ET-1 and TNF-alpha in HPS: analysis in prehepatic portal hypertension and biliary and nonbiliary cirrhosis in rats

Common bile duct ligation (CBDL) triggers a molecular cascade resulting in the hepatopulmonary syndrome (HPS). Both increased hepatic endothelin-1 (ET-1) production and pulmonary vascular ET(B) receptor expression with stimulation of endothelial nitric oxide synthase and TNF-alpha mediated inducible nitric oxide synthase and heme oxygenase-1 expression in pulmonary intravascular macrophages occur. Whether biliary cirrhosis is unique in triggering ET-1 and TNF-alpha alterations and HPS is unknown. We evaluated for HPS in rat prehepatic portal hypertension [partial portal vein ligation (PVL)], biliary (CBDL) and nonbiliary [thioacetamide treatment (TAA)] cirrhosis, and assessed ET-1 infusion in normal and PVL animals. Control, PVL, CBDL, TAA-treated, and ET-1-infused PVL animals had ET-1 and TNF-alpha levels measured and underwent molecular and physiological evaluation for HPS. HPS developed only in biliary cirrhosis in association with increased plasma ET-1 and TNF-alpha levels and the development of established molecular changes in the pulmonary microvasculature. In contrast, PVL did not increase ET-1 or TNF-alpha levels and TAA treatment increased TNF-alpha levels alone, and neither resulted in the full development of molecular or physiological changes of HPS despite portal pressure increases similar to those after CBDL. Exogenous ET-1 increased TNF-alpha levels and triggered HPS after PVL. Combination of ET-1 and TNF-alpha overproduction is unique to biliary cirrhosis and associated with experimental HPS. ET-1 infusion increases TNF-alpha levels and triggers HPS in prehepatic portal hypertension. ET-1 and TNF-alpha interact to trigger pulmonary microvascular changes in experimental HPS.     

Quantitation of fibrosis in liver biopsies

After 15 years of subjectively estimating the degree of fibrosis in liver biopsies stained with Sirius red F3BA, the amounts of Sirius red-stained fibrotic areas were quantitated with the help of a Cortex IQ 130 image quantifier. The percentage of Sirius red-stained areas was lowest in normal livers, increasing significantly in fatty livers, with stepwise higher values in chronic persistent hepatitis, toxic damage, chronic active hepatitis, fatty livers with obvious fibrosis, biliary cirrhosis and portal cirrhosis. Cases of portal cirrhosis had tenfold higher values than did normal livers. The results indicate that Sirius red is a reliable histochemical stain that can be used to quantitatively demonstrate the various degrees of fibrosis in liver biopsies.     

Biliary secretion of endotoxin and pathogenesis of primary biliary cirrhosis.

Previous studies suggested endotoxin, derived from the intestine through the portal blood to the liver, was predominantly metabolized by Kupffer cells. In the present study, fluorescent-labeled endotoxin injected into the rat portal vein was demonstrated not only in Kupffer cells but also in hepatocytes. Furthermore a great amount of labeled endotoxin was recovered in bile. In the livers of patients with primary biliary cirrhosis (PBC), immunohistochemistry demonstrated significant retention of endotoxin in the biliary epithelial cells, and treatment with ursodeoxycholic acid significantly reduced the retention in those cells. The study for detection of apoptosis demonstrated increased rates of apoptosis in hepatocytes and biliary epithelial cells in PBC liver, and the rate of apoptosis in biliary epithelial cells was significantly reduced after treatment with ursodeoxycholic acid. Immunohistochemistry in PBC liver demonstrated significant reduction of fluorescence intensity for a 7H6 antigen in biliary epithelial cells, indicating the increased paracellular permeability of bile ducts, because cellular immunolocalization of that antigen has been shown to be inversely correlated with the paracellular permeability of the tight junction. These results suggest that, in biliary epithelial cells, retention of endotoxin, increased apoptosis, and increased permeability of tight junctions may be involved in the pathogenesis of PBC.     

Possible role of endogenous retinoid (Vitamin A) toxicity in the pathophysiology of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic, cholestatic disease of unknown etiology commonly affecting women. It is characterized by progressive destruction of the small intrahepatic bile ducts and portal inflammation, leading to fibrosis and cirrhosis. The major signs and symptoms of PBC, which include pruritus, lethargy, the sicca syndrome, and osteoporosis, closely resemble the manifestations of hypervitaminosis A. Based on a review of the literature and other observations connecting PBC with retinoid metabolism (vitamin A and its derivatives), the hypothesis is proposed that exposure to excess endogenous retinoids contributes to the pathogenesis of PBC and may be to the cause of some of the signs and symptoms associated with the disease.     

Changes in the hepatic vessels in different types of cirrhosis

Changes in the vessels at hepatic cirrhosis have been studied in 96 persons and in 20 guinea pigs after carbon tetrachloride had been administered 6 and 18 times. At the initial stage of cirrhosis in the guinea pigs some narrowing of the sinusoid vessels is observed as a result of their constriction by hepatic cells with adipose degeneration. At the terminal stage of cirrhosis, especially the postnecrotic one in people, the distribution of the hepatic vessels is rather uneven. The angioarchitectonics is altered. At the portal and biliary cirrhosis, the vessels go round the cirrhotic nodes forming peculiar rings and "baskets". At the postnecrotic cirrhosis, the vascular rings are less pronounced and the angioarchitectonic picture resembles the one at the hepatic fibrosis. The vascular diameter at cirrhosis is larger than the normal one. The thickness of the interlobular artery wall is often uneven and in general is less than the normal; it can result from dilatation of the lumen, and the latter--from the vascular hypertonus. No outgrowth of the intima is observed.     

Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated state in advanced liver cirrhosis.

Advanced cirrhosis is associated with generalized vasodilation of unknown origin, which contributes to mortality. Cirrhotic patients are endotoxemic, and activation of vascular cannabinoid CB1 receptors has been implicated in endotoxin-induced hypotension. Here we show that rats with biliary cirrhosis have low blood pressure, which is elevated by the CB1 receptor antagonist SR141716A. The low blood pressure of rats with CCl4-induced cirrhosis was similarly reversed by SR141716A, which also reduced the elevated mesenteric blood flow and portal pressure. Monocytes from cirrhotic but not control patients or rats elicited SR141716A-sensitive hypotension in normal recipient rats and showed significantly elevated levels of anandamide. Compared with non-cirrhotic controls, in cirrhotic human livers there was a three-fold increase in CB1 receptors on isolated vascular endothelial cells. These results implicate anandamide and vascular CB1 receptors in the vasodilated state in advanced cirrhosis and indicate a novel approach for its management.     

Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl₄ administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl₄-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12?h after acute CCl₄-intoxication. Tissue levels of ET-1, ET_A- and ET_B receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl₄-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.     

Portal and arterial free and conjugated noradrenaline in two models of portal hypertension in rats

Free and conjugated noradrenaline concentrations were measured in portal-venous and arterial plasma from sham-operated rats or rats with portal hypertension. Two types of portal hypertension in rats were evaluated: in portal vein stenosed rats, the liver was normal, whereas cirrhosis developed in bile duct ligated rats. In cirrhotic rats, arterial free noradrenaline level was higher than in both sham-operated and portal-stenosed rats, this indicating that enhanced sympathetic nervous activity depends on the development of cirrhosis. In all groups of rats, portal venous plasma free noradrenaline was higher than arterial plasma level, indicating a production of noradrenaline by splanchnic organs. Arterial noradrenaline level may be mainly dependent on this splanchnic production in case of portal hypertension. Sulfoconjugated and glucuronoconjugated noradrenaline plasma levels were similar in the three groups of rats. This shows that alteration in conjugation is not likely to be a major factor in the abnormal circulating levels of free noradrenaline observed in cirrhotic rats.     

Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved

Fibrosis, defined as the excessive deposition of extracellular matrix in an organ, is the main complication of chronic liver damage. Its endpoint is cirrhosis, which is responsible for significant morbidity and mortality. The accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of fibroblasts, which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver. They are produced by the activation of precursor cells, such as hepatic stellate cells and portal fibroblasts. These fibrogenic cells are distributed differently in the hepatic lobule: the hepatic stellate cells resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes, whereas the portal fibroblasts are embedded in the portal tract connective tissue around portal structures (vessels and biliary structures). Differences have been reported between these two fibrogenic cell populations, in the mechanisms leading to myofibroblastic differentiation, activation and "deactivation", but confirmation is required. Second-layer cells surrounding centrolobular veins, fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved in fibrogenesis. It is now widely accepted that the various types of lesion (e.g., lesions caused by alcohol abuse and viral hepatitis) leading to liver fibrosis involve specific fibrogenic cell subpopulations. The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of fibrosis/cirrhosis.     

Increased incidence of menstrual abnormalities and hysterectomy preceding primary biliary cirrhosis.

A study was performed to assess the incidence of previous hysterectomy and dilatation and curettage among women with primary biliary cirrhosis. In 87 patients with primary biliary cirrhosis hysterectomy or dilatation and curettage had been performed significantly more often than among 100 age matched normal controls and 80 age matched patients with chronic active hepatitis or alcoholic liver disease. Among the 47 patients with primary biliary cirrhosis who had undergone hysterectomy or dilatation and curettage operations had been performed at a mean of 10.7 years and 13.2 years, respectively, before the onset of disease. The main indication for hysterectomy among patients with primary biliary cirrhosis and controls was menorrhagia. These menstrual disorders may be a consequence of high concentrations of oestrogens in patients with primary biliary cirrhosis.     

Transport systems in cholangiocytes: their role in bile formation and cholestasis.

Formation of bile requires the coordinated function of two epithelial cell types: hepatocytes, that are responsible for secretion of the major osmolytes and biliary constituents and cholangiocytes that regulate the fluidity and alkalinity of bile through secretion of osmolytes such as Cl- and HCO3- Studies in isolated cholangiocyte preparations have elucidated the basic transport mechanisms involved in constitutive and stimulated secretory activities in the biliary epithelium. Basolateral Na+/H+ exchanger and Na+:HCO3- symporter mediate HCO3- uptake, while an apical cAMP-activated Cl-/HCO3- exchanger secretes bicarbonate into the lumen. Cholangiocytes also possess a cAMP-stimulated Cl- conductance (CFTR) and a Ca-activated Cl- channel, both likely located at the apical membrane. Cholangiocyte secretory functions are regulated by a complex network of hormones mainly acting via the cAMP system. In addition, recent data indicate that part of the regulation of ductular secretion may take place at the apical membrane of the cholangiocyte through factors present into the bile, such as ATP, bile acids and glutathione. Primary damage to the biliary epithelium is the cause of several chronic cholestatic disorders (cholangiopathies). From a pathophysiological point of view, common to all cholangiopathies is the coexistance of cholangiocyte death and proliferation and various degrees of portal inflammation and fibrosis. Cholestasis dominates the clinical picture and, pathophysiologically, may initiate or worsen the process. Alterations in biliary electrolyte transport could contribute to the pathogenesis of cholestasis in primary bile duct diseases. Cystic Fibrosis-related liver disease represents an example of biliary cirrhosis secondary to a derangement of cholangiocyte ion transport. Most primary cholangiopaties recognize an immune-mediated pathogenesis. Cytokines, chemokines, and proinflammatory mediators released in the portal spaces or produced by the cholangiocyte itself, likely activate fibrogenesis, stimulate apoptotic and proliferative responses, and alter the transport functions of the epithelium.     

Hemodynamic effects of a combination of clonidine and propranolol in conscious cirrhotic rats

The hemodynamic effects of the combination of clonidine and propranolol were studied in conscious rats with portal hypertension owing to secondary biliary cirrhosis. Pressure and blood flow measurements (radioactive microsphere method) were performed in three groups of eight rats before and after drug administration. The combined effects of clonidine (2 micrograms/100 g body wt., i.v.) and propranolol (0.2 mg/min for 10 min) were compared with those observed after administration of either clonidine alone or propranolol alone. The association of clonidine and propranolol induced significant decreases in portal pressure (30%) and portal tributary blood flow (43%), the magnitude of these changes being significantly more marked than that after administration of either clonidine alone (12 and 20%, respectively) or propranolol alone (16 and 17%, respectively). After the combination, no significant change in arterial pressure was observed, but cardiac output significantly decreased and systemic vascular resistance significantly increased. Renal blood flow decreased to a similar extent (40%) in the three groups. These findings indicate that the combination of clonidine and propranolol is more effective for reversing splanchnic hemodynamic changes than clonidine alone or propranolol alone. The additive effects of this association are in agreement with the action of clonidine and propranolol at different levels (central and peripheral) and on different receptors (alpha and beta). It suggests that an increase in sympathetic activity may play a major role in hemodynamic changes observed in experimental cirrhosis.     

A novel canine model of portal vein stenosis plus thioacetamide administration-induced cirrhotic portal hypertension with hypersplenism

Current large animal models that could closely resemble the typical features of cirrhotic portal hypertension in human have not been well established. Thus, we aimed to develop and describe a reliable and reproducible canine cirrhosis model of portal hypertension. A total of 30 mongrel dogs were randomly divided into four groups: 1 (control; n = 5), 2 (portal vein stenosis [PVS]; n = 5], 3 (thioacetamide [TAA]; n = 5), and 4 (PVS plus TAA; n = 15). After 4-months modeling period, liver and spleen CT perfusion, abdominal CT scans, portal hemodynamics, gastroscopy, hepatic function, blood routine, the bone marrow, liver, and spleen histology were studied. The animals in group 2 (PVS) developed extrahepatic portosystemic collateral circulation, particularly esophageal varices, without hepatic cirrhosis and portal hypertension. Animals from group 3 (TAA) presented mild cirrhosis and portal hypertension without significant symptoms of esophageal varices and hypersplenism. In contrast, animals from group 4 (PVS + TAA) showed well-developed micronodular and macronodular cirrhosis, associated with significant portal hypertension and hypersplenism. The combination of PVS and TAA represents a novel, reliable, and reproducible canine cirrhosis model of portal hypertension, which is associated with the typical characteristics of portal hypertension, including hypersplenism.     

超声对原发性胆汁性肝硬化合并自身免疫性甲状腺炎的诊断价值和相关性分析

摘要 目的：探讨超声对原发性胆汁性肝硬化合并自身免疫性甲状腺炎的诊断价值以及相关性。方法：选取2017年1月~2019年12月我院收治的90例原发性胆汁性肝硬化合并自身免疫性甲状腺炎患者为研究组,根据患者病情的不同将其分为早期组（n=48）和晚期组（n=42）。另选取50例健康志愿者为对照组,对所有患者进行超声诊断,比较两组患者的门静脉血流参数、肝动脉血流参数、肝中静脉血流参数以及脾静脉血流参数。结果：晚期组的门静脉主干内径（portal vein diameter,PVD）明显高于早期组和对照组（P<0.05）,晚期组的平均流速（portal vein velocity,PVV）明显低于早期组和对照组（P<0.05）,三组患者的门静脉血流量（perceptual video quality,PVQ）差异无统计学意义（P>0.05）。晚期组的肝动脉内径（hepatic artery inner diameter,HAD）、阻力指数（resistance index,RI）、收缩期峰值流速（peak systolic velocity/end diastolic velocity,HAVmax）、血流量（blood flow,HAQ）均明显大于对照组（P<0.05）。晚期组的肝中静脉内径（middle hepatic vein diameter,MHAD）、最高平均流速（maximum average flow rate ,MHV Vm）、最大血流量（maximum blood flow,MHVQ）均较早期组和对照组减少（P<0.05）。晚期组的脾静脉内径（splenic vein diameter,SVD）明显高于对照组和早期组（P<0.05）,晚期组的脾静脉平均流速（splenic vein mean flow rate,SVm）、脾静脉血流量 (splenicveinflow, SVF)与对照组、早期组相比差异无统计学意义（P>0.05）;经过相关性分析肝脏超声指标与甲状腺超声指标均呈现正相关关系（P＜0.05）。结论：超声在原发性胆汁性肝硬化合并自身免疫性甲状腺炎患者中应用具有很高的价值,通过对原发性胆汁性肝硬化超声指标推测自身免疫性甲状腺炎病情程度,患者的超声图像具有一定的特征性,可以用以患者的诊断和病因分析,值得在临床中应用推广。     

Protective role of melatonin in early‐stage and end‐stage liver cirrhosis

The liver is composed of hepatocytes, cholangiocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells (HSCs) and dendritic cells; all these functional and interstitial cells contribute to the synthesis and secretion functions of liver tissue. However, various hepatotoxic factors including infection, chemicals, high‐fat diet consumption, surgical procedures and genetic mutations, as well as biliary tract diseases such as sclerosing cholangitis and bile duct ligation, ultimately progress into liver cirrhosis after activation of fibrogenesis. Melatonin (MT), a special hormone isolated from the pineal gland, participates in regulating multiple physiological functions including sleep promotion, circadian rhythms and neuroendocrine processes. Current evidence shows that MT protects against liver injury by inhibiting oxidation, inflammation, HSC proliferation and hepatocyte apoptosis, thereby inhibiting the progression of liver cirrhosis. In this review, we summarize the circadian rhythm of liver cirrhosis and its potential mechanisms as well as the therapeutic effects of MT on liver cirrhosis and earlier‐stage liver diseases including liver steatosis, nonalcoholic fatty liver disease and liver fibrosis. Given that MT is an antioxidative and anti‐inflammatory agent that is effective in eliminating liver injury, it is a potential agent with which to reverse liver cirrhosis in its early stage.     

The interrelationship between histologic and ultrastructural changes in the liver in the presence of pathologic states]

A study was made of liver cells of cirrhosis-affected patients and those with portal hypertensia due to subhepatic block. It was shown that the extent of cellular injury was directly related to the pattern of pathological process in the liver. In the case of active cirrhosis, the necrobiotic processes observed might be due to changes in the structures providing cellular metabolism. With stable cirrhosis, the changes observed suggest abnormalities in cell synthesis, in addition to the rise in aerobic respiration. The analysis of the data obtained in the cases of subhepatic block without cirrhosis showed a sufficient functional activity of liver cells.     

Breast cancer in women with primary biliary cirrhosis.

The occurrence of extrahepatic malignancy was studied in 195 unselected patients who satisfied predetermined biochemical, immunological, and histological criteria for the diagnosis of primary biliary cirrhosis. The incidence of breast cancer in women with primary biliary cirrhosis was found to be significantly higher than in an age and sex matched control population from the same well defined geographical area (p less than 0.0015). The association of breast cancer and primary biliary cirrhosis remains unexplained, though diminished immunological surveillance, fat soluble vitamin deficiency, or endocrine dysfunction may play a part.     

肝硬化患者肝脏门静脉血流与肝叶萎缩之间关系的研究

目的：探讨肝硬化患者肝脏右叶、左叶体积变化,检测肝硬化患者门静脉血流情况,分析二者之间的关系,以及门静脉血流与肝功能之间关系。方法：本研究纳入54例肝硬化患者和40例正常人,采用超声多普勒方法分析这些受试者的肝脏体积和门静脉主干及左右分支的内径、血流速、流量数据,并通过静脉血检测白蛋白、胆红素、胆碱酯酶水平等评估患者肝功能水平。结果：肝硬化组平均年龄46.3岁,男性32例,其中childA级患者16例,childB级患者27例,childC级患者11例;正常对照组平均年龄41．8岁,男性24例。肝硬化组患者右左肝叶之比明显低于正常对照组（p〈0．05）,门静脉内径和血流量明显高于正常对照组（p〈0．05）．随着child分级升高,门静脉血流量也明显升高。肝硬化组门静脉右支血流量明显低于左支血流量（p〈0．05）;此外肝硬化患者门静脉右支和左支血流量之比明显低于正常人群门静脉右左支之比（p〈0．05）;而且肝硬化患者门静脉右左支血流量之比与右左肝叶具有明显的相关性与右左肝叶之比具有明显的相关性（r=0．64,p〈0．05）。结论：评估肝硬化病人门静脉血流情况,对于判断肝脏病理变化程度,评价治疗效果,以及选择治疗方案方面都具有重要的临床价值     

Primary biliary cirrhosis--an attempt to evaluate the usefulness of selected tests in differential diagnosis]

Seven cases of the primary biliary cirrhosis are presented. The diagnostic problems in early stages of the primary biliary cirrhosis as well as the value of both biochemical and serologic test have been discussed with particular attention to the value of liver biopsy.