Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl(4)

AIM:To investigate effect of losartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCl(4); and to determine whether or not AT1 receptors are expressed on hepatic stellate cells. METHODS AND RESULTS:Fifty male Sprague-Dawley rats, weighing (180 plus minus20)g, were randomized into five groups (control group, model group, and three losartan treated groups), in which all rats were given the subcutaneous injection of 40% CCl(4)(every 3 days for 6 weeks) except for rats of control group. Rats of losartan-treated groups were treated with losartan (20 mg/kg, 10 mg/kg, 5 mg/kg, daily gavage). After 6 weeks liver tissue and serum samples of all rats were examined. Serum hyaluronic acid (HA), procollagen type III (PC III) were detected by radioimmunoassays. van Giesion collagen staining was used to evaluate the extracellular matrix of rats with liver fibrosis. The expression of AT1 receptors, transforming growth factor-beta (TGF-beta), and alpha-smooth muscle actinalpha-SMA) in liver tissue were determined by immunohistochemical techniques. Compared with model group, serum ALT and AST of losartan-treated groups were significantly reduced (italic>t = 4.20,P < 0.01 and italic>t = 4.57,P < 0.01). Serum HA and PC III also had significant differences (italic>t = 3.53,P<0.01 and t=2.20, P<0.05). The degree of fibrosis was improved by losartan and correlated with the expressions of AT1 receptors, TGF-beta, and alpha-SMA in liver tissue.CONCLUSION:AT1 receptor antagonist, losartan, could limit the progression of the hepatic fibrosis induced by CCl(4). The mechanism may be related to the decrease in the expression of AT1 receptors and TGF-beta, ameliorating the injury of hepatocytes; activation of local renin-angiotensin system might relate to hepatic fibrosis; and during progression of fibrosis, activated hepatic stellate cells might express AT1 receptors.     

The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CCl(4)

AIM:To assess the effect of ACE inhibitor and Ang II type 1 (AT1) receptor antagonist in preventing hepatic fibrosis caused by CCl(4) administration in rats;to investigate whether or not there are expression of AT 1 receptors on hepatic stellate cells; and to observe the effect of Ang II on proliferation and ECM synthesis of cultured HSCs.METHODS:Studies were conducted in male Sprague-Dawley rats. Except for the hepatofibrotic model group and the control group, in three treated groups, either enalapril (5mg/kg), or losartan (10mg/kg), or enalapril + losartan were given to the fibrotic rats by daily gavage, and saline vehicle was given to model and normal control rats. After 6 weeks, liver fibrosis was assessed directly by hepatic morphometric analysis, which has been considered the gold standard for the quantification of fibrosis. The expressions of AT 1 receptors and (alpha-mooth muscle actin,alpha-SMA) in liver tissue or isolated hepatic stellate cells (HSCs) were detected by immunohistochemical techniques. The effect of Ang II on HSC proliferation was determined by MTT method. Effect of Ang II on collagen synthesis of HSCs was determined by (3)H-proline incorporation.RESULTS:Contrasted to the fibrosis in rats of the model group, groups of rats treated with either enalapril or losartan, or a combination of two drugs showed a limited expansion of the interstitium (4.23 plus minus 3.70 vs 11.22 plus minus 4.79, P<0.05), but no difference was observed among three treated groups (5.38 plus minus3.43, 4.96 plus minus 2.96, 4.23 plus minus 2.70, P>0.05). Expression of AT 1 receptors was found in fibrotic interstitium of fibrotic rats, whereas in normal control rats they were limited to vasculature only to a very slight degree. AT 1 receptors were also expressed on activated HSCs in the culture. At concentrations from 10(-9) to 10(-5)mol/L, Ang II stimulated HSC proliferation in culture in a dose dependent manner. Increasing Ang II concentrations produced corresponding increases in (3)H-proline incorporation. Differences among groups were significant.CONCLUSION:Angiotensin converting enzyme inhibitors and AT 1 blocker may slow the progression of hepatic fibrosis;activated HSCs express AT 1 receptors, and Ang II can stimulate the proliferation and collagen synthesis of HSCs in a dose-dependent manner; and activation of RAS may be related to hepatic fibrogenesis induced by CCl(4).     

激动素抗大鼠肝纤维化的实验研究

目的:研究激动素对实验性肝纤维化的影响,探讨作用机制。方法:用CCl4诱导形成大鼠肝纤维化模型,使用0.1%激动素溶液0.5ml.100g-1.d-1,背部皮下注射,治疗12周,设正常对照组和模型组。血清ALT用全自动生化分析仪检测,血清透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PCⅢ)和Ⅳ型胶原(CⅣ)含量用RIA方法检测。肝组织学检查:采用苏木精-伊红染色观察肝组织炎症和纤维化程度。结果:激动素组ALT水平与模型组ALT水平分别为(57.40±17.49)U/L和(84.70±31.85)U/L,两组比较差异有显著性意义(P<0.05)。激动素组LN和CⅣ含量分别为(29.25±6.67)ng/ml和(14.84±5.84)ng/ml,较模型组(37.18±10.06)ng/ml、(35.69±30.84)ng/ml明显降低(P<0.05);激动素组HA和PCⅢ含量分别为(136.25±76.83)ng/ml和(11.43±3.67)ng/ml,较模型组(168.55±56.19)ng/ml、(14.64±8.00)ng/ml有不同程度降低,但无统计学意义。组织学检测结果表明,模型组肝脏炎症和纤维化程度明显高于激动素组。结论:激动素对实验性大鼠肝纤维化具有抑制作用。     

Effects of transmitters and interleukin-10 on rat hepatic fibrosis induced by CCl4

AIM: To study the effects of transmitters ET, AgⅡ, PGI2,CGRP and GG on experimental rat hepatic fbrosis and the antifibrogenic effects of IL-10.METHODS: One hundred SD rats were randomly divided into 3 groups: control group (N): intraperitoneal injection with saline 2 ml.kg^-1 twice a week; the fibrogenesis group (C): intraperitoneal injection with 50 % CC14 2 ml.kg^-1 twice a week; IL-10 treated group (E): besides same dosage of CC14 given, intraperitoneal injection with IL-10 4 ug.kg^-1 from the third week. In the fifth, the seventh and the ninth week,rats in three groups were selected randomly to collect plasma and liver tissues. The levels of ET, AgⅡ, PGI2, CGRP and GG were assayed by radioimmunoassay (RIA). The liver fibrosis was observed with silver staining.RESULTS: The hepatic fibrosis was developed with the increase of the injection frequency of CC14. The ET, AgⅡ, PGI2, CGRP and GG levels in serum of group N were 71.84&#177;60.2 ng.L^-1,76.21&#177;33.3 ng.L^-1, 313.03&#177;101.71 ng.L^-1, 61.97&#177;21.4 ng.L^-1 and 33.62&#177;14.37 ng.L^-1, respectively; the levels of them in serum of group C were 523.30&#177;129.3 ng.L^-1, 127.24&#177;50.0 ng.L^-1,648.91&#177;357.29 ng.L^-1, 127.15&#177;62.0 ng&#183;L^-1 and 85.26&#177;51.83ng.L^-1, respectively; the levels of them in serum of group E were 452.52&#177;99.5 ng.L^-1, 90.60&#177;44.7 ng.L^-1, 475.57&#177;179.70ng.L^-1, 102.2&#177;29.7 ng.L^-1 and 38.05&#177;19.94 ng.L^-1, respectively.The histological examination showed that the degrees of the rats liver fibrosis in group E were lower than those in group C.CONCLUSION: The transmitters ET, AgⅡ, PGI2, CGRP and GG play a significant role in the rat hepatic fibrosis induced by CCl4, IL-10 has the antagonistic action on these transmitters and can relieve the degree of the liver fibrosis.     

铁调素在CCl_4诱导的小鼠肝纤维化模型中的动态变化

目的研究铁调素(hepcidin)在CCl4诱导的小鼠肝纤维化中的动态变化。方法 C57BL/6小鼠给予CCl4灌胃建立肝纤维化模型,通过HE和Masson染色验证小鼠肝损伤及纤维化程度,并通过普鲁士蓝染色检测在肝纤维化进展过程中肝脏铁沉积情况;通过两步法胶原酶原位灌注与密度梯度离心分离肝脏原代肝细胞、肝星状细胞、Kupffer细胞;通过实时荧光定量PCR检测肝纤维化进展过程中hepcidin和肿瘤坏死因子(TNF)α、白细胞介素(IL)6、IL-1β的表达水平。计量资料两组间比较采用t检验。结果通过对肝组织进行HE和Masson染色发现,肝纤维化程度随造模时间的延长而逐渐加重,停止造模后肝纤维化出现自发性逆转;普鲁士蓝染色提示,随着造模时间的延长,肝脏铁沉积逐渐增加,造模4周时肝脏铁沉积面积较前增加,与正常对照组相比差异有统计学意义(t=4.772,P0.05),但在造模6周时无明显增加,仍高于正常对照,差异有统计学意义(t=10.32,P0.05);停止造模的自发逆转过程中铁沉积又出现下降;通过实时荧光定量PCR检测发现,肝脏中TNFα、IL-1β、IL-6在CCl4诱导的小鼠肝纤维化模型中持续增高,造模6周时达峰值,与正常对照组相比差异有统计学意义(t值分别为4.322、9.707、5.678,P值均0.05);肝脏hepcidin在此纤维化模型中表达早期升高,造模4周时已达峰值,与正常组相比差异有统计学意义(t=2.590,P0.05),但在自发逆转过程中表达降低,峰值早于炎症因子的表达。结论肝脏中铁沉积在CCl4诱导的小鼠肝纤维化进展过程中逐渐增加;hepcidin的表达在肝纤维化进展过程中逐渐升高,与炎症因子的表达相关。     

螺内酯预防大鼠实验性肝纤维化的动态研究

目的研究螺内酯对大鼠肝内纤维组织形成的动态变化.方法SD大鼠90只,随机分成正常对照组(8只)正常饮食,皮下注射花生油;肝纤维化模型组(42只)复合因素制成肝纤维化模型和螺内酯预防组(40只)造模方法同模型组,螺内酯每日100mg/kg灌胃.分别于第2、4、6、8周随机处死8只大鼠,取肝组织测定胶原面积和胶原定量,眼球采血检测透明质酸、Ⅲ型前胶原、Ⅳ型胶原和层粘连蛋白含量.结果螺内酯预防组较模型组同期肝组织胶原含量明显减少(P＜0 05),血清透明质酸、Ⅲ型前胶原、Ⅳ型胶原和层粘连蛋白水平均明显降低(P＜0.05).结论螺内酯有明显抑制大鼠肝纤维化形成的作用.     

Rating of CCl(4)-induced rat liver fibrosis by blood serum glycomics

BACKGROUND: Non-invasive staging of human liver fibrosis is a desirable objective that remains under extensive evaluation. Animal model systems are often used for studying human liver disease and screening antifibrotic compounds. The aim of the present study was to investigate the potential use of serum N-glycan profiles to evaluate liver fibrosis in a rat model. METHODS: Liver fibrosis and cirrhosis were induced in rats by oral administration of CCl(4). Liver injury was assessed biochemically (alanine aminotransferase [ALT] activity, aspartate aminotransferase [AST] activity and total bilirubin) and histologically. The N-glycan profile (GlycoTest) was performed using DNA sequencer-assisted-fluorophore-assisted carbohydrate electrophoresis technology. In parallel, the effect of cotreatment with antifibrotic interferon-gamma (IFN-gamma) was studied. RESULTS: The biopsy scoring system showed that CCl(4) induced early fibrosis (F < 1-2) in rats after 3 weeks of treatment, and cirrhosis (F4) after 12 weeks. Significant increases in ALT activity, AST activity and total bilirubin levels were detected only after 12 weeks of CCl(4) treatment. GlycoTest showed three glycans were significantly altered in the CCl(4)-goup. Peak 3 started at week 6, at an early stage in fibrosis development (F < 1-2), whereas peaks 4 and 5 occurred at week 9, at which time mild liver fibrosis (F = 1-2) had developed. The changes in the CCl(4)-IFN-gamma group were intermediate between the CCl(4)- and the control groups. CONCLUSION: The GlycoTest is much more sensitive than biochemical tests for evaluating liver fibrosis/cirrhosis in the rat model. The test can also be used as a non-invasive marker for screening and monitoring the antifibrotic activity of potential therapeutic compounds.     

地龙2号抑制大鼠肝纤维化的研究

目的 研究地龙2号抑制大鼠肝内纤维组织形成的作用。方法 雄性Wistar大鼠,随机分成6组:正常组:不做药物处理;阴性对照组:皮下注射花生油,单灌蒸水;模型组:皮下注射CCl4,每日单蒸水灌胃;阳性对照组:造模方法同模型组,用秋水仙碱每日0.1 mg/kg灌胃;地龙大剂量组:造模同时用地龙2号每日50mg/kg灌胃;地龙小剂量组:造模同时用地龙2号每日25mg/kg灌胃。于8周末处死大鼠,取血测血清ALT、AST、AST/ALT,放射免疫法检测透明质酸(HA)和层黏连蛋白(LN),取肝组织作HE染色,病理分级。结果 地龙2号组与模型组相比,病理示肝纤维化程度明显减轻(P<0.05),肝细胞损害亦轻(AST/ALT大剂量组P<0.05,小剂量组P<0.01),且反应肝纤维化的指标HA及LN均显著降低(P<0.01)。结论 地龙2号有明显抑制大鼠肝纤维化形成的作用。     

疏肝健脾方对四氯化碳诱导肝纤维化大鼠血清代谢组学的影响

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胆管结扎和四氯化碳诱导Wistar大鼠肝纤维化模型的建立及相关指标的对比分析

目的比较胆管结扎(BDL)、四氯化碳(CCl4)诱导大鼠肝纤维化的生化指标、肝脏病理学及纤维连接蛋白(FN)的表达。方法 90只健康雄性Wistar大鼠,分为CCl4肝纤维化模型组(44只)及其对照组(6只)和BDL肝纤维化模型组(30只)及其对照组(10只)。CCl4肝纤维化模型组是采用50%的CCl4橄榄油溶液对大鼠进行腹腔注射的方法来制备模型,而BDL肝纤维化模型组则采用结扎大鼠胆总管的方法来制备。观察大鼠一般情况,生化方法测血清ALT、AST、TBil、DBil;ELISA法测血清透明质酸(HA)和层黏连蛋白(LN)水平。HE和Masson染色观察肝组织的病理学变化,免疫组织化学染色观察肝组织FN表达。计量资料组间比较采用t检验。结果血清生化学结果显示,BDL组大鼠自胆管结扎术后第7天开始,TBil及DBil即上升到较高水平且随后一直保持此水平,CCl4肝纤维化模型组大鼠2周始逐渐升高,至8周达高峰;BDL大鼠纤维化指标HA、LN水平显著高于同期CCl4模型组;CCl4模型组大鼠可见肝细胞弥漫性脂肪变性,汇管区-汇管区或汇管区-中央静脉间纤维间隔形成极为显著,BDL大鼠则表现为肝内胆管显著增生,炎性细胞浸润及纤维间隔形成同时存在;BDL组中FN的表达呈分散无规则型,且纤维组织细小,而CCl4组的FN多集中表达于小叶之间的间隔处,纤维组织粗大。结论 BDL和CCl4均可诱导大鼠肝纤维化,BDL较早引起肝功能及肝纤维化指标升高,可见明显胆管增生,CCl4则以脂肪变性为主;FN在两种模型中表达分布不同。     

甘草酸对四氯化碳诱导大鼠肝纤维化肝组织p53蛋白表达的影响

目的：探讨甘草酸对四氯化碳诱导大鼠肝纤维化肝组织p53蛋白表达的影响。方法45只雄性SD大鼠随机均分为对照组、肝纤维化组和甘草酸干预组3组，每组各15只。肝纤维化组和甘草酸干预组构建四氯化碳诱导大鼠肝纤维化模型，对照组大鼠注射等量的橄榄油。甘草酸干预组大鼠以0．2％甘草酸溶液予腹腔注射，3 ml／只，3次／周；对照组大鼠予等量双蒸水替代。3组于实验第1、4、8周各处死5只大鼠，取肝组织行HE染色检测纤维化情况，采用免疫组织化学和Western印迹定量分析检测p53蛋白在大鼠肝组织内的表达情况。采用单因素方差分析比较各组大鼠实验第1、4、8周p53蛋白表达量差异，进一步组间两两比较采用LSD-t检验。结果实验第1周，3组大鼠肝组织p53蛋白表达量差异无统计学意义；实验第4、8周，肝纤维化组大鼠肝组织中p53蛋白表达量均较对照组大鼠升高，而甘草酸干预组大鼠肝组织中p53蛋白表达量均较肝纤维化组大鼠降低，且差异均有统计学意义（实验第4周：t值分别为2．39、2．74；实验第8周：t值分别为1．58、7．79；P值均为0．000）。结论甘草酸可减缓肝纤维化进程，其分子机制可能与p53蛋白的表达下调有关。     

初见端倪的肝纤维化靶向治疗

近年肝纤维化靶向治疗研究进展迅速,但如何将药物特异地投送至靶细胞并发挥理想效应仍然有诸多挑战。就肝纤维化靶向治疗研究中所涉及的靶细胞和靶分子及其选择原则、载体选择和修饰的一般原则及其方法、疗效和毒副作用检测等问题进行了阐述,并指出肝纤维化乃多细胞参与、多因子释放、多种信号通路激活的复杂病理过程,所以选择合适的靶点是靶向治疗的关键,而正确构建靶向投递系统是治疗成功的保证。     

莪术提取物对肝纤维化大鼠血管紧张素Ⅱ及其Ⅰ型受体的影响

中药莪术为姜科植物莪术的根茎,具行气破血、消积止痛作用,临床应用广泛,是治疗肝硬化的常用传统中药,资料显示莪术能促进肾脏细胞外基质的降解、对肝纤维化具有防治作用.研究证明,肝脏存在局部肾素-血管紧张素系统（RAS）,并在肝纤维化的发生发展中起重要作用.为了进一步阐明莪术治疗肝纤维化的作用机制,本文探讨了莪术对肝纤维化模型大鼠血管紧张素Ⅱ（ANGⅡ）及其Ⅰ型受体（AT1R）的影响.     

A distinct nitric oxide and adenosine A1 receptor dependent hepatic artery vasodilatatory response in the CCl4‐cirrhotic liver

Increase of portal venous vascular resistance is counteracted by decrease of hepatic arterial vascular resistance (hepatic arterial buffer response). This process is mediated by adenosine in normal livers. In cirrhosis, hepatic arterial vascular resistance is decreased but the involvement of adenosine in this process is unknown. The aim of our study was to identify the signalling pathway responsible for the decreased hepatic arterial resistance in cirrhotic livers. Methods: Cirrhosis was induced by CCl₄. Using a bivascular liver perfusion dose–response curves to adenosine of the HA were performed in the presence and the absence of pan‐adenosine blocker (8‐SPT), A1 blocker (caffeine) or nitric oxide synthase‐blocker (l ‐NMMA) after preconstriction with an α1‐agonist (methoxamine). Western blot of the HA were used to measure the density of the A1 and A2a receptors. Results: Adenosine caused a dose dependent relaxation of the hepatic artery of both cirrhotic and control animals that were blocked in both groups by 8‐SPT (P<0.02). The response to adenosine was greater in cirrhotic rats (P=0.016). Both l ‐NMMA (P=0.003) and caffeine reduced the response to adenosine in cirrhotic but not in control animals. Western blot analysis showed a higher density of A1 and a lower density of A2a receptor in cirrhotic animals (P<0.05). Conclusion: The adenosine‐induced vasodilatation of the HA is increased in cirrhotic rats suggesting a role for adenosine‐NO in the decreased hepatic arterial vascular resistance found in cirrhosis. This significantly greater response in cirrhosis by the A1 receptor follows the same pathway that is seen in hypoxic conditions in extra‐hepatic tissues.     

Expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by CCl4 or thioacetamide

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl₄ and thioacetamide (TAA) in rats. CCl₄ or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl₄-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl₄-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl₄-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21^WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl₄-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl₄ or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis. Received: May 1, 2000 / Accepted: August 25, 2000