硒对NO诱导的内皮细胞损伤的抑制机制研究

用外源性NO供体S－亚硝基谷胱甘肽（GSNO）处理人脐静脉内皮细胞系ECV－304细胞，研究其对细胞的损伤机制，并探讨硒在这一过程中的保护作用，通过MTT法测定细胞存活率，分光光度法[测定细胞LDH漏出率及细胞脂质过氧化水平，采用荧光标记技术研究细胞膜流动性变化，结果表明，NO可引起细胞脂质过氧化水平升高，细胞膜流动性下降，导致ECV－304细胞损伤，其作用具有浓度效应，细胞内硒可通过抑制细胞脂质过氧化水平及细胞膜流动性变化从而抑制NO诱导的细胞损伤。     

硒对NO诱导的内皮细胞损伤

用外源性NO供体S-亚硝基谷胱甘肽(GSNO)处理人脐静脉内皮细胞系ECV-304细胞,研究其对细胞的损伤机制,并探讨硒在这一过程中的保护作用.通过MTT法测定细胞存活率、分光光度法测定细胞LDH漏出率及细胞脂质过氧化水平,采用荧光标记技术研究细胞膜流动性变化.结果表明,NO可引起细胞脂质过氧化水平升高,细胞膜流动性下降,导致ECV-304细胞损伤,其作用具有浓度效应;细胞内硒可通过抑制细胞脂质过氧化水平及细胞膜流动性变化从而抑制NO诱导的细胞损伤.     

雪上一枝蒿醇提物体外抗炎作用的研究

为探讨雪上一枝蒿醇提物(EABD)对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞活性和细胞凋亡,分泌的一氧化氮(NO)和活性氧(ROS)的影响,经LPS诱导后以不同浓度雪上一枝蒿醇提物作用于小鼠腹腔巨噬细胞,用MTT法检测细胞活性,Hoechst33258荧光染色检测细胞凋亡,荧光探针DCFH-DA测定活性氧(ROS),Griess法检测NO.结果表明:EABD可显著抑制LPS造成的小鼠巨噬细胞损伤和细胞凋亡,20,100μg/mL EABD干预后使小鼠腹腔巨噬细胞内ROS水平降低,剂量依赖性减少NO表达(P<0.05),说明雪上一枝蒿通过抗氧化途径发挥体外抗炎作用.     

阿尔茨海默病异常黑胆质病证结合模型的氧化应激反应及方药干预实验

 为研究阿尔茨海默病(AD)异常黑胆质病证结合大鼠海马组织抗氧化酶的活性及脂质过氧化产物的浓度变化, 并观察方药对氧化应激的干预作用.选用雄性SPF级Wistar大鼠36只, 完全随机分成正常对照组、模型组、异黑颗粒高剂量干预组、异黑颗粒中剂量干预组、异黑颗粒低剂量干预组、多奈哌奇干预组.病证结合造模后3d开始药物干预15d, 行为学测试后应用紫外分光光度法检测大鼠大脑海马组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、单胺氧化酶(MAO)活性和丙二醛(MDA)含量.结果表明, 模型组大鼠海马组织SOD、GSH-Px、CAT活性明显减弱, MAO活性增强、MDA含量明显升高, 与空白对照组比较有显著性差异(P<0.01).经方药干预治疗后, 不仅生物表征有显著改善, 高剂量干预组大鼠海马组织SOD、GSH-Px、CAT活力较模型组明显提高, MAO活性减低、MDA含量明显下降, 与模型组比较有显著性差异(P<0.01), 与多奈哌奇干预组比较无明显差异(P>0.05).中剂量干预组SOD活性明显提高, 与模型组比较有显著性差异(P<0.01);GSH-Px、CAT活性提高、MAO活性降低, 与模型组比较有明显差异(P<0.05), MDA含量无明显改善, 与正常对照组比较有显著性差异(P<0.01).低剂量干预组SOD、CAT、MAO活性及MDA含量与模型组比较无显著性差异(P>0.05), 与正常对照组比较有显著性差异(P<0.01).GSH-Px活性与模型组比较有明显差异(P<0.05).由此得出, 异常黑胆质证结合双海马聚集态Aβ注射所建立的AD异常黑胆质病证结合大鼠模型, 存在明显的自由基损伤和氧化应激过度, 异常颗粒可能通过增强SOD、CAT、GSH-Px等抗氧化酶的活性, 抑制MAO活性减少单胺类神经递质的氧化分解, 抑制脂质过氧化反应而减少自由基对海马神经元损伤, 从而达到防治老年性痴呆的目的,方药高剂量疗效最突出.     

硫酸化茯苓多糖对小鼠腹腔巨噬细胞功能的影响

研究硫酸化茯苓多糖(SP)对脂多糖(LPS)诱导的体内外小鼠巨噬细胞氧化损伤的影响.体外实验中,制备小鼠腹腔巨噬细胞,加入不同质量浓度(0~100mg/mL)的SP和1mg/mL的LPS诱导剂,24h后检测细胞活力、一氧化氮(NO)、超氧阴离子自由基(O-2)及肿瘤坏死因子-α(TNF-α)含量.体内实验中,42只ICR小鼠随机分为空白组、模型组和预防组,采用腹腔注射法连续给药7天后收集腹腔巨噬细胞,检测多种生化指标.实验结果表明:LPS可降低巨噬细胞的活性,增加O-2的释放和NO、TNF-α的分泌;SP能够显著降低NO的水平,提高巨噬细胞抗O-2的活力,对于TNF-α的含量没有显著影响.SP可有效地改善LPS对小鼠腹腔巨噬细胞的氧化损伤作用.     

琐琐葡萄多糖对PC12细胞损伤的神经保护作用

 体外培养PC12细胞,采用20μmol/L β淀粉样蛋白25-35(Aβ25-35)作用24h诱导细胞损伤,建立阿尔茨海默病(AD)细胞模型,研究琐琐葡萄多糖(VTP)对PC12细胞损伤的神经保护作用。设立对照组、模型组和VTP保护组(20,40,80μg/mL),CCK-8法检测各组细胞的存活率,乳酸脱氢酶(LDH)法检测细胞膜通透性及完整性,化学比色法测定细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)含量,流式细胞术检测细胞凋亡率。结果显示,20,40,80μg/mL VTP可提高PC12细胞存活率,减少LDH渗漏,增加SOD活力,减少MDA含量,降低细胞凋亡率,与模型组比较有显著差异(P<0.01)。由此推论,VTP对Aβ25-35诱导的PC12细胞凋亡和氧化损伤具有明显的保护作用。     

儿茶素对溶血卵磷脂胆碱所致细胞损伤的保护作用

作者以体外培养的小牛主动脉内皮细胞为材料,研究了溶血卵磷脂胆碱（LPC）和氧自由基（OFR）对血管内皮细胞（VEC）的损伤,及儿茶素对VEC的保护。结果显示：当VEC与LPC（6ug/ml）或黄嘌呤／黄嘌呤氧化酶（X／XO）（10umol/L 200umol/L）共孵育24小时时,表现为细胞内乳酸脱氢酶（LDH）泄漏量增多,细胞内过氧化脂质丙二醛（MDA）含量升高,细胞生长减缓,存活率下降;当加入不同浓度的儿茶素后则可明显抑制LDH的泄量,降低MDA含量,细胞生长正常,存活率提高。表明LPC与X／XO对VEC有损伤作用,而儿茶素则能通过抗氧化途径抵抗LPC与X／XO至VEC的损伤。     

中药喘可治注射液对小鼠腹腔巨噬细胞的影响

目的:研究中药喘可治注射液(CKZ)对小鼠腹腔巨噬细胞凋亡、一氧化氮(NO)释放和细胞因子分泌的影响,并探讨其作用机制.方法:无菌分离小鼠腹腔巨噬细胞,制备单细胞悬液,加入CKZ(终体积分数20 μL/mL)孵育4 h后, 加入过氧化氢(H2O2)、放线菌酮(CHX)、环磷酰胺(CTX)诱导细胞凋亡,荧光酶标仪结合Sytox Green染色检测细胞凋亡;加药孵育4 h后,加入细菌脂多糖(LPS,终质量浓度10 μg/mL)和IFN-γ(终质量浓度80 ng/mL),24h后Grass试剂盒检测巨噬细胞NO的量;加药孵育4 h后,加入LPS(终质量浓度10 μg/mL)和IFN-γ(终质量浓度80 ng/mL),24h后流式细胞仪结合Cytometric Bead Array(CBA)技术检测细胞因子的释放.结果:CKZ能抑制H2O2、CHX、CTX诱导的细胞凋亡;促进非刺激状态下巨噬细胞NO和IL-6、IL-10、MCP-1、IFN-γ、TNF-α 5种细胞因子的产生, 抑制LPS和IFN-γ刺激的NO和IL-6、IL-10、MCP-1、IFN-γ、TNF-α5种细胞因子的产生.结论:CKZ可抑制巨噬细胞凋亡,并对巨噬细胞NO的分泌和IL-6、IL-10、MCP-1、IFN-γ、TNF-α5种细胞因子的生成具有双向调节作用.     

阿奇霉素-苦木注射液联用对炎症细胞因子释放影响的体外研究

实验研究阿奇霉素与苦木注射液体外联合用药对前炎症因子NO、TNF-α抑制作用的影响.采用脂多糖(LPS)体外刺激小鼠单核巨噬细胞RAW264. 7,刺激细胞释放TNF-α、NO前炎性因子.采用MTT法评价细胞毒性; Griess法检测培养液上清中NO含量;ELISA试剂盒法检测TNF-α含量;金正均法计算Q值进行药效联用评价.结果表明阿奇霉素联合苦木注射液在10～200μg/m L浓度范围内可明显抑制LPS诱导巨噬细胞RAW264. 7释放的炎性因子TNF-α、NO,并呈现良好的剂量依赖关系.阿奇霉素联合苦木注射液对炎症因子NO具相加抑制作用,对炎症因子TNF-α具协同抑制作用.     

腺梗豨莶提取物对脂多糖活化巨噬细胞释放一氧化氮的抑制作用

用脂多糖（LPS）诱导小鼠单核巨噬细胞RAW264．7释放一氧化氮（NO）,Griess法测定培养上清中NO释放量并计算抑制率,MTT法测定细胞存活率评价药物的细胞毒性,以检测腺梗豨莶不同溶剂萃取部位对活化巨噬细胞释放炎症介质NO的抑制作用．结果表明,石油醚及乙酸乙酯部位对活化巨噬细胞释放NO的抑制作用明显强于正丁醇部位及水层,即石油醚及乙酸乙酯部位为腺梗豨莶抑制活化巨噬细胞释放NO的主要活性部位,半数抑制浓度（IC50）值分别为6．0和6．5μg·mL^-1,其作用强度优于该植物的主要活性成分奇壬醇（IC50为37．5μg·mL^-1）,推断该活性部位中应含有奇壬醇以外的其他活性化合物．以上结果为进一步阐明腺梗豨莶的抗炎作用物质基础提供了一定的理论基础．     

一氧化氮参与附睾上皮细胞感染后的宿主防御

 以金黄色葡萄球菌Staphylococcus aureus感染附睾上皮细胞的体外感染模型,分别采用RT-PCR和western blot等 方法研究一氧化氮（NO）在附睾上皮细胞宿主防御中的作用。结果表明附睾上皮感染后上调iNOS RNA和蛋白水平的表达,并产生 大量的NO,采用iNOS的抑制剂L NMMA和AGE预处理后,NO水平显著降低,与空白处理组相比,抑制剂处理组的S aureus数量显著增加,这表明NO参与了附睾上皮细胞的宿主防御过程,在附睾上皮细胞的宿主防御中具有重要的作用。     

The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues--aspartate, glutamate and cysteine--are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.     

NO is necessary and sufficient for egg activation at fertilization

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.     

糖尿病大鼠血清一氧化氮浓度的变化

目的 :观察不同病程糖尿病大鼠血清一氧化氮 (NO)浓度变化 ,探讨糖尿病大鼠血清NO变化的规律和意义。方法 :建立链脲佐菌素诱导的糖尿病大鼠模型 ,分别在第 2、7、12周 3个时期取血清 ,葡萄糖酶法测定血清葡萄糖 ,硝酸还原酶法测定血清NO含量。结果 :(1)血糖变化　对照组各时期血糖值维持在正常水平 ;糖尿病组大鼠各时期血糖均明显高于对照组 (P <0 0 5 )。 (2 )血清NO浓度变化　对照组各时期血清NO浓度无显著性差异 ;糖尿病组大鼠 2周时血清NO浓度明显高于对照组 (P <0 0 5 ) ;7周、12周时糖尿病组大鼠血清NO浓度恢复到正常水平 ,与对照组相比无显著差异 (P >0 0 5 )。结论 :NO是糖尿病发生的重要因子之一 ,但可能与血糖的调节无关。     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.     

Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide

Studies of cultured bovine aortic endothelial cells using quantitative chemiluminescence techniques have shown that the amount of nitric oxide released under basal conditions, or in response to either bradykinin or the calcium ionophore A23187 is insufficient to account for the vasorelaxant activities of the endothelium-derived relaxing factor (EDRF) derived from the same source. This observation contradicts previous suggestions that nitric oxide and EDRF are the same compound, but may be explained if EDRF is a compound that contains nitric oxide within its structure but is a much more potent vasodilator than nitric oxide. Such a molecule could be one of several nitrosothiols which may yield nitric oxide after a one-electron reduction. The present experiments were carried out to test the possibility that the biological activities of the endothelium-derived relaxing factor might more closely resemble those of one of these compounds, S-nitrosocysteine, than nitric oxide. Nitric oxide release from cultured bovine aortic endothelial cells was detected by chemiluminescence and bioassay experiments compared the vasodilator potencies of nitric oxide, S-nitrosocysteine, and EDRF. The results suggest that EDRF is much more likely to be a nitrosylated compound such as a nitrosothiol than authentic nitric oxide.     

一氧化氮对紫外线-B辐射下小浮萍保护作用

以小浮萍为材料,研究紫外线-B(UV-B)辐射下NO对植物的保护作用.经紫外线处理过的小浮萍,叶绿素和可溶性蛋白质含量降低,而SOD、POD和CAT活性上升;加入NO后,可以阻止叶绿素含量下降,提高可溶性蛋白质含量,且增加SOD、POD和CAT的活性.NO可以提高小浮萍抗紫外线能力.     

Nitric oxide damages neuronal mitochondria and induces apoptosis in neurons

The cytotoxic effect of nitric oxide on primarily cultured rat cerebellar granule cells was studied, and the mechanisms were discussed. The results showed that nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP; 500 μmol/L) could induce apoptosis in immature cultures of cerebellar granule cells. Flow cytometry and HPLC analyses revealed that after treatment with SNAP, the mitochondrial transmembrane potential and the cellular ATP content decreased significantly. Nitric oxide scavenger hemoglobin could effectively prevent the neuronal mitochondria from dysfunction and attenuate apoptosis. The results suggested that nitric oxide activated the apoptotic program by inhibiting the activity of mitochondrial respiratory chain and thus decreasing the cellular ATP content.     

Nitric oxide mediates the fungal elicitor-induced Taxol biosynthesis of Taxus chinensis suspension cells through the reactive oxygen species-dependent and -independent signal pathways

Taxol (paclitaxel, NSC-125973), a secondary me- tabolite of the Taxus species, has been recognized as one of the best anticancer drugs emerging in the last decade[1]. The production of Taxol by various Taxus spp. cells in culture has been one of the most …     

溃疡性结肠炎大鼠模型的结肠电及NO变化

目的制作大鼠溃疡性结肠炎(UC)模型,研究结肠电异常和一氧化氮(NO)变化。方法以二硝基氯苯法制作溃疡性结肠炎模型,埋藏电极记录结肠电,以标准对照比色法检测其大肠洗液一氧化氮产物亚硝酸盐(NOP)的浓度。结果造模大鼠结肠形成典型的溃疡,并发现其结肠电活动明显异常,具体表现为基本电节律(BER)减慢及收缩性复合肌电(CEC)的幅值显著降低,负载峰电的BER和CEC百分数明显减少,大肠洗液NOP浓度则显著升高,两者呈显著负相关。结论NO在溃疡性结肠炎的病理过程及结肠电改变中具有重要的作用。