胆总管结扎诱导大鼠肝肺综合征模型的建立初步研究

目的 探讨胆总管结扎术诱导大鼠肝肺综合征(HPS)模型的建立.方法 40只SD大鼠被随机分为实验组30只和对照组(假手术组)10只,采用胆总管结扎术建立肝硬化和HPS模型.使用血气分析仪测定PaO2、PaCO2和pH.结果 在术后1 w,腹部超声检查显示实验组肝脏回声增粗,胆总管轻度扩张.在术后2 w,肝脏被膜不平整,...     

Participation of bone marrow cells in biliary fibrosis after bile duct ligation

BACKGROUND AND AIM: Bone marrow derived cells are involved in the process of hepatic fibrosis secondary to chronic injury. However, it is not yet known how quickly this event occurs in acute fibrosis models. The purpose of this study was to determine the role of bone marrow cells in rapid fibrosis following bile duct ligation in mice using green fluorescent protein (GFP) expressing bone marrow cells. METHOD: After whole body irradiation, 1 x 10(6) donor whole bone marrow cells from green fluorescent protein(+/-) mice were transplanted into C57BL/6 recipients via the tail vein. Four weeks after bone marrow transplantation, chimeric mice were subjected to common bile duct ligation, and livers of these animals were histologically examined after bile duct ligation using anti-fibroblast specific protein (FSP)-1 antibody and anti-alpha-smooth muscle actin (alpha-SMA) antibody. RESULTS: Periductal fibrosis consisting of fibroblast specific protein-positive cells was demonstrated histologically as early as day 7. Most of the fibrotic cells were green fluorescent protein-negative, however, a significant number of cells were green fluorescent protein-positive and some were also anti-FSP or alpha-SMA-positive. CONCLUSION: Differentiation of bone marrow derived cells into activated fibroblast and myofibroblast-like phenotypes occurs in the very early course of periductal fibrosis following bile duct ligation, suggesting a new strategy for prevention of biliary fibrosis by inhibiting migration of bone marrow cells to liver.     

Successively postadministered melatonin prevents disruption of hepatic antioxidant status in rats with bile duct ligation

We have reported that orally administered melatonin exerts a therapeutic effect on cholestatic liver injury in rats treated with bile duct ligation (BDL) possibly through its antioxidant and anti-inflammatory actions. Herein, we examined whether successively postadministered melatonin prevents the disruption of hepatic antioxidant status in BDL-treated rats. Wistar rats with BDL were killed 5 and 13 days after BDL. Melatonin (10 or 100 mg/kg body weight) was orally administered to rats with and without BDL everyday for 8 days, starting 5 days after BDL. The hepatic concentrations of thiobarbituric acid reactive substances, an index of lipid peroxidation, and reduced glutathione increased 5 days after BDL and further increased at 13 days. Hepatic vitamin E concentration and catalase and Se-glutathione peroxidase (Se-GSH-Px) activities were similarly reduced at 5 and 13 days after BDL. Hepatic ascorbic acid concentration and the hepatic activities of Cu,Zn- and Mn-superoxide dismutases, glutathione reductase, and glucose-6-phosphate dehydrogenase decreased 13 days after BDL. Melatonin postadministered to BDL-treated rats attenuated all these changes observed at 13 days after the treatment more effectively at the higher dose than at the lower dose. Melatonin administered to BDL-untreated rats increased the hepatic Se-GSH-Px activity at both doses and the hepatic activities of Cu,Zn- and Mn-superoxide dismutases at the higher dose. These results indicate that successively postadministered melatonin at pharmacological doses prevents the disruption of hepatic antioxidant status in rats with BDL through its direct and indirect antioxidant action, which may contribute to its therapeutic effect of BDL-induced cholestatic liver injury.     

Melatonin prevents increased asymmetric dimethylarginine in young rats with bile duct ligation

Abstract: Identifying and treating kidney injury in cirrhosis is important. Bile duct ligation (BDL) is a commonly used cholestatic liver disease model. We hypothesized that asymmetric dimethylarginine (ADMA) is involved in BDL‐induced oxidative stress and kidney injury, which can be prevented by melatonin. We also intended to elucidate whether increased ADMA is due to increased protein arginine methyltransferase‐1 (PRMT1, ADMA‐synthesizing enzyme) and/or decreased dimethylarginine dimethylaminohydrolase (DDAH, ADMA‐metabolizing enzyme). Three groups of young rats were studied, sham (N = 7), untreated BDL rats (N = 9), and melatonin‐treated BDL rats (N = 6, BDL + M). Melatonin‐treated BDL rats received daily melatonin 1 mg/kg/day via intraperitoneal injection. One‐third of the young BDL rats died compared with none in the BDL + M group. All surviving rats were killed 14 days after surgery. BDL rats had higher plasma aspartate aminotransferase, alanine aminotransferase, direct and total bilirubin, and ammonia levels than shams. They also had kidney injury characterized by increased tubulointerstitial injury scores and plasma creatinine and symmetric dimethylarginine levels, which melatonin prevented. Plasma ADMA levels were elevated in BDL rats, combined with increased hepatic PRMT1 and decreased renal DDAH activity. In addition, melatonin increased hepatic DDAH2 expression, increased DDAH activity and concomitantly decreased ADMA contents in both the liver and kidney. In conclusion, melatonin therapy decreased mortality and prevented kidney injury induced by BDL via reduction of ADMA (by increasing DDAH activity) and oxidative stress.     

Effects of secretin on bile production in two kinds of cholestatic models by choledocho-caval fistula and bile duct ligation in rats

Secretin is known to stimulate bile flow from the bile duct epithelium. To investigate the effects of secretin in cholestasis, we studied the response of the bile flow and the excretion of biliary components to secretin using two cholestatic models with or without damage to the bile duct epithelium. The model without bile duct epithelial damage was a choledocho-caval (CC) fistula over a 24-hour period, and the model with bile duct damage was a bile duct ligation over a 48-hour period. Secretin was administered by intravenous infusion for 30 minutes and bile was collected for 120 minutes. Controls were given saline similarly. The bile flow and biliary bicarbonate excretion rate were significantly increased after secretin infusion in the CC fistula rats when compared with the control rats, but no stimulation by secretin was observed in the ligated rats. These data indicate that secretin-induced bile production was enhanced under cholestatic conditions with no bile duct epithelial disturbance.     

新型生长抑素类似物SOM230减轻胆总管结扎大鼠肝纤维化

目的 应用胆总管结扎大鼠肝纤维化模型,观察长效生长抑素类似物SOM230对胆管结扎所致大鼠肝纤维化的治疗作用.方法 雄性SD大鼠80只,随机分为模型组、高剂量预防组(80 mg/kg SOM230)、低剂量预防组(8 mg/kg SOM230)、假手术组及正常对照组.分别在术后第2周和第4周取心脏血及肝组织,观察肝组织...     

Comparison of murine cirrhosis models induced by hepatotoxin administration and common bile duct ligation

AIM: To build up the research models of hepatic fibrosis in mice. METHODS: Inbred wild-type FVB/N mice were either treated with alpha-naphthyl-isothiocyanate (ANIT), allyl alcohol (AA), carbon tetrachloride (CCI4), 3,5-diethoxycarbonyl-l,4-dihydrocollidine (DDC), and silica, or subjected to common bile duct ligation (CBDL) to induce hepatic injury. Liver biopsies were performed every 4 wk to evaluate hepatic fibrosis over a period of 6 mo. Cumulative cirrhosis and survival curves were constructed by life table method and compared with Wilcoxon test. RESULTS: Under the dosages used, there was neither mortality nor cirrhosis in AA and silica-treated groups. DDC and ANIT caused cirrhosis within 4-12 and 12-24 wk, respectively. Both showed significantly faster cirrhosis induction at high dosages without significant alteration of survival. The duration for cirrhosis induction by CCU ranged from 4 to 20 wk, mainly dependent upon the dosage. However, the increase in CCU dosage significantly worsened survival. Intraperitoneal CCU administration resulted in better survival in comparison with gavage administration at high dosage, but not at medium and low dosages. After CBDL, all the mice developed liver cirrhosis within 4-8 wk and then died by the end of 16 wk. CONCLUSION: CBDL and administrations of ANIT, CCI4, and DDC ensured liver cirrhosis. CBDL required the least amount of time in cirrhosis induction, but caused shortened lives of mice. It was followed by DDC and ANIT administration with favorable survival. As for CCU, the speed of cirrhosis induction and the mouse survival depended upon the dosages and the administration route.     

Dose-related effects of dexamethasone on liver damage due to bile duct ligation in rats

INTRODUCTIONObstruction of bile ? ow through the extrahepatic biliary system results in development of oxidant injury, hepatic fibrosis, biliary cirrhosis, and portal hypertension[1,2]. Patients with obstructive jaundice are at significant risk for severe…     

Effect of selective cyclooxygenase-2 inhibitor meloxicam on liver fibrosis in rats with ligated common bile ducts.

Aim: Cholestasis triggers fibrogenesis in the liver. Hepatic cyclooxygenase-2 (COX-2) expression increases in various chronic liver diseases caused either by viruses or toxins. We hypothesized that selective COX-2 inhibitor meloxicam could suppress inflammation and fibrogenesis in a rat model of cholestasis induced by bile duct ligation (BDL). Methods: Forty-three Sprague-Dawley rats were assigned to one of four treatment groups (sham-operation, BDL, daily meloxicam injections following BDL, and daily meloxicam injection without BDL). Liver histopathology was analyzed with hematoxylin-eosin and Masson's trichrome staining. The expression of alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta1 (TGF-beta1), and COX-2 were measured with immunohistochemical staining. The levels of COX-2, TGF-beta1, and matrix metalloproteinase-9 (MMP-9) production were measured with the Western blot method and an enzyme immunoassay. Results: Meloxicam treatment attenuated the expression of alpha-SMA, TGF-beta1, and COX-2 in rats that were treated with BDL for 3 weeks. This was associated with a marked reduction in collagen accumulation and histological improvement. In addition, meloxicam treatment was found to downregulate the levels of hepatic COX-2, TGF-beta1, and MMP-9 production. Conclusion: Cholestasis in BDL rats induces hepatic COX-2 expression. Selective COX-2 inhibitor meloxicam reduces BDL-induced hepatic fibrosis, and this is associated with reduced hepatic TGF-beta1 expression as well as decreased cyclooxygenase activity in the liver.     

Divergent effects of irritants on the gastric mucosa in rats during various stages after bile duct ligation

Background and Aim: Controversy exists as to whether rats after bile duct ligation (BDL) are more susceptible to gastric mucosal damage (GMD) induced by irritants. In the present study we characterize GMD after intragastric instillation of either ethanol or hydrochloric acid (HCL), 3 and 21 days after the surgical procedure. Methods: Bile duct ligation and sham operated (SO) rats were studied. Results: Three days after surgery, BDL rats exhibited a reduction in gastric mucosal nitric oxide synthase (NOS) activity but an increase in ethanol‐induced GMD. Twenty‐one days after surgery gastric mucosal prostaglandin (PG) E₂ generation in BDL rats was increased while NOS activity in both groups was similar. Ethanol‐induced GMD in SO rats was higher. Pretreatment with NG‐nitro‐L‐arginine methyl ester, prior to ethanol administration was associated with an increase in gastric mucosal PGE₂ generation: (147% in SO and 104% in BDL rats) and in GMD (176% in SO and 303% in BDL rats). HCL induced GMD was of similar magnitude in both groups in both time periods. Conclusions: The gastric resistance to damage by irritants in rats with BDL is not a static phenomenon. This may result from sequential changes that occur in the gastric mucosal defense mechanisms during the evolution of liver disease.     

Segmental cholangitis impairs hepatic regeneration capacity after partial hepatectomy in rats

Background:

Patients with hilar cholangiocarcinoma or hepatolithiasis often develop segmental cholangitis (SC), but it is unclear whether hepatectomy for patients with SC can be performed safely.

Methods:

Rats were subjected to segmental bile duct ligation (SBDL) with LPS (SC group) or a saline (Sham group) infusion into the bile duct of the ligated lobes. The rats were sacrificed at 3, 24 and 48 h after the SBDL. For another experiment, the rats were subjected to partial hepatectomy (PHx) for the ligated lobes. Hepatic regeneration rates and the expression of regeneration-associated genes were evaluated.

Results:

In the SC group, severe parenchymal damage was observed in the acute phase (3 h). Altered gene expression in the liver in response to biliary infection occurred not only in the infected lobes but also in the non-infected lobes. In the rats of the SC group, both the hepatic regeneration rate and serum HGF levels were significantly lower than in the Sham group.

Conclusion:

These results clearly demonstrate that SC impairs the regeneration capacity of the contralateral remnant liver. Therefore, hepatectomy should be avoided for patients with SC even if it occurs in the part of the liver to be resected.     

Dietary glycine blunts liver injury after bile duct ligation in rats

AIM： To investigate the effects of （dietary） glycine against oxidant-induced injury caused by bile duct ligation （BDL）.
METHODS： Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley （SD） rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid （DCA） in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase （LDH） activity was determined in the supernatant of cultivated hepatocytes.
RESULTS： Serum alanine transaminase levels increased to about 600 U/L 1 d alter BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed.
CONCLUSION： These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.     

Effects of simvastatin, pentoxifylline and spironolactone on hepatic fibrosis and portal hypertension in rats with bile duct ligation

Aims/Methods: Our aim was to the antifibrotic and hemodynamic effects of simvastatin (SMV), pentoxifylline (PTX) and spironolactone (SPN), three drugs which may have antifibrotic and/or portal hypotensive properties, in a model of hepatic fibrosis and portal hypertension induced in rats by bile duct ligation. A blind study was performed in five groups of 53 Sprague-Dawley rats: sham, placebo (PL), SMV (2.5 mg·kg⁻¹·J⁻¹, PTX (50 mg·kg⁻¹·J⁻¹) and SPN (100 mg·kg⁻¹·J⁻¹). Drugs were administered by daily gavage over a 4-week period as soon as bile duct ligation was performed. At day 28, the following parameters were evaluated: area of hepatic fibrosis by image analysis after staining collagen with picrosirius and plasma concentrations of hyaluronate, splanchnic and systemic hemodynamics (radiolabeled microspheres).Results: Portal venous pressure (PL: 15.5±1.5, SMV: 15.8±2.5, PTX: 15.9±1.8, SPN: 13.5±2.1 mmHg, p<0.05) and porto-systemic shunts (PL: 30±31, SMV: 18±27, PTX: 25±24, SPN: 5±4%, p<0.05) were significantly reduced in the SPN group; other hemodynamic parameters were not significantly altered. There was a significant correlation between porto-systemic shunts and portal pressure (r_s=0.47, p<0.01). The area of fibrosis was not significantly different among the four groups of bile duct ligated rats (PL: 8.7±3.9, SMV: 7.1±3.6, PTX: 7.8±2.7, SPN: 6.6±3.3%) but was higher than in sham rats (1.5±0.5%, p<0.001). Hyaluronate was significantly higher in bile duct ligated rats (from 374±162 to 420±131 μg/l, among the four groups) than in sham rats (52±16 μg/l, p<0.0001).Conclusions: In this model, none of the drugs prevented hepatic fibrosis. On the other hand, spironolactone decreased portal pressure and prevented porto-systemic shunts. Therefore, this drug may have beneficial effect in patients with early portal hypertension.     

胆管结扎大鼠肝纤维化模型中HSC的分离和培养及PARs的表达

目的探讨胆管结扎诱导的大鼠肝纤维化模型的制备和改良的Friedman方法分离和培养肝星状细胞(HSC),以及HSC蛋白酶活化受体(PARS)的表达与HSC活化的关系。方法采用结扎大鼠胆管诱导肝纤维化动物模型,应用改良的Friedman方法和Nycodenz一步密度梯度离心法分离肝纤维化大鼠HSC。病理学检测大鼠肝纤维化分级,免疫组化检测Desmin、α-SMA和PARS在HSC的表达,实验同时设立阴性对照。结果模型组大鼠肝纤维化病理学分级第1周为0,第2~3周多为1~2级,第4、5周为2~3级,第6周肝组织结构破坏,肝细胞变性坏死,炎性细胞浸润,病理学分级多为4级。改良的Friedman方法大鼠肝脏可获得5×106~1×107个细胞,存活率在96%以上,纯度平均为95%~96%。免疫组化检测α-SMA和PARS在培养的HSC于7、14天其表达逐渐增多,二者表达呈一致关系。结论结扎大鼠胆管建立的肝纤维化模型形成时间较短,实验中无有毒物质接触,是肝纤维化的理想模型;改良的Friedman方法分离和培养HSC,在保证HSC纯度的同时,提高了产量和活率,是研究HSC的有效方法;PARS的表达与HSC活化相关。