制可能与清除耳蜗组织中氧自由基,提高SOD及NOS活力有关。