云芝多糖对巨噬细胞GPx基因表达的影响

细胞内存在两种谷胱甘肽过氧化物酶;硒谷胱甘肽过氧化物酶和非硒谷肽甘肽过氧化物酶,它们在保护细胞免受氧化损伤等过程中起重要作用。为揭示云芝多糖作用与细胞抗氧化酶的关系,采用酶活性测定,斑点杂交等方法,探讨云芝多糖对小鼠腹腔巨噬细胞过氧化物酶表达的影响。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞的两种过氧化物酶活性,并使其ｍＲＮＡ含量增加,应用阻断剂的研究发现,云芝多糖对巨噬细胞ＳｅＧＰｘ及Ｇ     

原核表达的萝卜PHGPx和谷胱甘肽对NIH3T3细胞氧化损伤的联合保护作用

将大肠杆菌中高效表达的萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)及其底物还原型谷胱甘肽(GSH)联合作用于H2O2、过氧化叔丁基(t-BHP)以及磷脂氢过氧化物(PLPCOOH)损伤的小鼠NIH3T3成纤维细胞,研究其对于细胞过氧化损伤的保护作用。发现单独加入10 μg/ml RsPHGPx并不能明显保护细胞应对过氧化损伤,但是10 μg/ml RsPHGPx与3 mg/ml GSH共同作用,可显著提高GSH对细胞过氧化损伤的保护效果,提高细胞存活率,降低细胞膜脂质过氧化水平和胞内活性氧(ROS)水平,保护细胞维持形态完整和抑制细胞凋亡。这一联合作用结果说明RsPHGPx的催化作用可以快速清除细胞内多种过氧化物,高效地保护细胞免受过氧化损伤,为RsPHGPx的应用提供了实验依据。     

脱氧核糖核酸对谷胱甘肽过氧化物酶活性影响的初步研究

谷胱甘肽过氧化物酶是机体内重要抗氧酶系之一。它的活力和含量,反映机体清除自由基的能力。自由基对细胞结构的损伤很大,随着年龄的增长,抗氧化酶活力逐渐下降,从而引起自由基及脂质过氧化产物日益增多,最终导致机体衰老和老年性疾病的发生^[1]。本试验试图探讨DNA对谷胱甘肽过氧化物酶活性影响从而探索DNA对抗自由基的作用。     

谷胱甘肽磷脂氢过氧化物酶研究进展

谷胱甘肽磷脂氢过氧化物酶(PHGPx)是生物体内一种重要的抗氧化酶。它是一种硒依赖性蛋白,在谷胱甘肽(GSH)的参与下能特异性地还原磷脂氢过氧化物(PLOOH)和胆固醇氢过氧化物(ChOOH),从而保护生物膜免受过氧化损伤。它还是核酸等生物大分子的重要保护剂,并且在细胞凋亡调控中发挥作用。     

壳寡糖缓解甲萘醌诱导巨噬细胞损伤机制初探

目的:研究壳寡糖对甲萘醌诱导的巨噬细胞氧化损伤的保护作用.方法:通过MTT实验检测相应处理的细胞活力,并通过相应试剂盒检测细胞氧化还原体系中某些相关酶的活力及相应产物含量.结果:壳寡糖能够缓解甲萘醌诱导的细胞损伤,并且发现壳寡糖可以缓解甲萘醌导致的胞内超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-PX)活力的降...     

异质性荧光染色的巨噬细胞功能研究──Ⅱ．异质性荧光染色的巨噬细胞过氧化物酶活性

细胞化学研究文献报导,巨噬细胞中过氧化物酶的活性有明显的差异,只有部分巨噬细胞过氧化物酶呈阳性反应。本文用淀粉、白喉类毒素和卡介苗分别诱导和活化小鼠腹腔巨噬细胞,进行过氧化物酶反应,并以７６９０－Ｘｕ荧光染液复染后证明,酶反应阳性细胞呈蓝色荧光,而酶反应阴性细胞为淡蓝绿色和黄色荧光。实验表明,过氧化物酶阳性的巨噬细胞是分化程度低的幼稚细胞,因此,过氧化物酶的活性可作为低分化的巨噬细胞的一种标志酶。同时,本文用免疫荧光单克隆抗体间接染色法观察了三种物质诱导和活化的异质性荧光染色的巨噬细胞的分泌功能。     

动物抗氧化系统中主要抗氧化酶基因的研究进展

抗氧化系统是机体清除体内多余的活性氧、保护自身免受氧化损伤的重要体系,其中超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶等起主要作用。本文将对动物抗氧化系统中,超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶基因的种类、分布、结构及表达进行综述。     

云芝提取物对改善小鼠胃肠功能的作用

以大黄水煎剂建立小鼠胃肠功能损害模型,探讨云芝提取物和其中的白桦脂酸对小鼠胃肠功能的影响以及白桦脂酸对小鼠在脂质过氧化方面的影响。研究结果表明云芝提取物和白桦脂酸能够较为显著地提高胃蛋白酶活性,增加胃泌素和胃酸分泌量（P<0.01）;同时白桦脂酸高低剂量组能够明显降低小鼠血浆中丙二醛的含量,提高超氧化物歧化酶、谷胱甘肽过氧化物酶的活性以及肝脏匀浆中谷胱甘肽过氧化物的含量（P<0.01）。说明云芝提取物和白桦脂酸能够有效地改善小鼠的胃肠功能,且抗脂质过氧化是云芝活性成分白桦脂酸治疗作用机制之一。     

牛磺酸对虎纹蛙非特异性免疫及血清氧化还原状态的影响

为探讨牛磺酸在无尾两栖类免疫调节和抗氧化能力方面的功能和作用,通过灌胃方式研究了不同浓度牛磺酸对虎纹蛙(Hoplobatrachus rugulosus)脾巨噬细胞呼吸爆发、外周血吞噬活力、胃溶菌酶活力以及血清丙二醛(MDA)和谷胱甘肽(GSH)含量的影响。结果表明:连续7 d的牛磺酸灌胃可显著提高脾巨噬细胞的呼吸爆发强度和外周血细胞的吞噬活力并呈现出明显的剂量效应。当灌胃浓度达到0.8 g/L时两者均达到峰值,但高于0.8 g/L时则表现出条件毒性。胃溶菌酶活力在各浓度下无明显变化。此外,血清MDA含量随牛磺酸浓度的升高而降低,GSH含量在一定的浓度范围内呈现出明显的剂量效应,在1 g/L时达到峰值。研究结果证明牛磺酸可以明显提高虎纹蛙的非特异性免疫功能和机体氧化防御能力,但摄入过量则表现出条件毒性。基于上述指标的评估,虎纹蛙对牛磺酸的适宜需求量按体重计算约为16~20 mg/kg。     

猫爪草多糖对小鼠腹腔巨噬细胞活力的调节作用

此次研究旨在探讨猫爪草多糖对体外培养的正常状态下的原代小鼠腹腔巨噬细胞活性的调节作用,以及小鼠腹腔巨噬细胞在体外培养条件下的活力变化情况。以原代培养的小鼠腹腔巨噬细胞为研究对象,设对照组(加入100μL DMEM培养基)和实验组(分别加入25μg/mL, 50μg/mL, 100μg/mL, 200μg/mL,400μg/mL的猫爪草多糖),分别采用噻唑蓝(MTT)比色法、CCK-8法、乳酸脱氢酶释放法和中性红吞噬实验检测不同浓度的猫爪草多糖对体外培养的小鼠腹腔巨噬细胞活力的调节作用;同时设置24 h、36 h、48 h、60 h和72 h的不同培养时间,观察在体外培养条件下,小鼠腹腔巨噬细胞活力的变化情况。结果表明:与对照组相比,不同浓度的猫爪草多糖均能增强小鼠腹腔巨噬细胞的活力,且猫爪草多糖浓度在100~400μg/mL的细胞活力极显著增强(p<0.01)。此外,各处理组的巨噬细胞在体外培养24~72 h不更换培养液的条件下,48 h处活性最佳。体外培养条件下,一定浓度的猫爪草多糖可以激活小鼠腹腔巨噬细胞,通过猫爪草多糖激活巨噬细胞,可能是猫爪草发挥提升机体免疫力的作用机制之一。此外,体外培养的巨噬细胞虽能存活长达一个月,但仍有一个最佳活力时间。     

Phagocytosis and stimulation of the respiratory burst by phorbol diester initiate S-thiolation of specific proteins in macrophages.

Addition of chemical oxidants to cells in culture has been shown to induce binding of low-molecular-weight thiols to reactive sulfhydryls on proteins in a process termed S-thiolation. We found that stimulation of the respiratory burst in mouse macrophages, with release of O2-, resulted in S-thiolation of several proteins, most prominently three with molecular weights of 74, 33, and 22 kDa. One protein (28 kDa) was S-thiolated without addition of an exogenous stimulus. Exposure of cells to concentrations of hydrogen peroxide like those released in the respiratory burst induced S-thiolation of these same proteins. S-thiolation and release of O2- began at approximately the same time. Stimulation of LPS-elicited macrophages induced prominent S-thiolation of three different proteins (38, 30, and 21 kDa). Under the conditions of these experiments, there was no detectable increase in glutathione disulfide and a negligible decrease in glutathione, which suggests that S-thiolation can occur without significant perturbation of the glutathione peroxidase/reductase cycle. S-thiolation of proteins could help protect the macrophage against the autoxidative damage associated with the respiratory burst. Modification of specific proteins by S-thiolation might serve to modulate cellular metabolic events.     

Oxygen-dependent microbicidal systems of phagocytes and host defense against intracellular protozoa

The role of oxygen-dependent microbicidal systems of leukocytes in the host defense against the major nonerythrocytic intracellular protozoa which infect man--Toxoplasma gondii, Trypanosoma cruzi, and the Leishmania species--is reviewed. The hydrogen peroxide-halide-peroxidase microbicidal system is uniformly cidal to these organisms in vitro. Peroxidase-independent oxygen product(s) toxicity is more variable. Studies to data indicate that phagocytes which contain granule peroxidase and which have the capacity to generate a vigorous respiratory burst; eg, neutrophils and monocytes, possess substantial activity against these protozoa. The absence of granule peroxidase together with the markedly attenuated respiratory burst of resident macrophages leaves these cells with a severe microbicidal defect. These protozoa can enter resident macrophages in the absence of antibody and survive and replicate within the intracellular environment. Enhancement of the antiparasite activity of resident macrophages can be accomplished either by activation of these cells by exposure to sensitized T-cell products, or by the introduction of exogenous peroxidase into the vacuole. Other factors influencing the ability of protozoa to survive intracellularly include the capacity of these organisms to avoid effective triggering of the macrophage respiratory burst and the levels of endogenous scavengers of oxygen products within the parasite.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.     

Macrophage control of mycobacterial growth induced by picolinic acid is dependent on host cell apoptosis

The effects of picolinic acid (PA) on the intramacrophagic growth of Mycobacterium avium were studied. PA reduced M. avium growth inside mouse macrophages and led to a complete control of mycobacterial growth when added together with IFN-gamma. The mechanism involved did not require TNF-alpha, NO, or the respiratory burst, and was not dependent on either iron or zinc withholding. The mycobacteriostatic activity of the macrophages was associated with the induction of morphological changes that culminated in apoptosis at day 4 of treatment. PA alone induced apoptosis in macrophages, and this effect was increased by IFN-gamma treatment. Apoptosis at day 4 of infection was reduced by inhibiting macrophage activation with the prostaglandin 15 deoxy-prostaglandin J2 or by treating the cells with the antioxidant N-acetylcysteine. Mycobacterial growth was partially restored in macrophages treated with PA and IFN-gamma when 15 deoxy-prostaglandin J2 was added, concomitant with a delay in apoptosis. N-Acetylcysteine or glutathione could also completely revert the mycobacteriostatic effects of PA or PA plus IFN-gamma.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Effect of vitamin E on the oxidative metabolism of macrophages

The oxidative metabolism of macrophages in vitamin E deficiency was studied on Aug-Lac strain rats. Vitamin E deficiency was shown to enhance luminol-dependent chemiluminescence of macrophages stimulated by opsonized zymosan. There was also an increase in microviscosity of macrophage membrane lipid phase, that was estimated with a fluorescent probe. The incubation of macrophages with dl-alpha-tocopherol led to the inhibition of macrophage chemiluminescence. Superoxide dismutase, glutathione peroxidase and glutathione reductase activity was not affected by vitamin E deficiency.     

A novel selenocystine-beta-cyclodextrin conjugate that acts as a glutathione peroxidase mimic

A novel artificial glutathione peroxidase mimic consisting of a selenocystine-di-beta-cyclodextrin conjugate (selenium-bridged-6, 6'-amino-selenocystine-6,6'-deoxy-di-beta-cyclodextrin), in which selenocystine is bound to the primary side of beta-cyclodextrin through the two amino nitrogen groups of selenocystine, was synthesized. The glutathione peroxidase activities of the mimic-catalyzed reduction of H(2)O(2), tert-butylhydroperoxide, and cumene hydroperoxide by glutathione are 4.1, 2.11, and 5.82 units/micromol, respectively. The first activity was 82 and 4.2 times as much as that of selenocysteine and ebselen, respectively. Studies on the effect of substrate binding on the glutathione peroxidase activity suggest that it is important to consider substrate binding in designing glutathione peroxidase mimics. The detailed steady-state kinetic studies showed that the mimic-catalyzed reduction of H(2)O(2) by glutathione followed a ping-pong mechanism, which was similar to that of the native glutathione peroxidase.     

Unsaturated fatty acids as modulators of macrophage respiratory burst in the immune response against Rhodococcus equi and Pseudomonas aeruginosa

In this paper, using the monocyte/macrophage cell line RAW264.7, we systematically investigate the impact of macrophage enrichment with unsaturated fatty acids on cellular radical synthesis. We found that the intracellular production of reactive nitrogen and oxygen intermediates depends on the activation status of the macrophages. For unstimulated macrophages PUFA enrichment resulted in an increase in cellular radical synthesis. For stimulated macrophages, instead, an impeding action of unsaturated fatty acids on the respiratory burst could be seen. Of particular importance, the impact of unsaturated fatty acids on the macrophage respiratory burst was also observed in RAW264.7 cells cocultivated with viable bacteria of the species Rhodococcus equi or Pseudomonas aeruginosa. PUFA supplementation of macrophages in the presence of R. equi or P. aeruginosa reduced the pathogen-stimulated synthesis of reactive nitrogen and oxygen intermediates. Furthermore, the unsaturated fatty acids were found to impede the expression of the myeloperoxidase gene and to reduce the activity of the enzyme. Hence, our data provide indications of a possible value of PUFA application to people suffering from chronic infections with R. equi and P. aeruginosa as a concomitant treatment to attenuate an excessive respiratory burst.     

Selenium supplementation sensitizes renca cells to tert-butylhydroperoxide induced loss of viability

Mouse renal carcinoma (renca) cells growing exponentially in foetal bovine serum (1%) supplemented with selenium (1 microM, sodium selenite) were exposed to oxidative insult. It was found that glutathione peroxidase activity increased (44%), while the activities of catalase, glutathione disulfide reductase, and level of total glutathione did not change due to selenium supplementation. Selenium supplementation made renca cells susceptible to tert-butylhydroperoxide induced cell death, while it did not affect the viability when the cells were exposed to hydrogen peroxide. It suggested that the contribution of glutathione peroxidase in antioxidant defense mechanism of renca cells was possibly not crucial and the function of catalase might be important especially against hydrogen peroxide.