血管活性物质在大鼠肝肺综合征发病机制中的作用

目的:探讨内皮素-1(endothelin-1,ET-1)、一氧化氮(nitric oxide,NO)、降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在大鼠肝肺综合征(hepatopulmonary syndrome,HPS) 发病机制中的作用.方法:健康Wistar大鼠32只随机平均分为假手术组、CBDL术后3 wk组、4 wk组、5 wk组.采用胆总管结扎(common bile duct ligation, CBDL)术制备大鼠HPS模型成功后,行肝功能及肝肺病理检查.放射免疫分析法检测大鼠血浆和肝组织、肺组织匀浆中ET-1和CGRP的水平,利用硝酸还原酶法检测大鼠血清和肝、肺组织匀浆中NO的水平.结果:在大鼠HPS形成过程中,肝细胞变性、坏死,大量纤维组织增生,形成假小叶.肺泡毛细血管增生、扩张.伴肺泡间隔增宽、容量减小.术后3-5 wk,CBDL组大鼠血浆和肝、肺组织匀浆中ET-1、NO、CGRP水平显著升高,且与ALT水平呈正相关(血浆,ET-1:r=0.9889,P=0.0111:NO:r-=0.9935,P=0.0065;CGRP:r=0.9714.P=0.0286;肝组织:r=0.9969,P=0.0035;r=0.9993,P=0.0070;r=0.9507,P=0.0493;肺组织:r=0.9939,P=0.0061;r=0.9991,P=0.0009;r=0.9557,P=0.0443).结论:在大鼠HPS形成过程中,血浆和肝、肺组织匀浆中ET-1,NO和CGRP水平持续升高,提示血管活性物质ET-1,NO和CGRP在其发病机制中可能起着一定作用.     

肾上腺髓质素和内皮素-1在大鼠肝肺综合征发病机制中的作用

[目的]探讨肝肺综合征(HPS)的发病机制.[方法]采用胆总管结扎(CBDL)术制备大鼠HPS模型,观察肺组织肾上腺髓质素(ADM)、内皮素-1(ET-1)及其受体(ETRA和ETRB)的表达和分布.[结果]在大鼠HPS形成过程中,血浆和肺组织中ADM、ET-1水平动态升高,且与肺泡-动脉氧分压差(A-aDO2)正相关;HPS大鼠肺组织中ADM、内皮素前体原(ppET-1 mRNA)的表达较假手术组明显增强,差异均有统计学意义(P＜0.05).HPS大鼠肺血管ETRA的分布及染色强度与假手术组比较无明显变化,而ETRB在远端肺小动脉和小静脉内膜上表达明显增强.图像分析结果显示CBDL 5周(w)组大鼠ETRA染色面积、平均积分光密度值与假手术组比较差异无统计学意义(P＞0.05),而CBDL 5 w组大鼠ETRB染色面积和平均积分光密度值明显高于假手术组,差异均有统计学意义(P＜0.05).[结论]扩血管物质ADM和缩血管物质ET-1的共同作用可能参与HPS的发生,肺组织中升高的ET-1可能更多地通过与在肺血管表达增强的ETRB结合从而扩张肺血管.     

肝肺综合征大鼠肺组织内皮素-1及其受体基因的表达

目的探讨内皮素-1及其受体在大鼠肝肺综合征(HPS)发病机制中的作用。方法分别采用RT-PCR和原位杂交方法检测HPS大鼠肺组织内皮素前体原(ppET-1)mRNA水平及两种亚型内皮素-1(ET-1)受体ETRA和ETRB在HPS大鼠肺组织的分布和表达。结果HPS大鼠肺组织ppET-1mRNA表达显著升高(0.48±0.06),为假手术(Sham)组表达量(0.21±0.04)的2.3倍(t=2.4114,P<0.05)。HPS大鼠肺血管ETRA的分布及染色强度与Sham组比较无明显变化,染色面积和平均积分光密度值与Sham组比较差异无统计学意义(P>0.05),而ETRB在远端肺小动脉和小静脉内膜上表达明显增强,尤以静脉明显。ETRB染色面积和平均积分光密度值明显高于Sham组(P<0.001)。结论HPS大鼠肺组织ET-1基因表达水平升高。ET-1在HPS大鼠肺血管的总体反应可能由于ETRB表达的增高而表现为扩张。     

肝肺综合征大鼠肾上腺髓质素及其受体的表达

目的 探讨肾上腺髓质素(ADM)在肝肺综合征(HPS)发病机制中的作用。方法 采用Wistar大鼠行胆总管结扎术(CBDL)制备HPS动物模型，进行血气分析，使用放射免疫方法检测血浆和肺组织匀浆中ADM水平；采用RT-PCR方法检测肺组织中ADM mRNA及其特异性受体Ll mRNA的表达。结果(1)从术后第3周至第5周，CBDL组大鼠血浆和肺组织中ADM水平动态升高；(2)CBDL 5周组大鼠肺组织中ADM mRNA的表达较对照组升高，差异有显著性(P＜0．05)，而两组间Ll mRNA的表达差异无显著性(P＞0．05)；(3)相关分析表明，各阶段血浆和肺组织ADM水平均与天冬氨酸转氨酶(AST)、门静脉压力、肺泡-动脉氧分压差(A-aDO2)呈正相关，出现腹水者血浆和肺组织ADM水平高于未出现腹水者。结论 在HPS形成过程中，血浆和肺组织中扩血管物质ADM水平持续升高，与肝功能受损状态、门脉压力升高程度、腹水形成及PaO2降低有关，提示ADM增多可能参与HPS时肺内血管扩张(IPVD)的发生。     

ACEI类药物对糖尿病大鼠脂质过氧化、内皮素-1及血脂的影响

目的探讨血管紧张素转化酶抑制剂(ACEI)洛汀新、依那普利和卡托普利治疗对糖尿病大鼠脂质过氧化、NO、内皮素-1(ET-1)、TC、TG的影响。方法采用腹腔注射四氧嘧啶方法制备糖尿病大鼠模型,然后经腹腔注射ACEI类药物,剂量为5 mg/kg,1次/d,10周后取血测定TC、TG、NO和ET-1水平。用硫代巴比妥酸法测定肝组织匀浆中丙二醛(MDA)的含量,用比色法测定肝组织匀浆中超氧化物歧化酶(SOD)的水平。结果糖尿病模型组大鼠血浆中的TG、TC水平高于正常对照组(P<0.01);经ACEI类药物治疗后糖尿病大鼠血浆TG、TC水平低于糖尿病模型组(P<0.05)。糖尿病模型组较正常对照组肝脏组织MDA活性提高,SOD含量降低(P<0.05);与糖尿病模型组比较,经ACEI类药物治疗后可降低肝组织中MDA含量,升高SOD活性。糖尿病模型组大鼠血浆NO、ET-1水平低于正常对照组(P<0.05),经ACEI类药物治疗后糖尿病大鼠血浆NO、ET-1水平升高(P<0.05)。结论通过ACEI类药物治疗后的糖尿病大鼠降低了TG、TC水平和MDA活性,升高了NO、ET-1水平和SOD含量。     

高压氧治疗肝肺综合征大鼠的实验研究

目的 研究高压氧治疗对大鼠肝肺综合征(HPS)的作用.方法 采用胆总管结扎术(CBDL)制备HIS动物模型.44只雄性SD大鼠随机分为4组:假手术对照组(Sham组)8只、单纯造模组(CBDL5w组)12只、治疗1周组(HBOT1w组)12只、治疗2周组(HBOT2w组)12只.两治疗组在造模成功后送入高压氧舱治疗,分别治疗1周和2周.Sham组和CBDL5w组大鼠在术后5周,HBOT1w组和HBOT2w组在治疗结束后,进行血气分析、肝功能以及血清一氧化氮(NO)、内皮素-1(ET-1)浓度检测,肝、肺病理检查,并使用激光多普勒血流仪检测肝、肺微循环.结果 CBDL5w组大鼠肝功能检测中ALB降低,ALT、AST、ALP、γ-GT、TBIL及TBA均升高,血气分析中PaO2降低,PA-aO2增大,与Sham组相比均有显著差异(P＜0.01),且病理检查CBDL5w组大鼠的肝脏组织可见胆汁性肝纤维化表现,肺组织可见肺毛细血管扩张、充血,证实HPS大鼠模型制成;同时与Sham组相比,CBDL5w组大鼠血清NO及ET-1浓度均升高(P＜0.01),肝脏毛细血管血流量降低(P＜0.01),肺脏毛细血管血流量增大(P＜0.01).经高压氧治疗后,HBOT1w组大鼠与CBDL5w组相比,以上指标均好转(P＜0.01).HBOT2w组大鼠与CBDL5w组相比,除血清NO水平和肺脏毛细血管血流量无统计学差异(P＞0.05)外,其余指标的差异有显著性意义(P＜0.01).结论 高压氧治疗大鼠HPS可通过降低血清NO、ET-1浓度,提高组织氧含量及改善肝、肺微循环等,有效改善肝、肺功能及病理,且治疗前期效果更好.     

内皮素系统与门静脉高压症的病理生理和临床治疗

门静脉高压症(portal hypertension.PHT)是一种常见的临床综合征,且引起很多严重并发症.人体和动物实验都证实PHT时血浆和肝组织中内皮素-1(ET-1)浓度显著升高.ET-1在某种组织表现出的作用取决于在该组织ET-1受体的分布情况,而在PHT的不同阶段,即使同一组织内皮素受体的分布也不相同.ET-1参与PHT时多种病变,因此以内皮素系统作为靶点可能是治疗PHT的一项新的策略.     

纳络酮对油酸型ARDS大鼠的治疗作用

将30只健康的Wistar大鼠随机分为对照组、油酸组、纳络酮组,由股静脉注入油酸0.1 ml/kg建立急性呼吸窘迫综合征(ARDS)模型,测定血清肿瘤坏死因子(TNF-α)、白介素-1β(IL-1β)、内皮素-1(ET-1)和肺组织丙二醛(MDA),并计算左肺湿/干重比值,并行肺组织病理学观察.结果 显示,油酸组与对照组比较,左肺湿/干重比值、血清中TNF-α、IL-1β及血浆ET-1含量明显升高,肺组织中MDA含量明显升高,肺组织出现明显病理损害,纳络酮组动物肺损伤轻于油酸组.认为纳络酮可抑制TNF-α、IL-1β、ET-1、MDA升高,对实验性大鼠油酸ARDS有一定的治疗作用.     

肝肺综合征发病机制研究进展

肝肺综合征(HPS)是多见于肝硬化患者的一种综合病征,其发病机制复杂且未完全阐明。内皮源性血管物质一氧化氮(NO)和内皮素-1(ET-1)在其发病机制中的作用已成为研究热点。深入了解其发病机制可为HPS的治疗提供新的途径。     

肝肺综合征发病机制研究进展

肝肺综合征(HPS)是多见于肝硬化患的一种综合病征，其发病机制复杂且未完全阐明。内皮源性血管物质一氧化氮(NO)和内皮素-1(ET-1)在其发病机制中的作用已成为研究热点。深入了解其发病机制可为HPS的治疗提供新的途径。     

Arterial hypoxemia and intrapulmonary vasodilatation in rat models of portal hypertension

Background Rats with chronic bile duct ligation (CBDL) and portal vein ligation (PVL) are used as models of portal hypertension. CBDL rats show hypoxemia with intrapulmonary vasodilatation (IPVD), and are recognized as a model of hepatopulmonary syndrome (HPS), while PVL rats are normoxemic. We investigated the differences in arterial oxygenation between these models, and the key factors leading to HPS.Methods Forty-eight Sprague-Dawley rats were prepared as CBDL or PVL models, or as Sham rats. Arterial oxygenation, hemodynamics (reference sample method), and IPVD were simultaneously evaluated in conscious and unrestrained animals, using ¹⁴¹Ce- or ¹¹³Sn-labeled microspheres (15 µm in diameter), respectively. Endothelin-1 (ET-1) and nitrate/nitrite (end products of nitric oxide; NOx) production by the lung tissue (increment across the lungs) was also determined.Results The extent of IPVD was similar in both models, but hypoxemia was only observed in CBDL rats. The ET-1 level and the increment in NOx were significantly increased in CBDL rats, and the increment was directly correlated with impairment of oxygenation. Blood flow through the bronchial arteries (anatomical shunting) was increased in CBDL rats, reaching more than three times the level in PVL rats or Sham rats.Conclusions These results support the hypothesis that NO derived from the lung tissues contributes to hypoxemia, and IPVD appears to be a prerequisite for impaired oxygenation. The considerable increase of anatomical shunting may potentially contribute to impaired oxygenation in CBDL rats.     

Increased pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism in experimental hepatopulmonary syndrome

BACKGROUND/AIMS: In experimental hepatopulmonary syndrome (HPS), hepatic endothelin-1 (ET-1) release during common bile duct ligation (CBDL) and ET-1 infusion in pre-hepatic portal hypertension after portal vein ligation (PVL) initiate vasodilatation through an endothelin B receptor mediated increase in pulmonary endothelial nitric oxide synthase (eNOS). We evaluated if pulmonary ET receptor expression changes in experimental cirrhosis and portal hypertension and confers susceptibility to HPS.METHODS: In normal, PVL and CBDL animals, lung ET receptor expression and localization were assessed and ET receptor levels and functional analysis of ET-1 effects on eNOS levels were evaluated in intralobar pulmonary artery (PA) and aortic (AO) segments. Normal rats underwent evaluation for HPS after ET-1 infusion.RESULTS: There was a selective increase in ET(B) receptor expression in the pulmonary vasculature from PVL and CBDL animals. ET-1 stimulated NO production and an ET(B) receptor mediated increase in eNOS levels in PA segments from PVL and CBDL animals, but not normal animals. ET-1 did not alter lung eNOS levels or cause HPS in normal rats.CONCLUSIONS: ET(B) receptor expression and ET-1 mediated eNOS and NO production are enhanced in the lung vasculature in cirrhotic and portal hypertensive animals and correlate with in vivo susceptibility to ET-1 mediated HPS.     

The role of endothelin-1 and the endothelin B receptor in the pathogenesis of hepatopulmonary syndrome in the rat

Endothelin-1 (ET-1) stimulation of endothelial nitric oxide synthase (eNOS) via pulmonary endothelial endothelin B (ET(B)) receptors and pulmonary intravascular macrophage accumulation with expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are implicated in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Our aim was to evaluate the role of ET-1 in the development of experimental HPS. The time course of molecular and physiological changes of HPS and the effects of selective endothelin receptor antagonists in vivo were assessed after CBDL. Effects of ET-1 on intralobar pulmonary vascular segment reactivity and on eNOS expression and activity in rat pulmonary microvascular endothelial cells (RPMVECs) were also evaluated. Hepatic and plasma ET-1 levels increased 1 week after CBDL in association with a subsequent increase in pulmonary microvascular eNOS and ET(B) receptor levels and the onset of HPS. Selective ET(B) receptor inhibition in vivo significantly decreased pulmonary eNOS and ET(B) receptor levels and ameliorated HPS. CBDL pulmonary artery segments had markedly increased ET(B) receptor mediated, nitric oxide dependent vasodilatory responses to ET-1 compared with controls and ET-1 triggered an ET(B) receptor dependent stimulation of eNOS in RPMVECs. Pulmonary intravascular macrophages also accumulated after CBDL and expressed HO-1 and iNOS at 3 weeks. Selective ET(B) receptor blockade also decreased macrophage accumulation and iNOS production. In conclusion, ET-1 plays a central role in modulating pulmonary micovascular tone in experimental HPS.     

Biliary cyst fluid from common bile duct-ligated rats stimulates endothelial nitric oxide synthase in pulmonary artery endothelial cells: a potential role in hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from pulmonary microvascular dilatation in cirrhosis and is associated with increased pulmonary endothelial nitric oxide synthase (eNOS) levels. In the common bile duct ligation (CBDL) model, endothelin-1 (ET-1) released from the liver contributes to the rise in pulmonary eNOS and intrapulmonary vasodilatation. Whether substances, including ET-1, are found in the biliary tree and selectively enter the circulation after CBDL to influence the pulmonary vasculature is unknown. We assessed if control bile and fluid obtained from the obstructed biliary tree in CBDL animals contains ET-1 and alters eNOS expression and activity in bovine pulmonary artery endothelial cells (BPAECs). Control bile and biliary cyst fluid contained concentrations of ET-1 25- to 42-fold normal plasma levels, and hepatic venous concentrations of ET-1 were selectively increased after CBDL. Biliary cyst fluid caused a dose-dependent induction of eNOS messenger RNA (mRNA) (1.9-fold control), protein (2.5-fold control), and enzyme activity (2.2-fold control) maximal at a 1:10 dilution. The increases were associated with enhanced nitric oxide (NO) production (3.1-fold control) and were inhibitable with an ET(B) receptor antagonist. Bile from sham and portal vein-ligated animals did not increase eNOS expression and at dilutions of 1:100 and 1:10 caused cell toxicity. These results show that bile and biliary cyst fluid contain high concentrations of ET-1 that are specifically increased in hepatic venous blood after CBDL. Biliary cyst fluid increases eNOS expression and activity in an ET(B) receptor-dependent manner in BPAECs. The findings suggest a novel mechanism for the susceptibility of CBDL animals to the HPS.     

肝肺综合征大鼠血细胞因子和肺血管巨噬细胞观察

目的：观察肝肺综合征大鼠肺组织中肺血管巨噬细胞浸润情况和血浆内毒素水平。方法取 Wistar大鼠30只,随机分为假手术组和胆总管结扎肝纤维化模型组（CBDL 组）,于造模5 w 末处死动物,收集血清及肝肺组织;分别检测血气、内毒素（鲎试验）、一氧化氮（硝酸还原酶法）、肿瘤坏死因子-α（ELISA 法）,观察肝、肺组织病理学变化及肺组织肺血管内巨噬细胞的浸润情况。结果在 CBDL 组动物,5 w 时血气分析示 PaO2为（64.2±7.3） mmHg,显著低于假手术组[（97.7±4.5）mmHg,P〈0.05）];CBDL 组5 w 时血浆内毒素、一氧化氮和肿瘤坏死因子-α分别为（0.2297±0.0362）Eu/ml、（56.2±9.5）μmol/L 和（1.5±0.2）ng/ml,均明显高于假手术组[（0.0938±0.0309） Eu/ml、（32.9±4.3）μmol/L 和（1.5±0.2）ng/ml,P〈0.05）];病理学检查示5 w 时模型组大鼠肝组织可见纤维化表现,肺组织中肺毛细血管扩张、充血,有肺血管巨噬细胞存在。结论在胆总管结扎法诱导的大鼠肝硬化模型中,血清内毒素水平升高、肺血管巨噬细胞浸润和炎性细胞因子分泌增加,可能是肺血管扩张、低氧血症及肝肺综合征发生的重要机制。     

The association of lipopolysaccharide and inflammatory factors with hepatopulmonary syndrome and their changes after orthotopic liver transplantation

Background

The level of lipopolysaccharides (LPS) and inflammatory factors were higher in end stage liver disease patient than in normal person for the damage of intestinal mucosal barrier function. Hepatopulmonary syndrome (HPS) was a common pulmonary complication in end stage liver disease. But the association of LPS and inflammatory factors such as toll like receptor 2 (TLR2), TNF-α and ET-1 with the development of HPS was undefined.

Methods

Thirty-one HPS patients were researched (26 patients were performed liver transplantation, five were not). Ten healthy volunteers were recruited as negative control. Blood was collected from the 26 HPS patients before and 3, 7, 14, 21 and 28 days after orthotopic liver transplantation (OLT), and from five HPS patients without OLT and ten healthy volunteers once to detect TLR2 mRNA and iNOS mRNA in peripheral blood monocytes and plasma LPS, TNF-α and ET-1 level. Their levels before and after OLT were compared.

Results

TLR2 mRNA, iNOS mRNA, LPS, TNF-α and ET-1 before OLT in HPS patients were 336,594.1±366,901.1, 63,982.2±74,127.5 copies/ugRNA, 4.3±3.3, 90.1±76.0 and 319.9±124.4 ng/L, respectively. They were 10,338.3±3,814.6, 19,168.5±2,417.4 copies/ugRNA, 0.94±0.69, 2.7±0.1 and 84.2±10.6 ng/L in normal control group. They were significantly higher in HPS patients than those in control group (P<0.05). After OLT, liver function improved to normal. Also TLR2 mRNA, TNF-α and ET-1 decreased in HPS patients after OLT compared with those before OLT. And PaO₂ and PaO₂/FiO₂ improved greatly with intrapulmonary shunt decreased to normal after OLT.

Conclusions

Lipopolysaccharides at the end stage of liver disease with the release of series of inflammatory factors may be associated with the development of HPS.     

Intrapulmonary vascular dilatation and nitric oxide in hypoxemic rats with chronic bile duct ligation

BACKGROUND/AIMS: Nitric oxide (NO) has been suggested as the major cause of pulmonary vascular dilatation and hypoxemia in hepatopulmonary syndrome (HPS). The aim of this study was to assess the effect of NO on arterial oxygenation in rats with common bile duct ligation (CBDL rats), a model of HPS. METHODS: Arterial blood gases were measured in 44 CBDL rats and 44 Sham rats under unrestrained conditions. Intrapulmonary shunting was assessed with (141)Ce-labeled microspheres (15-mum diameter) and serum nitrate/nitrite levels were measured by HPLC. The effect of NOS inhibition on A-aDO(2) was studied using L-NAME. RESULTS: A decrease of PaO(2) below 82.7 mmHg (the mean value-2sigma in Sham rats) was seen in 43% of CBDL rats. Intrapulmonary shunting was greater in CBDL rats than in Sham rats (P<0.001). A correlation between the extent of shunting and A-aDO(2) was found in all animals studied (r=0.89, P<0.001, n=16). Serum levels of nitrate/nitrite increased significantly across the lungs, and the increase was significantly correlated with A-aDO(2) in the total population of animals studied. Administration of L-NAME to CBDL rats achieved a significant improvement of A-aDO(2). CONCLUSIONS: These results suggest that pulmonary vascular dilatation due to NO leads to hypoxemia in CBDL rats.     

大鼠实验性肝硬化门脉高压形成过程中内皮素及其转化酶的动态变化

目的 观察大鼠实验性肝硬化门脉高压形成过程中,外周血内皮素(ET-1)含量和肝脏前内皮素原(ppET-1)和内皮素转化酶(ECE)mRNA表达的动态变化,以期对其在门脉高压中动态变化的特点有进一步的了解。方法 通过四氯化碳(CCl4)介导大鼠实验性肝硬化门脉高压模型,放免法检测ET-1,半定量RT-PCR法观察不同时期肝组织中ppET-1和ECEmRNA的表达差异。结果 与对照组相比,模型组中外周血ET-1含量和肝脏ppET-1的表达分别从第8周和第6周开始显著升高,且ET-1的升高与门脉高压形成呈显著相关,而肝脏中ECE的表达无明显差异。结论 外周血中ET-1含量的增高和肝脏中ppET-1表达的增加可能是形成肝硬化门脉高压的重要原因之一。