不同运动负荷大鼠主要器官各型NOS表达水平的研究

[目的]检测不同运动负荷时大鼠主要器官如心、脑、股外肌等器官组织NOS各亚型的mRNA表达水平和蛋白表达水平的比较,从基因水平探讨一氧化氮在运动中对机体的影响。[方法]用反转录聚合酶链式反应(RT-PCR)检测3个器官组织的NOS3种亚型(eNOS、nNOS、iNOS)的mRNA表达。Western blot方法检测各组织的蛋白表达。[结果]RT-PCR检测发现:eNOS、nNOS、iNOS在心肌、脑及股外肌组织中均有mRNA表达。股外肌在中等强度运动时eNOS、nNOS、iNOS的mRNA表达明显高于对照组,股外肌的iNOS的mRNA表达在运动疲劳后则比中等强度运动时明显降低(P﹤0.05)。心肌组织在不同的运动负荷状态下各组织eNOS、nNOS的mRNA表达无明显差异,心肌组织在中等强度和疲劳运动后iNOS的mRNA均明显高于对照组(P﹤0.01)。脑组织在中等强度运动后nNOS、iNOS的mRNA表达明显增强(P﹤0.05),nNOS的mRNA表达在运动疲劳后比中等强度运动时明显降低(P﹤0.05)。Westernblot方法检测:股外肌组织在进行中等强度运动时eNOS nNOS、iNOS蛋白表达均明显强于对照组和运动疲劳组,疲劳组蛋白表达均减弱。脑组织在进行中等强度运动时nNOS、iNOS蛋白表达明显强于对照组,疲劳组nNOS蛋白表达最弱。心肌组织在中等强度和疲劳运动后iNOS蛋白表达增强,疲劳组iNOS蛋白表达最强。[结论]中等强度运动上调eNOS、nNOS、iNOS的mRNA表达和蛋白表达;疲劳运动可导致nNOS、eNOS的mRNA表达和蛋白表达减弱或有减弱趋势。     

The effect of cadmium toxicity on renal nitric oxide synthase isoenzymes

The aim of this study was to assess the cadmium (Cd) toxicity on renal nitric oxide synthase (NOS) isoenzymes. The study was carried out on 18 inbred male (Cd group: 10 and control group: 8) Wistar rats. Cd group received drinking water containing 15 mg/L Cd for 30 days; and at the end of the 30 days, plasma Cd was analysed. One kidney was snap frozen to assess the endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) expressions by Western blot analyses, and the other kidney was preserved for histopathological examination. Plasma Cd levels were significantly elevated in the Cd group. The Western blot analyses found higher levels of eNOS, iNOS and nNOS in the Cd group but only eNOS and nNOS levels were statistically significant. There was no difference in pathological assessment of the renal tissues. Cd toxicity increases NOS isoenzyme levels and may affect renal physiology.     

铅对大鼠脑细胞一氧化氮合酶表达的影响

目的观察实验性铅中毒对大鼠脑细胞一氧化氮合酶(NOS)表达的影响,为进一步揭示铅的神经毒作用机制提供科学依据。方法取健康成年雄性SD大鼠24只,随机分为4组,每组6只,经腹腔注射醋酸铅连续染毒5d,剂量分别为25、50、100mg/kg体重,分别取海马、皮层部位脑组织,用免疫组化方法分别测定海马、皮层组织中神经元型一氧化氮合酶(nNOS)和依赖型一氧化氮合酶(iNOS)的蛋白含量。结果各剂量染铅组皮层、海马组织中iNOS表达与对照组相比,明显升高(P均<0.05),并有良好的剂量-反应关系(r=-0.727,P<0.05)。各剂量染毒组海马nNOS表达明显升高(P均<0.05),并有良好的线性关系(r=0.847,P<0.05),皮层组织nNOS表达无明显变化(P均>0.05)。结论铅所引起的神经细胞毒性可能通过铅诱导NOS的高表达,使得NO生成过多进而造成神经损害。     

牛磺酸对糖尿病大鼠视网膜NO及其合酶影响

目的 探讨牛磺酸对糖尿病大鼠视网膜一氧化氮(NO)及其合酶(NOS)表达的影响.方法 链脲佐菌素制备大鼠糖尿病模型,24只成年雄性SD大鼠随机分成对照组、糖尿病组、牛磺酸干预组,每组8只.牛磺酸干预组饲料添加1.2%牛磺酸.12周处死,取视网膜标本进行实验.硝酸还原酶测定NO含量,免疫组化法测定神经型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(iNOS)的表达.结果 糖尿病组与牛磺酸干预组NO含量表达为(2.138±0.260)和(1.053±0.159)μmol/L,差异有统计学意义(P<0.01);糖尿病组与牛磺酸干预组iNOS吸光度分别为(0.390±0.022)和(1.101±0.006),后者明显低于前者(P<0.01);牛磺酸干预组nNOS吸光度为(0.429±0.035),明显高于糖尿病组的(0.125±0.024)(P<0.01).结论 牛磺酸通过减少视网膜NO含量和影响iNOS、nNOS表达,改变糖尿病性视网膜病变(DR).     

Expression of NOS isoforms in internal spermatic veins of infertile men with varicocele

Although varicocele is a relatively common entity encountered in the evaluation of infertile men, the exact pathophysiology still remains unclear. Recently, as previously widely investigated in various parts of human circulatory system, nitric oxide synthase (NOS) and its product, nitric oxide (NO) have been thought to play a role in the development of varicocele and thus male infertility. In this study, we determined the concentration of NO metabolite and the expression of NOS isoforms in the internal spermatic (ISV) and superficial branch of inferior epigastric veins of infertile men with varicocele. The study included 60 infertile men with clinically unilateral or bilateral varicocele. Expression of inducible and endothelial NOS (iNOS and eNOS) isoforms were investigated in tissue arrays of internal spermatic and superficial branch of inferior epigastric veins with immunohistochemistry. NO metabolite (nitrite) levels were measured using the calorimetric method. A significantly higher expression of eNOS was observed in the varicose veins (mean score: 2.25 and 1.55, respectively; p?=?0.0001). However, statistically, there was no significant difference for expression of iNOS between varicose and control veins (p?=?0.094). The nitrite concentration and NOS expression were not found to be correlated with clinical variables (varicocele grade, maximum varicose vein diameter, and sperm concentration, motility, and morphology) (p?>?0.05). As a result, the significantly higher expression of eNOS in ISV may be responsible for the development of varicocele, although this finding is not accompanied by an increase in NO concentration. Still, the pattern of the relationship between varicocele and increased eNOS expression warrants further investigation.     

Expression of NOS isoforms in internal spermatic veins of infertile men with varicocele

Although varicocele is a relatively common entity encountered in the evaluation of infertile men, the exact pathophysiology still remains unclear. Recently, as previously widely investigated in various parts of human circulatory system, nitric oxide synthase (NOS) and its product, nitric oxide (NO) have been thought to play a role in the development of varicocele and thus male infertility. In this study, we determined the concentration of NO metabolite and the expression of NOS isoforms in the internal spermatic (ISV) and superficial branch of inferior epigastric veins of infertile men with varicocele. The study included 60 infertile men with clinically unilateral or bilateral varicocele. Expression of inducible and endothelial NOS (iNOS and eNOS) isoforms were investigated in tissue arrays of internal spermatic and superficial branch of inferior epigastric veins with immunohistochemistry. NO metabolite (nitrite) levels were measured using the calorimetric method. A significantly higher expression of eNOS was observed in the varicose veins (mean score: 2.25 and 1.55, respectively; p?=?0.0001). However, statistically, there was no significant difference for expression of iNOS between varicose and control veins (p?=?0.094). The nitrite concentration and NOS expression were not found to be correlated with clinical variables (varicocele grade, maximum varicose vein diameter, and sperm concentration, motility, and morphology) (p?>?0.05). As a result, the significantly higher expression of eNOS in ISV may be responsible for the development of varicocele, although this finding is not accompanied by an increase in NO concentration. Still, the pattern of the relationship between varicocele and increased eNOS expression warrants further investigation.     

妊娠肝内胆汁淤积症孕妇血清和脐血清NO变化及胎盘NOS的表达

目的:通过测定妊娠肝内胆汁淤积症(ICP)患者血清、脐血清一氧化氮(NO)水平及胎盘一氧化氮合酶(NOS)的表达强度,探讨NO在ICP围产儿不良结局中的作用。方法:以ICP孕妇31例为ICP组,正常孕妇30例为对照组,采用亚硝酸盐法测定母血、脐血NO水平,免疫组化检测胎盘iNOS和eNOS表达强度,对染色强度进行图像分析。结果:ICP组脐血NO水平低于对照组(P<0.01),胎盘iNOS和eNOS表达强度均显著低于对照组(分别为P<0.001和P<0.01),两组孕妇血清NO水平无显著差异(P>0.05)。结论:ICP患者胎盘绒毛组织iNOS和eNOS表达降低,可能导致胎儿-胎盘循环阻力升高,是ICP患者发生胎儿宫内窘迫和早产的原因之一。     

Nitric oxide activity in platelets of dengue haemorrhagic fever patients: the apparent paradoxical role of ADMA and l-NMMA

Dengue haemorrhagic fever (DHF) is a prevalent acute disease that occurs in patients infected by an arbovirus in tropical and subtropical regions. We have previously shown increased intraplatelet nitric oxide (NO) production in patients with dengue fever associated with reduced platelet aggregation. In this study, l-arginine transport as well as expression and activity of nitric oxide synthase (NOS) isoforms in the presence or absence of l-arginine analogues were examined in 23 DHF patients. l-arginine transport and NOS activity in platelets were increased in patients with DHF compared with controls. However, platelet endothelial NOS (eNOS) and inducible (iNOS) protein levels did not differ between healthy controls and DHF patients. Endogenous or exogenous analogues did not inhibit platelet NOS activity from DHF patients. In contrast, endogenous l-arginine analogues [N(G)-monomethyl-l-arginine (l-NMMA) and asymmetric dimethylarginine (ADMA)] inhibited NOS activity in platelets from healthy subjects. These results show the first evidence that the intraplatelet l-arginine-NO pathway is activated in DHF patients. The lack of inhibition of NO formation in vitro by all l-arginine analogues tested in DHF platelets may suggest another mechanism by which NOS activity can be regulated.     

The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS

The up-regulation of transcobalamins [hitherto posited as indicating a central need for cobalamin (Cbl) in inflammation], whose expression, like inducible nitric oxide synthase (iNOS), is Sp1- and interferondependent, together with increased intracellular formation of glutathionylcobalamin (GSCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), may be essential for the timely promotion and later selective inhibition of iNOS and concordant regulation of endothelial and neuronal NOS (eNOS/nNOS.) Cbl may ensure controlled high output of nitric oxide (NO) and its safe deployment, because: (1) Cbl is ultimately responsible for the synthesis or availability of the NOS substrates and cofactors heme, arginine, BH(4) flavin adenine dinucleotide/flavin mononucleotide (FAD/FMN) and NADPH, via the far-reaching effects of the two Cbl coenzymes, methionine synthase (MS) and methylmalonyl CoA mutase (MCoAM) in, or on, the folate, glutathione, tricarboxylic acid (TCA) and urea cycles, oxidative phosphorylation, glycolysis and the pentose phosphate pathway. Deficiency of any of theNOS substrates and cofactors results in 'uncoupled' NOS reactions, decreasedNO production and increased or excessive O(2) (-), H(2)O(2), ONOO(-) and other reactive oxygen species (ROS), reactive nitric oxide species (RNIS) leading to pathology. (2) Cbl is also the overlooked ultimate determinant of positive glutathione status, which favours the formation of more benign NO species, s-nitrosothiols, the predominant form in which NO is safely deployed. Cbl status may consequently act as a 'back-up disc' that ensures the active status of antioxidant systems, as well as reversing and modulating the effects of nitrosylation in cell signal transduction.New evidence shows that GSCbl can significantly promote iNOS/ eNOS NO synthesis in the early stages of inflammation, thus lowering high levels of tumour necrosis factor-a that normally result in pathology, while existing evidence shows that in extreme nitrosative and oxidative stress, GSCbl can regenerate the activity of enzymes important for eventual resolution, such as glucose 6 phosphate dehydrogenase, which ensures NADPH supply, lactate dehydrogenase, and more; with human clinical case studies of OHCbl for cyanide poisoning, suggesting Cbl may regenerate aconitase and cytochrome c oxidase in the TCA cycle and oxidative phosphorylation. Thus, Cbl may simultaneously promote a strong inflammatory response and the means to resolve it.     

硫酸镍对雌性大鼠卵巢的毒性作用及其机制

选健康性成熟Wistar雌性大鼠,每次以5．00mg/kg、2．50mg/kg、1．25mg/kg硫酸镍(NiSO4)腹腔注射染毒,0．2ml／100g动物体重,1次／d,连续21d。于最后一次染毒结束次日心脏采血放免法测雌二醇(E2)、孕酮(P),同时测定卵巢NOS酶活力、NO含量及卵巢和子宫镍含量。结果 染毒各剂量组动物子宫、卵巢镍含量及卵巢NOS酶活力与对照组比较差异有显著性;5．00mg/kg、2．50mg/kg染毒组血清E2、P含量,卵巢NO含量与对照组比较差异有显著性。提示腹腔NiSO4染毒引起雌性大鼠卵巢损伤,血清E2、P下降,其机制可能与卵巢镍含量升高诱导NOS、NO水平增高有关。     

增强型体外反搏对心脏骤停犬血清一氧化氮系统的影响

目的观察探讨增强型体外反搏(EECP)对心脏骤停(CA)犬血清一氧化氮系统的影响。 方法将16只雄性杂种犬(体重10～15 kg,犬龄18～30个月)根据随机数字表法随机分为EECP组和对照组,每组8只。采用心外膜电致颤诱发CA,经心肺复苏(CPR)至自主循环恢复(ROSC)后,EECP组行连续EECP干预3 h,对照组不予EECP干预。于自主循环恢复(ROSC)后15 min、4 h和96 h采静脉血,用酶联免疫吸附剂测定(ELISA)法测血清一氧化氮(NO)、内皮型一氧化氮合酶(eNOS)、神经元型一氧化氮合酶(nNOS)、诱导型一氧化氮合酶(iNOS)的浓度变化。 结果EECP组在ROSC后4 h、96 h的血清NO、eNOS浓度高于对照组[ROSC后4 h：(34.41±10.01) nmol/ mL>(19.65±5.84) nmol/ mL,(63.86±17.54 ) nmol/mL>(41.01±8.86) nmol/mL,P<0.01;ROSC后96 h：(40.64±12.94) nmol/ mL>(25.80±6.04) nmol/ mL,(47.43±19.40) nmol/ mL>(27.73±11.41) nmol/ mL,P<0.05]; EECP组在ROSC后4 h、96 h的血清iNOS浓度分别为(73.11±32.82) U/ mL和(56.36±33.30) U/ mL,低于对照组的(129.88±45.49) U/ mL和(110.38±53.69) U/ mL(P均<0.05)。 结论EECP可升高CA-ROSC犬血清NO浓度,可能与上调血清eNOS水平并下调血清iNOS水平有关,利于CA后脑复苏。     

牛磺酸锌对染铅大鼠海马NOS活性和nNOS蛋白及基因表达的影响

目的:探讨不同剂量牛磺酸锌(TZC)拮抗铅对大鼠海马一氧化氮合酶(NOS)活性和神经元型一氧化氮合酶(nNOS)蛋白及基因表达的损害。方法:用NADPH-黄递酶(NADPH-d)组化、ABC免疫组化和半定量逆转录-聚合酶链式反应(RT-PCR)法,研究饮用含0.2 g/L醋酸铅(PbAc)饮水和含不同剂量(5.9、17.7g/kg)TZC饲料喂养的大鼠海马NOS活性、nNOS阳性神经元数目以及nNOS基因表达的变化。结果:与对照组大鼠海马CA1区NOS、nNOS阳性神经元数(56.80±6.69,60.67±13.48)和海马nNOS mRNA相对含量(0.454±0.045)相比,染铅组大鼠海马CA1区NOS、nNOS阳性神经元数(42.60±6.1 7,46.67±9.04)和海马nNOS mRNA相对含量(0.071±0.025)明显减少(P<0.05)。而铅加5.9 g/kg TZC 组大鼠海马CA1区NOS、nNOS阳性神经元数(61.60±7.30,56.87±6.64)和海马nNOSmRNA相对含量(0.412±0.061)均较染铅组明显增加(P<0.05)。铅加牛磺酸(Tau)组和铅加17.7 g/kgTZC组海马nNOS mRNA相对含量(0.138±0.026和0.137±0.019)较染铅组明显增加(P<0.05)。各组大鼠海马齿状回NOS和nNOS阳性细胞数的变化与CA1区变化基本一致,而CA3区无明显差别。结论:5.9 g/kg TZC能明显增强慢性染铅大鼠海马NOS活性和nNOS蛋白及基因表达。     

苦瓜对胰岛素抵抗鼠肾组织中一氧化氮合酶的影响

目的　研究苦瓜对胰岛素抵抗大鼠 (IR)肾组织细胞中一氧化氮合酶 (NOS)的变化 ,探讨苦瓜治疗胰岛素抵抗的作用机制。方法　将 IR大鼠随机分成 IR+苦瓜组和 IR+蒸馏水组。分别用苦瓜汁和蒸馏水每天灌胃 1次 ,连续 7天 ,然后处死动物 ,测试总 NOS及诱生型一氧化氮合酶 (i NOS)活性。结果　胰岛素抵抗大鼠经苦瓜灌胃后其总 NOS和 i NOS与试验对照组比较 ,差异无显著性 (P>0 .0 5 ) ,经苦瓜灌胃后的 IR大鼠与正常大鼠比较 ,总 NOS和 i NOS则明显增高 (P<0 .0 1)。结论　苦瓜对 IR大鼠肾组织中总 NOS或 i NOS的影响不明显 ,其机理有待进一步研究。     

Total antioxidant status in the blood serum of rats exposed to N-nitroso compounds and nitric oxide synthase inhibitors

In this study, the total antioxidant status (TAS) was assayed in the blood serum of rats pretreated per os with either N-nitrosodiethylamine (NDEA) (0.1 mg/kg b.w./day) or N-methyl-N-nitrosourea (NMU) (0.1 mg/kg b.w./day) for 30 days. The animals were also dosed per os with spermidine (SPR) (10 mg/kg b.w./day) for a first 21 day period, and Nomega)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg b.w./day) given to animals for 3 days (days 22-24), respectively. Nitric oxide synthase (NOS) inhibitor, L-NAME was found to mitigate TAS levels in the blood serum of rats pretreated with NDEA and NMU. No such changes were found in animals dosed with L-NAME only nor even with L-NAME and spermidine, respectively. Since spermidine, also known as an inhibitor of iNOS synthesis, elevated TAS levels in rats dosed with L-NAME and NDEA/NMU, the polyamine was suggested to modify the NOS/NO origin to serve the physiological level of the total anti-oxidant status in rat blood serum.     

锌对急性缺氧小鼠大脑皮层NOS活力和nNOS蛋白表达的影响

目的观察锌对急性缺氧小鼠大脑皮层一氧化氮合酶 (NOS)和神经元型一氧化氮合酶 (nNOS)阳性神经元的变化 ,探讨锌抗脑缺氧的作用机制。方法制备小鼠急性缺氧模型 ,采用NADPH -d组织化学和nNOS免疫组织化学方法 ,研究给锌组和不给锌组急性缺氧小鼠大脑皮层NOS和nNOS阳性神经元数量的变化。结果给锌组比不给锌组小鼠的耐缺氧时间延长 33.0 6 %(P <0 .0 5 ) ,大脑皮层NOS和nNOS阳性神经元数量分别减少 18.0 3%和 2 1.6 3% ,(P <0 .0 5 )。结论急性缺氧时锌通过减少皮层NOS活力和nNOS水平而发挥其抗脑缺氧作用。     

染矽尘大鼠肺组织一氧化氮合酶的表达

目的　研究染矽尘大鼠早期炎性肺损伤过程中一氧化氮合酶 (NOS)的表达规律 ,为矽肺纤维化机制提供理论依据。方法　气管暴露法建立矽肺动物模型。测定支气管肺泡灌洗液 (BALF)中诱导型NOS(iNOS)的表达和总NOS的活力。在组织芯片上使用SP法检测大鼠肺组织iNOS的表达 ,并用Image ProPlus图像分析法对iNOS的表达进行定量测定。结果　iNOS主要表达在巨噬细胞和中性粒细胞胞浆内。与对照组相比 ,染矽尘大鼠肺组织中iNOS积分光密度于染尘后 3、7d时分别增加了1.47× 10 5 、2 .73× 10 5 ,2 8d时降低了 1.11× 10 5 ,差异均有显著性 (P <0 .0 5或P <0 .0 1)。大鼠BALF中iNOS活力在染尘后 3、7、14d时分别增加了 0 .86、1.89、0 .92U/ml ,差异均有显著性 (P <0 .0 5或P <0 .0 1)。BALF总NOS活力在染尘后 1、3、7、14d时分别增加了 1.43、2 .0 5、2 .61、2 .19U/ml,差异均有显著性 (P <0 .0 5或P <0 .0 1)。结论　在矽尘诱导下 ,大鼠肺组织中表达iNOS的细胞主要是肺泡巨噬细胞和中性粒细胞 ,其肺组织中iNOS的表达从染尘后 1d至 2 8d有一抛物线型的过程     

Differential modulation by vascular nitric oxide synthases of the ethanol-evoked hypotension and autonomic dysfunction in female rats

We recently reported that chronic exposure to ethanol lowers blood pressure (BP) via altering cardiac contractility and autonomic control in female rats. In this investigation we conducted pharmacological and molecular studies to elucidate the role of constitutive and inducible nitric oxide synthase (NOS) in these hemodynamic effects of ethanol. Changes caused by selective inhibition of eNOS [N⁵-(1-iminoethyl)-l-ornithine; l-NIO], nNOS (N^ω-propyl-l-arginine; NPLA), or iNOS (1400W) in BP, heart rate (HR), myocardial contractility index (dP/dt_max), and power spectral indices of hemodynamic variability were evaluated in telemetered female rats receiving ethanol (5%, w/v) or control liquid diet for 8 weeks. Ethanol increased plasma nitrite/nitrate (NOx) and enhanced the phosphorylation of eNOS and nNOS, but not iNOS, in the tail artery. Ethanol also reduced BP, +dP/dt_max, low-frequency bands of interbeat intervals (IBI_LF, 0.25–0.75 Hz) and IBI_LF/HF ratio while high-frequency bands (IBI_HF, 0.75–3 Hz) were increased, suggesting parasympathetic overactivity. l-NIO (20 mg/kg i.p.) caused greater increases in BP in control than in ethanol-fed rats but elicited similar reductions in IBI_LF/HF and +dP/dt_max both groups. NPLA (1 mg/kg i.p.) caused minimal effects in control rats but exacerbated the reductions in BP, +dP/dt_max, and IBI_LF/HF in ethanol-fed rats. No hemodynamic modifications were caused by 1400W (5 mg/kg i.p.) in either rat group. Together, these findings suggest that nNOS acts tonically to offset the detrimental cardiovascular actions of ethanol in female rats, and the enhanced vascular NO bioavailability may explain the blunted l-NIO evoked pressor response in ethanol-fed rats.     

酒精对大鼠胚胎发育及脑nNOS表达的影响

采用整体动物模型观察器官形成期酒精暴露对宫内胚胎发育影响,结合酶组织化学和免疫组织化学技术,探讨酒精对脑组织中海马区NOS(nirtic oxide synthase活性和nNOS(neuronal nitricoxide synthase)表达影响.结果显示10g/kg/d组即可出现FAS形态特征;酒精能导致脑海马区NOS活性和nNOS表达增强,提示NO产量增高.结果说明酒精具有发育毒性,酒精引起胚胎脑发育异常可能与酒精引起的脑海马区NO增加有关.