5′-磷酸腺苷对小鼠脾细胞氧化应激损伤的保护作用

目的研究5′-磷酸腺苷（5′-AMP）体外抗氧化和对体外氧化损伤脾细胞的损伤修复能力。方法用化学比色法测定5′-AMP体外清除二苯代苦味酰基自由基（DPPH自由基）的能力;建立过氧化氢（H2O2）氧化损伤体外培养小鼠脾细胞模型,用MTT法检测5′-AMP修复受损伤脾细胞的作用,并分析其对细胞抗氧化体系及抗氧化能力的影响。结果5′-AMP具有剂量依赖性的体外抗氧化和清除活性氧能力,添加0.5mmol/L、1mmol/L、5mmol/L和10mmol/L5′-AMP均能显著修复H2O2诱导的脾细胞氧化损伤（P〈0.05）,总抗氧化能力和抗氧化酶类活力（P〈0.01）,5′-AMP添加量大于1mmol/L时,可显著降低丙二醛（MDA）含量（P〈0.01）。其细胞培养液的氧自由基（ROS）水平逐渐降低,5′-AMP添加量为10mmol/L时,ROS水平接近对照组水平。结论5′-磷酸腺苷能显著修复氧化损伤,具有显著的抗氧化作用。     

鸟苷酸二钠体外抗氧化能力的研究

本文研究了鸟苷酸二钠（GMPNa2）对羟自由基和DPPH·自由基的清除效果,同时建立了H2O2氧化损伤体外培养脾细胞模型,用MTT法检测GMPN％的修复及增殖作用,并分析了GMPNa2对细胞抗氧化体系及抗氧化能力的影响,以评价其抗氧化活性。结果表明：GMPNa2具有剂量依赖性的体外抗氧化和清除活性氧能力,10mmol／LGMPNa2的羟自由基清除能力高达96．644％,但DPPH清除率为6．589％。GMPNa2清除羟自由基与同浓度Vc相近,而其清除DPPH自由基的能力很弱。添加0．5、1、5和10mmol／LGMPNa2均能显著修复H2O2诱导的脾细胞氧化损伤（P〈0．05）,提高总抗氧化能力和抗氧化酶类活力（P〈0．01）。GMPNa2添加量大于1mmol／L时,可显著降低MDA含量（P〈0．01）。说明GMPNa2具有显著的抗氧化作用。     

茶多糖的抗氧化活性及对细胞氧化损伤的保护机制

茶多糖是一种从茶叶中提取的酸性糖蛋白, 具有良好的抗氧化活性。以自由基清除率为指标, 分析皖西南地区夏秋茶多糖的抗氧化活性, 基于H₂O₂和EDTA-Fe²⁺建立的外源性羟基自由基(·OH)损伤细胞模型和PMA诱导内源性羟基自由基损伤模型, 进一步探讨茶多糖对自由基损伤的修复作用机制。结果表明, 茶多糖具有良好的体外抗氧化活性, 对DPPH·和·OH均具有较强的清除效果, EC₅₀值分别为209.5和535.2 µg∙mL^-1, 最大清除效率与Vc相当。细胞增殖实验表明, 外源性和内源性自由基氧化损伤模型中细胞存活率均随着茶多糖浓度的增加而升高, 在茶多糖浓度为800 µg∙mL^-1时细胞存活率分别高达87.41%和85.84%, 且显著高于模型组(47.67%和48.03%)。在修复机制上, 利用激光共聚焦显微镜显影细胞内活性氧(ROS)分布以及荧光强度, 分析结果显示, 与模型组相比, 茶多糖对于细胞模型中外源和内源性ROS均具有明显的清除效果, 与体外抗氧化实验结果一致。茶多糖在体外表现出良好的自由基清除效率, 可在细胞水平上改善自由基损伤。该研究在细胞水平上揭示了茶多糖清除自由基的抗氧化损伤机制, 为后续进一步阐明茶多糖抗衰老作用奠定了基础。     

长角血蜱唾液腺中腺苷三磷酸双磷酸酶的纯化及其酶切机制

利用染料亲和层析(Cibacorn Blue柱)和离子交换层析(Macrosphere WCX柱)对长角血蜱Haemaphysalis longicornis唾液腺的腺苷三磷酸双磷酸酶进行纯化,经SDS-PAGE证实其分子量为66 kD。腺苷三磷酸双磷酸酶可以水解ATP和ADP,但对AMP无水解作用,水解ATP和ADP的K_m值均为0.2 μmol/L,V_max值分别为12.5和15.6 μmol/(min·mg)。腺苷三磷酸双磷酸酶水解ATP的中间产物是ADP,最终产物是AMP和正磷酸。表明腺苷三磷酸双磷酸酶水解ATP的位点是5'-核苷酸的γ-磷酸键,水解ADP的位点是5'-核苷酸的β-磷酸键。     

H2O2致体外培养动脉内皮细胞损伤及山莨菪碱保护

目的:实验以过氧化氢(H2O2)损伤体外培养动脉内皮细胞(AEC),对过氧化氢介导内皮细胞损伤、凋亡的机制进行探讨,观察山莨菪碱(Ani)是否抑制H2O2介导的AEC损伤、凋亡,并探讨Ani保护损伤、凋亡的机制.方法:体外培养AEC随机分组:对照组(正常培养AEC);H2O2损伤组(0.5 mmol/L H2O2损伤12 h);Ani组(不同浓度Ani:0.05,0.1,0.2 mg/mL处理45 min后加0.5 mmol/L H2O2损伤12 h).结果:1.低浓度H2O2(0.5 mmol/L)可以损伤AEC并诱导凋亡.H2O2损伤组台盼蓝着染率及LDH释放率均高于对照组(P<0.01);细胞总抗氧化能力(T-AOC)下降(比对照组P<0.01);琼脂糖凝胶电泳发现凋亡细胞的梯状电泳条带;细胞荧光染色见凋亡形态特征.2.Ani对H2O2诱导的内皮细胞损伤具保护作用.Ani组与H2O2损伤组比较,台盼蓝着染率及LDH释放率下降;T-AOC上升;在Ani 0.2 mg/mL组DNA琼脂糖凝胶电泳未见凋亡细胞特征性的梯状电泳条带;荧光染色未见凋亡细胞特征.结论:1.低浓度过氧化氢使AEC总抗氧化能力明显下降,耗竭细胞抗氧化能力,可致AEC的损伤、凋亡.2.山莨菪碱能减少AEC抗氧化能力的消耗,维持抗氧化能力平衡,保护0.5 mmol/L H2O2 介导的AEC损伤、凋亡.     

γ-聚谷氨酸的抗氧化及对UV辐射致皮肤细胞光老化的修复作用

寻找天然抗氧化物用于修复皮肤光老化问题是目前的研究热点之一。γ-聚谷氨酸(γ-PGA)是由微生物发酵制备的一种天然高分子聚合物材料,具有清除自由基的能力,但其抗氧化、抗皮肤光老化效果及与γ-PGA分子量的关系尚未明确。本文中,笔者考察不同分子量的γ-PGA对超氧阴离子自由基(·O~-_2)、羟基自由基(·OH)和1,1-二苯基-2-三硝基苯肼(·DPPH)的清除能力。结果显示:不同分子量的γ-PGA对以上3种自由基均表现出一定程度的自由基清除活性,清除率可分别达31.36%、97.55%和74.64%。通过构建紫外线诱导小鼠皮肤成纤维细胞(L929)氧化损伤光老化模型,确定不同分子量的γ-PGA都能一定程度修复光老化细胞损伤并能减少光老化细胞中氧自由基(ROS)、氮自由基(NO)的增加,其中分子量3.0×10~5的γ-PGA修复光老化细胞损伤的效果最佳。     

氯仿对非洲绿猴肾细胞损伤机理的研究

目的　实验研究氯仿染毒 2 4h对非洲绿猴肾细胞的损伤及其机理。方法　运用显微荧光术测定了非洲绿猴肾细胞 (Vero细胞 )内活性氧 (ROS)含量及游离Ca2 + 浓度 ,同时 ,测定Vero细胞培养上清液乳酸脱氢酶(LDH)活力用于检查Vero细胞受损情况。结果　接触浓度为 4 0mmol L氯仿的Vero细胞内ROS含量及游离Ca2 +浓度与对照组比较无显著性差异 (P >0 0 5 ) ,同时 ,表示Vero细胞受损指标 (LDH活力 )也无显著性差异 (P >0 0 5 ) ;而接触浓度为 8 0mmol L、12 0mmol L氯仿的Vero细胞内ROS含量显著高于对照组 (P <0 0 1) ,其Vero细胞受损也显著增加 (P <0 0 5、P <0 0 1)。结论　较高浓度的氯仿能损伤Vero细胞 ,其损伤的可能途径是通过提高Vero细胞内ROS含量     

铜藻多糖对H2O2诱导的HaCaT细胞氧化应激的保护作用

为探究铜藻多糖(Sargassum horneri polysaccharides, SHP)对H₂O₂诱导的人角质形成细胞(HaCaT)氧化应激损伤的保护作用,测定了SHP对总抗氧化能力(T-AOC)、DPPH自由基、羟自由基(·OH)和超氧阴离子自由基(O₂^·-)的清除作用,以评价SHP的体外抗氧化能力,并建立H₂O₂诱导HaCaT细胞氧化损伤模型;通过测定细胞存活率、细胞活性氧以及酶活,评价SHP对HaCaT细胞氧化损伤的保护作用。结果表明,当SHP为1 mg/mL时,DPPH的清除率为68%、·OH清除能力65.48 U/mL;在SHP为3 mg/mL时,O₂^·-清除能力为84.86 U/mL,T-AOC为33.55。SHP能显著提高H₂O₂诱导氧化损伤的HaCaT细胞活力,其中经100μg/mL SHP处理后,HaCaT细胞存活率由56.85%提高到80.57...     

胞外三磷酸腺苷对铅胁迫下植物细胞损伤和过氧化氢及其清除酶水平的调节

细胞外三磷酸腺苷（extracellular adenosine-5'-triphosphate）是植物细胞的重要信号分子。以烟草悬浮细胞BY-2（Nicotiana tabacum L.cv.Bright Yellow-2）为材料,探讨了胞外三磷酸腺苷对铅胁迫下细胞损伤、H₂O₂（过氧化氢）含量及H₂O₂清除酶活性的影响。结果显示,随着Pb（NO₃）₂浓度的不断提高（30~400 μmol·L^-1）,细胞外三磷酸腺苷含量呈现出逐渐下降的趋势,但胞内三磷酸腺苷含量及细胞的受损伤程度逐渐增大;同时,H₂O₂含量和过氧化氢酶的活性均有所上升,并在200 μmol·L^-1 Pb（NO₃）₂处理下达到最大值,而过氧化物酶的活性则不断降低。较之Pb（NO₃）₂胁迫下的细胞,对Pb（NO₃）₂胁迫的细胞加入外源三磷酸腺苷使得细胞受损伤程度显著降低,H₂O₂含量减少,过氧化氢酶活性减弱,而过氧化物酶活性增强。实验结果表明,Pb（NO₃）₂胁迫诱导的植物细胞损伤和H₂O₂及其清除酶水平的变化能受到细胞外三磷酸腺苷水平的调节。     

腺苷脱氨酶抑制巨噬细胞增殖迁移的作用研究

目的:探讨腺苷脱氨酶对鼠源巨噬细胞RAW264.7增殖、迁移、细胞周期、细胞凋亡的影响。方法:用不同浓度(0、0.25、1.25、2.5、5U/m L)的腺苷脱氨酶处理RAW264.7细胞后,用实时细胞分析系统检测细胞增殖能力,用流式细胞术检测腺苷脱氨酶对细胞凋亡和周期的影响,划痕修复实验检测RAW264.7细胞迁移能力。结果:与对照组相比,高浓度腺苷脱氨酶(2.5 U/m L、5 U/m L)处理可以显著抑制RAW264.7细胞的增殖能力,且抑制效果随腺苷脱氨酶浓度升高而增强(P<0.05)。流式细胞术检测结果显示,相较于对照组,高浓度腺苷脱氨酶(2.5 U/m L)处理可以诱导RAW264.7细胞凋亡,并导致细胞周期G2/M期阻滞(P<0.05)。此外,细胞划痕实验表明,高浓度腺苷脱氨酶(2.5 U/m L)处理可以显著抑制RAW264.7巨噬细胞的迁移能力(P<0.05)。结论:高浓度腺苷脱氨酶对巨噬细胞增殖和迁移具有抑制作用,并可诱导细胞凋亡和细胞周期阻滞。     

The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Fate of Cyclic Nucleotides in PC12 Cell Cultures: Uptake, Metabolism, and Effects of Metabolites on Nerve Growth Factor-Induced Neurite Outgrowth

The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate [(Sp)-cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable. Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2-cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth. 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific. In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5'-AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds.     

Ethanol extract of Scutellaria baicalensis Georgi prevents oxidative damage and neuroinflammation and memorial impairments in artificial senescense mice

Aging is a progressive process related to the accumulation of oxidative damage and neuroinflammation. We tried to find the anti-amnesic effect of the Scutellaria baicalens Georgia (SBG) ethanol extract and its major ingredients. The antioxidative effect of SBG on the mice model with memory impairment induced by chronic injection of D-galactose and sodium nitrate was studied. The Y-maze test was used to evaluate the learning and memory function of mice. The activities of superoxide dismutase, catalase and the content of malondialdehyde in brain tissue were used for the antioxidation activities. Neuropathological alteration and expression of bcl-2 protein were investigated in the hippocampus by immunohistochemical staining. ROS, neuroinflammation and apoptosis related molecules expression such as Cox-2, iNOS, procaspase-3, cleaved caspase-3, 8 and 9, bcl-2 and bax protein and the products of iNOS and Cox-2, NO, PGE2, were studied using LPS-activated Raw 264.7 cells and microglia BV2 cells. The cognition of mice was significantly improved by the treatment of baicalein and 50 and 100 mg/kg of SBG in Y-maze test. Both SBG groups showed strong antioxidation, antiinflammation effects with significantly decreased iNOS and Cox-2 expression, NO and PGE2 production, increased bcl-2 and decreased bax and cleaved caspase-3 protein expression in LPS induced Raw 264.7 and BV2 cells. We also found that apoptotic pathway was caused by the intrinsic mitochondrial pathway with the decreased cleaved caspase-9 and unchanged cleaved caspase-8 expression. These findings suggest that SBG, especially high dose, 100 mg/kg, improved the memory impairments significantly and showed antioxidation, antiinflammation and intrinsic caspase-mediated apoptosis effects.     

Effect of 5'-adenosine monophosphate and cyclic 3',5'-adenosine monophosphate on the ability of guinea pig bone marrow precursor cells to form fibroblast-like cell colonies in vitro

The effect of 5'-AMP and cyclic 3',5'-AMP on the ability of cells--precursors of bone marrow to form colonies of fibroblast-like cells in vitro was studied in the guinea pig. No reliable effect of both substances (within the limits of 10(-5) to 10(-8) M) on the formation of fibroblast colonies in vitro by the cells--precursors of bone marrow was shown. No differences were established in the size of colonies between the experimental and control variants.     

Suppression of tyrocidine production by purine nucleotides and related substances in Bacillus brevis

Bacillus brevis (ATCC 8185) produces an antibiotic peptide, tyrocidine. We found that adenosine or 5'-AMP suppressed the production of tyrocidine with half-maximum inhibition at 100-300 microM. This inhibition was specific to the production of tyrocidine since neither adenosine nor 5'-AMP showed any effect on bacterial growth. Cyclic nucleotides had no effect. These results suggest that adenosine, 5'-AMP or its metabolite was specifically involved in the regulation of tyrocidine production.     

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.