家兔动脉粥样硬化进程中内皮依赖性因子的变化及其相互关系研究

目的　探讨血红素氧合酶-1(HO -1) /一氧化碳(CO)系统与一氧化氮合酶(NOS) /一氧化氮(NO)在动脉粥样硬化中的变化、相互关系及对动脉粥样硬化进程的影响。方法　家兔予以高胆固醇饮食(n=8)以及在高胆固醇饮食的同时经饮水给予L -精氨酸(n=8)或亚硝基左旋精氨酸甲酯(n=8),或经腹腔注射血红素L -赖氨酸盐(n=8)或锌原卟啉9 (ZnPP- IX) (n=8 ),共10周。结果与对照组比较,胆固醇组主动脉NO生成量显著减少,CO生成则明显增加,NOS活性显著降低(P均<0 .01),而HO -1表达升高,主动脉斑块面积达(40. 2±8 .9)% 。与胆固醇组比较,外源性血红素L -赖氨酸盐干预组的主动脉内膜斑块面积[ (26. 6±9 .2)% ]明显缩小,主动脉CO的生成量和HO- 1表达明显升高(P<0. 01),但NOS活性与NO产量较正常对照组显著降低(P<0 .01),与胆固醇组比较则无显著差异;与胆固醇组比较,外源性L 精氨酸显著升高主动脉cNOS活性,增加NO生成量,主动脉斑块面积[ (28. 1±7 .7)% ]明显缩小(P均<0. 01),而HO -1的表达和CO的生成较正常对照组显著升高,与胆固醇组相比差异无统计学意义。与胆固醇组比较,血红素L 赖氨酸盐干预组、L 精氨酸组的主动脉组织内c- myc及c- fos的mRNA和蛋白表达均显著降低,而ZnPP组和亚硝基左旋精氨酸甲酯组则无明显差异。结论　动     

实验性肝硬化大鼠肺脏血红素氧合酶的表达与定位

目的 观察肝硬化大鼠肺脏中血红素氧合酶(HO)的表达与定位,探讨血红素氧合酶-内源性一氧化碳在肝硬化时肺脏病变的作用。方法 建立四氯化碳肝硬化模型,应用免疫组织化学法和免疫印迹法对肝硬化大鼠和正常对照大鼠肺脏中血红素氧合酶的表达与定位进行比较。 结果 肝硬化模型制备成功。正常对照组大鼠HO-1在肺组织的表达较低,而肝硬化组大鼠的肺组织中HO-1蛋白表达明显增高(0.062±0.021与0.185±0.044,t=11.24,P<0.01 ;0与5 294.92±46.02,t=11.45,P<0.01),主要在血管和支气管的平滑肌细胞,内皮细胞及气道上皮细胞的胞浆内,而HO-2在两组大鼠的肺组织中表达差异无显著性(0.218±0.048与0.238±0.079,t=0.99,P>0.05; 5984.17±35.64与6040.75±42.43,t=0.02,P>0.05)。 结论 血红素氧合酶-内源性一氧化碳途径可能在肝硬化肺脏病变的发生机制中起一定的作用。     

哮喘豚鼠血红素氧合酶1的表达与调节

目的　探讨血红素氧合酶１（ＨＯ１） 在哮喘中的表达与作用机制。方法　７０ 只豚鼠完全随机分为７ 组,每组１０ 只,其中两组分别应用ＨＯ１ 特异性激动剂血晶素和抑制剂锡原卟啉处理豚鼠哮喘模型,另外５ 组分别为哮喘发作前、发作、自然缓解、地塞米松预防和正常对照组,应用分光光度计和放射免疫竞争分析法等,检测血及肺组织的ＨＯ１ 活性、一氧血红蛋白（ＣＯＨｂ）、环磷酸鸟苷（ｃＧＭＰ） 、血浆ＩｇＥ 水平,观察支气管肺泡灌洗液（ＢＡＬＦ） 中细胞数、肺组织病理学和ＨＯ１ 免疫组织化学染色的变化。结果　哮喘发作组和血晶素激动组的各项指标与正常对照组比较,差异均有非常显著性（ｔ＝４－７５４～１０－１８８,Ｐ＜０－０１） ,血晶素组更为显著,如肺ＨＯ１ 活性：每小时每毫克蛋白生成胆红素量为（１４４９±４２６）ｐｍｏｌ;肺ＣＯＨｂ：每毫克蛋白含（０－８３ ±０－２９） ％ ;肺ｃＧＭＰ：每毫克蛋白含（１－９６ ±０－６５）ｐｍｏｌ;血浆ＩｇＥ：（７３±２１） ｋＵ／Ｌ;ＨＯ１ 蛋白表达≥４ 级;ＢＡＬＦ细胞总数：（２１±４） ×１０８／Ｌ。地塞米松预防组和锡原卟啉抑制组除ＣＯ 含量和肺ＨＯ１ 活性略高外,余项指标与正常对照组比较差异无     

肝硬化大鼠食管一氧化氮合酶分布定位研究

目的探讨一氧化氮（ＮＯ）在肝硬化食管静脉曲张及血流动力学改变中的作用。方法应用还原型辅酶Ⅱ－黄递酶（ＮＡＤＰＨ－ｄ）组化染色观察ＣＣＩ４所致肝硬化大鼠食管下段一氧化氮合酶（ＮＯＳ）分布；应用荧光法检测大鼠食管组织匀浆及血清中ＮＯ含量；应用５７Ｃｏ标记微球测定大鼠血流动力学指标。结果肝硬化鼠食管粘膜及粘膜下血管内皮ＮＯＳ呈强阳性反应，而正常鼠则呈弱阳性或阴性反应；肝硬化大鼠食管及血清中ＮＯ含量显著升高。且全部出现门脉高压和高动力循环状态。结论ＮＯ在肝硬化大鼠食管静脉曲张及血流动力学改变中可能起重要作用。     

Temporal expression of hepatic inducible nitric oxide synthase in liver cirrhosis

AIM: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. We have found inducible nitric oxide synthase (iNOS) can be induced in hepatocytes of cirrhotic liver. This study further investigated the temporal expression and activity of hepatic iNOS in cirrhosis development. METHODS: Cirrhosis was induced in rats by chronic bile duet ligatjon (BDL). At different time points after the operation, samples were collected to examine NO concentration, liver function, and morphological changes. Hepatocytes were isolated for determination of iNOS mRNA, protein and enzymatic activity. RESULTS: Histological examination showed early cirrhosis 1-2 wk after BDL, with advanced cirrhosis at 3-4 wk. Bilirubin increased dramatically 3 d after BDL, but decreased by 47% on d 14. Three weeks after BDL, it elevated again. Systemic NO concentration did not increase significantly until 4 wk after BDL, when ascites developed. Hepatocyte iNOS mRNA expression was identified 3 d after BDL, and enhanced with time to 3 wk, but reduced thereafter. iNOS protein showed a similar pattern to mRNA expression. iNOS activity decreased from d 3 to d 7, but increased again thereafter till d 21. CONCLUSION: Hepatic iNOS can be induced in the early stage, which increases with time as cirrhosis develops. lts enzymatic activity is significantly correlated with protein expression and histological alterations of the liver, but not with systemic NO levels, nor with absolute values of liver function markers.     

肝硬化大鼠全胃肠外营养时一氧化氮和一氧化氮合酶活性的变化及意义

目的 观察一氧化氮（NO）、一氧化氮合酶（NOS）活性在肝硬化大鼠全胃肠外营养（TPN）时的变化,并探讨其意义。方法 Wistar大鼠分为3组：正常组8只,肝硬化对照组6只,肝硬化TPN组7只。测定血清NO浓度、肝脏NO含量和肝脏NOS比活性,观察肝脏β－BADPH黄递酶组化染色。结果 除TPN组一只大鼠因输液过快,肺水肿死亡外,其余大鼠均成活。血清NO浓度、肝脏NOS比活性,肝硬化对照组比正常组升高明显（P＜0．05）,肝硬化TPN级比肝硬化对照组升高明显（P＜0．05）。肝脏NO含量,肝硬化对照组比对照组升高明显（P＜0．05）,但肝硬化TPN组比肝硬化对照组降低明显（P＜0．05）。肝脏β－NADPH黄递酶组化染色,正常组为－,肝硬化对照组为＋,肝硬化TPN组为＋＋。结论 NO可能是一种抗肝损伤因子,损伤因子存在时,表现为肝脏NOS活性增强,当肝脏NO含量减少时,表现为肝脏损害的加重。     

Nitric oxide synthase distribution in esophageal mucosa and hemodynamic changes in rats with cirrhosis

SubjectheadingsesOPhagUs;nitricoxidesynthase;livercirrhosis;hemodynamicsINTRODUCTIONCirrhosiswithportalhypertensionisassociatedwithhyperdynamiccirculationcharacterizedbygeneralizedvasodilationandincreasedcardiacoutputandsplanchnicregionalbloodflows.EndogenousNO,averypotentvasodilatorfactor,mayplayaveryimportantroleinthepathogenesisofhemodynamicchangesincirrhosis.ItisnuclearwhetherNOisinvolvedinthepathogenesisofesophagealvaricesasoneofseverecomplicationsofhepaticcirrhosis.Thepresentstud…     

Increased heme oxygenase activity in splanchnic organs from portal hypertensive rats: role in modulating mesenteric vascular reactivity

BACKGROUND/AIMS: We have recently demonstrated that heme oxygenase-1 is upregulated in splanchnic organs of portal hypertensive rats. In the present study, we assessed whether heme oxygenase enzymatic activity is increased in splanchnic organs of portal hypertensive rats, and the relative contribution of heme oxygenase and nitric oxide synthase to the vascular hyporeactivity in portal hypertension. METHODS: Heme oxygenase activity was measured in splanchnic organs of portal hypertensive and sham-operated rats. The effects of heme oxygenase and nitric oxide synthase inhibition on pressure responses to potassium chloride and methoxamine were assessed in perfused mesenteric vascular beds of portal hypertensive and sham-operated rats. RESULTS: Heme oxygenase activity was increased in the mesentery, intestine, liver, and spleen of portal hypertensive rats. The hyporeactivity to potassium chloride in portal hypertensive rats was overcome after simultaneous inhibition of both heme oxygenase and nitric oxide synthase, but only partially attenuated after nitric oxide synthase inhibition alone. The hyporeactivity to methoxamine was completely reversed after nitric oxide synthase blockade. CONCLUSIONS: These results demonstrate that heme oxygenase activity is increased in splanchnic organs of portal hypertensive rats. They also suggest that heme oxygenase contributes to the hyporeactivity to potassium chloride, but not to methoxamine, in portal hypertensive rats.     

慢传输型便秘大鼠结肠诱导型一氧化氮合酶和血红素氧合酶2的变化

目的 探讨慢传输型便秘(STC)大鼠结肠内诱导型一氧化氮合酶(iNOS)和血红素氧合酶2(HO-2)的变化.方法 健康Wistar大鼠32只,随机分为STC组和对照组,每组16只.采用复方苯乙哌啶灌胃法制备STC大鼠模型,饲养100 d后,采用活性炭灌胃法测定肠道传输速度确定模型建立.用免疫组织化学方法分别检测iNOS和HO-2在大鼠结肠的表达情况.结果 STC组大鼠日均粪便粒数、日均粪便干重、日均粪便质量均比对照组明显减少;检测大鼠肠道传输速度,STC组较对照组明显减慢,首粒黑便排出时间较对照组显著延长.iNOS和HO-2在STC大鼠结肠的表达明显强于对照组.结论 iNOS和HO-2在STC大鼠结肠的表达异常,表明iNOS和HO-2在慢传输型便秘发病机制中可能起着重要的作用.     

肝病患者一氧化氮合酶表达异常及其临床意义

目的 探讨肝病患者抗氧化能力及一氧化氮合酶（NOS） 表达的临床价值。方法 收集26例急性肝炎（AH）、36例慢性肝炎（CH）、13例肝炎后肝硬化（LC）和23例原发性肝癌（HCC）患者的血清,分别检测其总抗氧化能力（TAO）、NOS浓度及一氧化氮（NO）水平,分析它们在肝脏疾病中的改变机制。结果 患者血清TAO在AH、CH中异常率为80％,在LC和HCC为50％;血清中NOS活性在肝病患者中的异常率在70％ 左右;NO水平在AH、CH和LC中异常率为70％,HCC组为48％。AH组、CH组TAO平均水平明显高于对照组,但LC和HCC组的差异不明显;肝病患者NO和NOS平均浓度显著高于对照组,但HCC患者NOS和NO平均浓度均低于AH、CH和LC组患者。结论 肝病患者血清NOS水平与NO浓度呈高度正相关,NO增加可能 对肝细胞起保护作用。     

病毒性肝炎患者血浆一氧化氮和内皮素水平及意义

各型病毒性肝炎及活动性肝硬化患者共２１３例,采用镉柱还原法及重氮化法检测ＮＯ＾－２／ＮＯ＾－３水平,放免法检测了ＥＴ－１水平。急性肝为,慢性活动型肝炎,肝炎肝硬化等各型肝病患者血清ＮＯ＾－２／ＮＯ＾－３及ＥＴ－１水平均有不同程度的升高,肝炎急性期ＮＯ就有明显升高,慢性肝病ＮＯ与ＥＴ随病情进展水平升高,二者水平良好的相关性。     

血红素氧合酶与肝脏疾病

血红素氧合酶-一氧化碳(HO-CO)系统可在应激状态下激活,有抗氧自由基、抗炎性介质损伤,舒张血管平滑肌等作用,在人体各系统中发挥重要作用.此文就血红素氧合酶在肝病中的研究进行综述.     

诱导型和内皮型一氧化氮合酶基因多态性与肝硬化门静脉高压的相关性

目的探讨诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)基因多态性与肝硬化门静脉高压的相关性。方法采用病例对照和聚合酶链反应-限制性片段长度多态性技术,检测106 例乙型肝炎后肝硬化患者(其中门静脉高压症65例)和108名健康对照者iNOS基因启动子-969G→C多态性及eNOS基因第七外显子894G→多态性,比较等位基因及基因型频率,并进行综合分析。结果在iNOS启动子969G→C多态性中,门静脉高压症组C等位基因和GC基因型频率比对照组显著升高(x2- 5.93,P<0.05)。GC基因型启动子的活性比GG基因型活性强。在eNOS的894G→T多态性中,T等位基因频率明显高于对照组(x2-3.91,P<0.05)。采用Logistic多元回归显示iNOS基因启动子-969G→C多态性及cNOS的894G→T多态性是门静脉高压症新的危险因素。结论iNOS启动子969G→C多态性和eNOS的894G→T多态性与门静脉高压症相关,是形成门静脉高压症新的危险因素。iNOS启动子-969G→C多态性可导致该基因启动子功能活性增强。     

内皮细胞型一氧化氮合酶基因多态性与糖尿病肾病的相关性研究

目的：探讨内皮细胞型一氧化氮合酶（eNOS)基因第7外显子894G→T点突变,及其第4内含子的1个27bp的插入／缺失（a/b)多态性,与2型糖尿病肾病（DN）之间的关系。方法：894G→T点突变采用聚合酶链反应限制性片段长度多态性（PCR－RFLP）技术,27bp的a/b多态性采用聚合酶链反应结合4％琼脂糖凝胶电泳分离技术。比较各组间的等位基因频率与基因型频率。结果：（1）早期糖尿病肾病组（DN^ 组）T等位基因及TG基因型频率显著高于糖尿病非肾病患者（DN^-组,P＜0．05）。（2）DN^ 组a等位基因及ab基因型频率显著高于DN^-组（P＜0．05）。（3）DN^ 组的TGab基因型频率亦显著高于DN^-组（P＜0．05）。（4）糖基化血红蛋白（GHbA1c),收缩压（SBP）,总胆固醇（TC）,eNOS基因第7外显子894G→T基因点突变及第4内含子a/b多态性均属糖尿病肾病的独立危险因素。结论：糖尿病患者eNOS基因第7外显子T等位基因及第4内含子a等位基因与DN^ 的发生密切相关,两种等位基因同时存在者,DN^ 发病风险更高。     

Nitric oxide synthase activity in rat cardiac mitochondria

Abstract. Recent publications shown mitochondrial localization of the enzyme nitric oxide synthase (NOS) in a number of tissues. However, conflicting results about mitochondrial NOS (mtNOS) immunoreactivity and enzymatic activity are available to date in the literature. In this study we purified mitochondria from rat hearts and analysed these preparations for NOS immunoreactivity and activity, showing the presence of either a constitutive (the endothelial isoform) and an inducible NOS immunoreactivity. A basal NOS activity (64.2 ± 5.1 pmol/mg protein/30 min) was detectable. 1 mM N^G-Monomethyl-L-arginine (L-NMMA), a competitive inhibitor of all NOS isoforms, caused a drop in NOS activity to 33.8 ± 1.9 pmol/mg protein/30 min. Simultaneous administration of 10 µM (S)-2-amino-(1-iminoethylamino)-5- thiopentanoic acid (GW274150), a specific NOS2 inhibitor, together with removal of Ca²⁺ and calmodulin (CaM) from the assay buffers, known to interfere with the activity of constitutive NOS isoforms, caused a reduction in NOS activity (17.4 ± 1.2 pmol/mg protein/30 min). 10 µM GW274150 reduced NOS activity to 41.6 ± 4 pmol/mg protein/30 min, while Ca²⁺/CaM withdrawal reduced basal NOS activity to 45.8 ± 5 pmol/mg protein/30 min. This dual mtNOS machinery is suggested to be involved in modulating cardiac O₂ consumption in different (patho)physiological conditions.     

Analysis of two constitutive forms of microsomal heme oxygenase in different rat tissues

ＡｎａｌｙｓｉｓｏｆｔｗｏｃｏｎｓｔｉｔｕｔｉｖｅｆｏｒｍｓｏｆｍｉｃｒｏｓｏｍａｌｈｅｍｅｏｘｙｇｅｎａｓｅｉｎｄｉｆｅｒｅｎｔｒａｔｔｉｓｓｕｅｓＸＩＡＺｈｅｎＷｅｉ１,ＬＩＹｕｎＺｈｕ１,ＣＨＥＮＳｈｕｎＮｉａｎ１,ＳＨＥＮＱｉｎｇＸ...     

肝硬化大鼠内脏血管一氧化氮合酶基因表达及活性的动静脉差异

目的观察肝硬化内脏血管一氧化氮合酶(NOS)表达及活性的动静脉差异在门静脉高压形成机制中的意义.方法以四氯化碳皮下注射制备大鼠门静脉高压模型,应用免疫组织化学、化学发光以及RT-PCR分别检测大鼠门静脉(PV)和肠系膜动脉(MA)组织NOS的分布、活性及基因表达.结果肝硬化组大鼠PV和MA血管各层均有iNOS分布,而eNOS则局限于内皮层.肝硬化大鼠内脏血管NOS活性[pmol.min-1.mg-1.蛋白(PV 23.82±2.48,MA 43.46±4.93)]及mRNA表达均较对照组(PV 16.48±1.54,MA 16 95±2.34)显著升高(P＜0.05与0.01);同时肝硬化大鼠MA的总NOS活性和eNOS活性及eNOS mRNA表达均显著高于PV(P＜0.01).结论内脏血管eNOS亚型活性及表达增加可能在肝硬化时NO产生增多中起主要作用,而NOS活性及表达在MA与PV之间的动静脉差异,可能是NO参与门脉高压形成的重要机制之一.     

Expression of nitric oxide synthase protein and gene in the splanchnic organsof liver cirrhosis and portal hypertensive rats

AIM To investigate the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS)protein and eNOS mRNA gene in the splanchnic organs of liver cirrhosis and portal hypertensive rats.METHODS In control and CCl4-induced liver cirrhotic rats, the expression of eNOS and iNOS proteins wasdetected by immunohistochemical method, and eNOS mRNA was detected by in situ hybridization.RESULTS The expression of eNOS protein and eNOS mRNA increased in most organs of the cirrhotic rats,including bronchial and alveolar epithelial cells, renal tubular epithelial cells and mesenchyma, endothelialand adventitial cells of aorta and superior mesenteric artery, whereas no significant increase of iNOS proteinwas found. In the hepatic tissue, NOS protein and eNOS mRNA were present in mesenchymal cells and vesseladventitial cells, no difference was observed in the expression between control and cirrhotic rats.CONCLUSION The expression of NOS varied in region. In splanchnic organs and vasculars there was anincreased expression of eNOS which induced aplanchnic vasodilation and increased the inflow of portal vein,while in the liver tissue and blood vessel showed no increased expression, which may be associated withincreased intrahepatic vascular resistance.     

Vascular effects of a heme oxygenase inhibitor are enhanced in the absence of nitric oxide

BACKGROUND: Vascular endothelium and smooth muscle express heme oxygenase (HO) that metabolizes heme to biliverdin, iron and carbon monoxide (CO). Carbon monoxide promotes endothelium-independent vasodilation, but also inhibits nitric oxide formation. This study examines the hypothesis that an inhibitor of HO promotes endothelium-independent vasoconstriction, which is attenuated in the presence of unabated nitric oxide formation. METHODS: In vivo studies were conducted in anesthetized male Sprague-Dawley (SD) rats instrumented with flow probes and arterial catheters. In vitro experiments were performed on pressurized first-order gracilis muscle arterioles isolated from male SD rats superfused with oxygenated modified Krebs buffer. RESULTS: Vascular smooth muscle and endothelium showed positive HO-1 and HO-2 immunostaining. In anesthetized rats the HO inhibitor chromium mesoporphyrin (CrMP; 45 micromol/kg intraperitoneally) had minimal effect on hindlimb resistance. However, in animals pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 mg/kg intraperitoneally), CrMP substantially increased hindlimb resistance. In contrast, in rats infused with phenylephrine to increase blood pressure and vascular tone, CrMP had no effect on hindlimb resistance. In isolated arterioles denuded of endothelium, CrMP (15 micromol/L) caused a powerful vasoconstriction, which was abolished in the presence of a functional endothelium. In arterioles with intact endothelium pretreated with L-NAME (1 mmol/L), or with L-NAME and sodium nitroprusside (10 to 30 nmol/L), CrMP promoted a similarly powerful vasoconstriction as in vessels denuded of endothelium. CONCLUSIONS: These results suggest that smooth muscle-derived CO may contribute to endothelium-independent regulation of vascular tone by providing a vasodilatory influence. Furthermore, the dilatory effects of endogenous CO are offset by a unique interaction between the CO and nitric oxide systems.     

内源性H2S对大鼠实验性肝硬化门脉高压的影响

目的:研究新型气体信号分子硫化氢(hydrogensulfide,H<,2>S)对大鼠肝硬化门脉高压的影响及其调节机制.方法:对照组(8只),肝硬化(Cirrhosis,C)组(8只),C+NaHS(C+S)组(8只),C+左旋硝基精氨酸甲酯(L-NAME+锌原卟啉(ZnPP)(C+L+Zn)组(8只),C+NaHS+...