一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氢(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组(1)阴性对照组(Normal组);(2)H2O2组H2O2(0.1mmol/L)与心肌细胞共育4h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组NO供体SNAP(0.5mmol/L)处理心肌细胞10min后,加入H2O2与心肌细胞共育4 h.用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量.通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化.结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2%,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P＜0.01),细胞存活率明显下降(58.3±7.6%,P＜0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7%;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P＜0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P＜0.01).SNAP预处理细胞可显著提高心肌细胞存活率(79.7±9.3%,P＜0.01),降低LDH活性和细胞凋亡率(分别为957.8±110.9 U/L和9.1±3.3%,P＜0.01);并提高细胞抗氧化能力,表现为较H2O2处理组的SOD活性增高(21.36±3.11 NU/ml,P＜0.01),MDA含量下降(9.12±1.47 μmol/L,P＜0.01).激光共聚焦显微镜检测结果表明,H2O2可升高细胞内钙,而SNAP则可降低细胞内钙,SNAP预处理细胞后可取消H2O2升高细胞内钙的作用.上述结果提示,NO供体SNAP可对抗H2O2对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力和对抗H2O2引起的细胞内钙超载有关.     

Protective and therapeutic effects of resveratrol on acetic acid-induced gastric ulcer

Sprague Dawley rats of both sexes were injected with either saline or RVT (10 mg/kg) either before or after acetic acid ulcer induction and decapitated 3, 5 or 10 days after ulcer. In the saline-treated ulcer groups, macroscopically evident ulcers were observed, while RVT-pretreated or RVT-treated groups had lower macroscopic ulcer scores. Likewise, the microscopic damage scores were lower for the RVT-administered groups. Gastric myeloperoxidase activity, malondialdehyde, collagen and tumour necrosis factor-alpha levels, as well as luminol- and lucigenin-enhanced chemiluminescence levels that were elevated in the saline-administered ulcer groups, were depressed with both RVT-pretreatment and RVT-treatment. Moreover, depleted glutathione levels in the ulcer groups were increased back to control levels by both pre- and post-treatments of RVT. Results demonstrate that resveratrol has both protective and therapeutic effects on oxidative gastric damage by suppressing pro-inflammatory cascades, including the activation of pro-inflammatory cytokines, accumulation of neutrophils and release of oxygen-derived free radicals.     

The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 µg/kg caerulein, L-arginine used for NO induction and N^ω-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.     

The involvement of nitric oxide in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes

Oxidative stress plays a crucial role in the manifestations of maneb (MB) and paraquat (PQ)-induced toxicity including MB+PQ-induced Parkinson's disease (PD). Polymorphonuclear leukocytes (PMNs) actively participate in the oxidative stress-mediated inflammation and organ toxicity. The present study was undertaken to investigate the MB- and/or PQ-induced alterations in the indices of oxidative stress in rat PMNs. Animals were treated with or without MB and/or PQ in an exposure time dependent manner. In some sets of experiments, the animals were pre-treated with NOS inhibitors N^G-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG) along with respective controls. A significant increase in myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide, iNOS expression and lipid peroxidation (LPO) was observed in PMNs of MB- and/or PQ-treated animals, while catalase and glutathione S-transferase (GST) activities were attenuated. L-NAME and AG significantly reduced the augmented nitrite content, iNOS expression and MPO activity to control level in MB and PQ exposed animals. Although the augmented LPO was also reduced significantly in L-NAME and AG treated rat PMNs, the level was still higher as compared with controls. Alterations induced in SOD and GST activities were not affected by NOS inhibitors. The results thus suggest that MB and/or PQ induce iNOS-mediated nitric oxide production, which in turn increases MPO activity and lipid peroxidation, thereby oxidative stress.     

Role of nitric oxide in the reperfusion induced injury in hyperthyroid rat hearts

We recently reported that hyperthyroidism affects the heart response to ischemia/reperfusion. A significant tachycardia during reperfusion was associated with an increase in the oxidative stress of hearts from T₃-treated animals. In the present study we checked the possible role of nitric oxide (NO) in this major stress induced by the hyperthyroid state. We compared the functional recovery from ischemia/reperfusion of Langendorff preparations from euthyroid (E) and hyperthyroid (H, ten daily intraperitoneal injections of T₃, 10 μg/100 g body weight) rats, in the presence and in the absence of 0.2 mM N^ω-nitro-L-arginine (L-NNA). At the end of the ischemia/reperfusion protocol (10 min preischemic perfusion, 20 min global ischemia, 30 min reperfusion) lipid peroxidation, antioxidant capacity (C_A) and susceptibility to in vitro oxidative stress were determined on heart homogenates. The main effect of hyperthyroidism on the reperfusion functional response was confirmed to be a strong tachycardic response (154% recovery at 25 min reperfusion) accompanied by a low recovery in both left ventricular diastolic pressure (LVDP) and left ventricular dP/dt_max. This functional response was associated with a reduction in C_A and an increase in both lipid peroxidation and susceptibility to oxidative stress. Perfusion of hearts with L-NNA per se had small but significant negative chronotropic and positive inotropic effects on preischemic performance of euthyroid rat hearts only. More importantly, L-NNA perfusion completely blocked the reperfusion tachycardic response in the hyperthyroid rats. Concomitantly, myocardium oxidative state (lipid peroxidation, C_A and in vitro susceptibility to oxidative stress) of L-NNA perfused hearts was similar to that of E animals. These results suggest that the higher reperfusion-induced injury occurring in hyperthyroid animals is associated with overproduction of nitric oxide.     

Inhibitory effect of resveratrol dimerized derivatives on nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells

Four types of resveratrol dimerized analogues were synthesized and evaluated in vitro on LPS-induced NO production in RAW 264.7 cells. The results showed that several compounds, especially those containing 1,2-diphenyl-2,3-dihydro-1H-indene core (type I), exhibited good inhibitory activities. Among 25 analogues, 12b showed a significant inhibitory activity (49% NO production at 10 μM, IC₅₀ = 3.38 μM). Further study revealed that compound 12b could suppress LPS-induced iNOS expression, NO production, and IL-1β release in a concentration-dependently manner. The mechanism of action (MOA) involved for its anti-inflammatory responses was through signaling pathways of p38 MAPK and JNK1/2, but not ERK1/2.     

一氧化氮在炎性疼痛中的作用

一氧化氮（nitric oxide,NO）是细胞内重要的信使分子和神经递质,它参与多种生命活动,包括炎性疼痛.NO对炎性疼痛的发展和维持起到了重要的作用.研究NO在疼痛中所起到的作用及其机制有利于阐明痛觉生理和发现疼痛治疗的新手段.目前研究表明,脊髓水平NO参与炎性疼痛调制的可能机制主要有NO/cGMP途径、参与调控即刻早期基因、与其他神经递质的协同作用.另外研究表明,3种类型的一氧化氮合酶（nitric oxide synthases,NOS）在炎性疼痛过程中被激活或者有不同程度的增强表达.     

一氧化氮在铁诱导的大鼠心肌损伤中的作用

采用Langendorff灌流大鼠心脏和酶解分离的心肌细胞为实验模型,研究铁负荷下心肌损伤情况以及一氧化氮（NO）在铁诱导的心肌损伤中的地位。结果显示:（1）心肌铁负荷（Fe-HQ）可使分离心肌细胞舒张期细胞长度缩短、收缩幅度和速度降低,离体灌流心脏左室发展压（LVDP）、±dp/dtmax、冠脉流量呈现双相变化;冠脉流出液中乳酸脱氢酶（LDH）、肌酸激酶（CK）释放量和心肌丙二醛（MDA）增高。（2）NO的前体L-精氨酸（L-argi-nine,L-Arg）引起心肌细胞舒张期细胞长度缩短、收缩幅度降低。离体灌流心脏LVDP、冠脉流量、和±dp/dtmax增高,用K-H液复灌后可恢复正常。（3）L-Arg预处理,再行Fe-HQ灌流,与单纯的L-Arg或Fe-HQ组相比,心肌细胞舒张期细胞长度、收缩幅度和速度减小;离体灌流心脏LVDP、±dp/dtmax、心率和冠脉流量明显下降,冠脉流出液中LDH、CK增加。（4）Nω-硝基-L-精氨酸甲酯（L-NAME）和Fe-HQ合并灌流后,与单纯Fe-HQ组相比,心肌细胞舒张期细胞长度、收缩幅度和速度增加。L-NAME可阻断Fe-HQ引起的LVDP、左室舒张末压（LVEDP）和±dp/dtmax降低,冠脉流出液中LDH、CK增高。（5）用Triton X-100短暂处理以去除冠脉内皮后,与保留冠脉内皮的心肌相比,Fe-HQ引起的LVDP和±dp/dtmax的一过性增高现象被抑制,但     

一氧化氮对强光胁迫下高羊茅叶片抗氧化系统的调节效应

在强光胁迫下,采用水培法,使用外源NO和去除内源NO措施,研究了NO对2个高羊茅(Arid3和Houndog5)品种的抗氧化系统的调节作用.结果表明,Arid3的内源NO比对照增加201.5%,而Houndog5仅增加21.1%.Houndog5发生严重氧化损伤,电解质渗出率和丙二醛(MDA)含量分别比对照增加72.6%和85.1%,而Arid3受到的伤害较轻,分别比对照增加36.1%和30.1%.使用O.2 mmol·L~(-1)的NO专一清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(PTIO)清除内源NO,加剧强光胁迫对Arid3与Houndog5氧化伤害.应用0.1 mmol·L~(-1)的外源NO供体硝普钠(SNP)能降低强光胁迫造成的Arid3和Houndog5 叶片脂氧合酶(LOX)活性,降低超氧自由基(O_2)产生速率和质膜相对透性的增加以及MDA和H_2O_2的累积;同时,O.1 mmol·L~(-1)的SNP还能够诱导过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸氧化物酶(APX)和谷胱甘肽还原酶(GR)活性.而外源NO的保护作用被PTIO逆转.上述结果暗示,NO可能作为一个活性分子诱导抗氧化物酶的活性,减轻强光胁迫下2个高羊茅品种氧化损伤.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

The effect of shock wave lithotripsy on nitric oxide and malondialdehyde levels in plasma and urine samples

Shock wave lithotripsy (SWL) is accepted as the first treatment choice for most urinary stones, but it has adverse effects on the kidneys. The mechanisms underlying shock wave-induced renal injury have been discussed and include shear stress, thermal and cavitation effects and free radical formation. We investigated the effects of SWL on plasma and urinary nitrite, a stable metabolite of nitric oxide (NO), and malondialdehyde (MDA) concentrations. Between February and October 2004, 12 men and 8 women with renal calculi were treated using a Dornier MPL-9000 lithotriptor. The ages ranged from 22 to 45 years (average age: 33.7 years). Plasma and urinary NO and MDA levels were analysed before, immediately after, 30 and 60 min and 24 h after SWL. Plasma NO levels were higher than baseline levels immediately, and at 30, 60 min and 24 h after treatment (p = 0.016, p = 0.031, p = 0.033 and p = 0.045, respectively). Simultaneously, the mean urinary NO levels also showed significant elevation after SWL compared with baseline values, except for 24 h (p = 0.021, p = 0.023 and p = 0.048, respectively). The mean levels of plasma MDA showed statistically significant elevation immediately, and 30 and 60 min after SWL termination compared with pre-SWL values (p = 0.012, p = 0.008 and p = 0.012, respectively). Urinary MDA levels obtained immediately (p = 0.035), and 30 (p = 0.006) and 60 (p = 0.045) min after SWL were increased compared to pre-SWL values. We speculate that SWL treatment causes oxidative stress caused by renal ischemia-reperfusion (I/R) injury. Additionally, the increase of NO production may have prevented renal damage caused by vasoconstriction.     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

一氧化氮对过氧化氢所致听力损失的保护作用

通过全耳蜗灌流法在体观察一氧化氮(N0)能否通过一氧化氮/环磷酸鸟苷(NO/cGMP)途径对抗过氧化氢这种氧自由基所致的听力损失。实验选用耳廓反射灵敏、无耳毒性药物使用史的健康杂色豚鼠(250-350 g)50只,雌雄不拘,随机分为5组,每组10只动物,分别行全耳蜗灌流人工外淋巴液;过氧化氢(H2O2);L-精氨酸(合成NO的底物);H2O2+L-精氨酸;H2O2+L-精氨酸+L-NNA(一氧化氮合成酶的抑制剂),均灌流2 h。通过圆窗龛电极,每隔30 min记录复合动作电位(compound action potential,CAP:由短声Click诱发)阈值,耳蜗微音器电位(cochlear microphonic,CM;由短纯音Tone Burst诱发)幅度,了解耳蜗功能的变化,并分离取出耳蜗基底膜并制备基底膜硬铺片,通过碘化毗啶(PI)和Hoecbst双染色方法,观察耳蜗组织各类细胞损伤情况。结果显示,灌流H2O2+L-精氨酸组的CAP阈移和CM下降幅度值明显低于单独灌流H2O2组,差异有显著性(氏P<0.05);前者形态学观察未见明显的细胞损伤,后者可见大量坏死红染的细胞。H2O1+L-精氨酸+L-NNA组CAP阈移和CM下降幅度与单独灌流H2O2组比较无统计学差异。实验结果提示NO可能通过NO/cGMP途径部分对抗过氧化氢所致的听力损失。     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

Dual effect of nitric oxide on phenoloxidase-mediated melanization

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO₂ at the active site of PO followed by oxidation of DOPA by NO₂. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.     

一氧化氮在大鼠肢体缺血再灌注后肺损伤中的作用

在大鼠肢体缺血再灌注（LIR）损伤模型上,观察应用一氧化氮合酶（NOS）抑制剂氨基胍（AG）及一氧化氮（NO）合成前体物质L－精氨酸（L－Arg)对大鼠骨骼肌和肺组织的NOS活性、NO含量、丙二醛（MDA）、髓过氧化物酶（MPO）和湿／干重（W／D）值的影响以及肺磷脂酰胆碱（PC）的改变,并观察了肺组织在光镜下形态学的变化。结果显示,与对照组比较,LIR组骨骼肌和肺组织NOS活性均增强,MDA值、MPO活性增加,W／D值增大,肺PC含量降低;光镜下,肺间质多形核粒细胞（PMN）聚集和浸润,肺间隔面密度值增加。给予AG后,与LIR组相比NOS活性降低,NO产生下降,而MPO活性、W／D比值增加,肺PC含量进一步降低;镜下PMN聚集和浸润增加,肺间隔面密度值增大。而给予L－Arg后能 减轻LIR引起的上述变化。上述结果提示,LIR后2h时,骨骼肌和肺组织NOS活性增加,NO产生增多;内源性NO可能在LIR所诱发的早期急性肺损伤中起保护作用。     

Nitric oxide synthase activity and nitric oxide level in erythrocytes of guinea pigs with experimental otitis media with effusion

Free radicals have been implicated in the pathogenesis of an increasing number of disease and inflammatory states. They may cause cell and tissue damage by chemical modification of proteins, carbohydrates, nucleotides and lipids. Under physiological conditions free radicals are parts of normal regulatory circuits and are neutralized by antioxidants. Infections are one cause of increased free radicals production. The aim of our study was to assess whether increased oxidative stress is reflected by erythrocyte nitric oxide synthase activity and nitric oxide levels in guinea pigs with experimental otitis media with effusion (n = 6) and in a control group (n = 6). Erythrocyte nitric oxide synthase activity and nitric oxide levels were measured in both groups. The nitric oxide synthase activity and nitric oxide level in the experimental otitis media with effusion were significantly higher than those of the control group. There was a significant positive correlation between the nitric oxide synthase activity and nitric oxide in the experimental otitis media with effusion group. Thus, increased nitric oxide levels may play an important role in cell and tissue damage due to experimental otitis media with effusion.     

Chemical constituents isolated from Polygala japonica leaves and their inhibitory effect on nitric oxide production in vitro

A methanolic extract of dried leaves of Polygala japonica Houtt (Polygalaceae) significantly attenuated nitric oxide production in lipopolysaccharide-simulated BV2 microglia. Five anthraquinones chrysophanol (1), emodin (2), aloe-emodin (3), emodin 8-O-β-D-glucopyranoside (4) and trihydroxy anthraquinone (5), and four flavonoids kaempferol (6), chrysoeriol (7), kaempferol 3-gentiobioside (8) and isorhamnetin (9) were isolated from the methanolic extract using bioactivity-guided fractionation. Among them, compounds 1–4, 6 and 7 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia at the concentrations ranging from 1.0 to 100.0 μM.     

Dehydroepiandrosterone inhibits lipopolysaccharide-induced nitric oxide production in BV-2 microglia

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimer's disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age-related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)-stimulated BV-2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS-stimulated BV-2 cell cultures. Pretreatment of BV-2 cells with DHEA reduced the LPS-induced iNOS mRNA and protein levels in a dose-dependent manner. The LPS-induced iNOS activity in BV-2 cells was decreased by the exposure of 100 microM DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS-stimulated BV-2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.