Role of hepatic vitamin A and lipocyte distribution in experimental hepatic fibrosis

ABSTRACT— Lipocytes are the major site of hepatic vitamin A storage, and they have been demonstrated to lose their vitamin A content in the process of hepatic fibrosis. To investigate the relationship between hepatic vitamin A content and the degree of hepatic fibrosis, we measured levels of retinyl palmitate and retinol in the CCl₄-induced fibrotic liver using high-performance liquid chromatography. We estimated hepatic collagen content using a spectrophotometric analysis with sirius red, and also by measuring hydroxyproline levels. Lipocytes were detected by an immunoperoxidase method with anti-desmin antibody, and were counted morphometrically through a Texture Analyzing System. A significant negative correlation was observed between the level of retinyl palmitate and collagen content (r = –0.64) as well as the hydroxyproline level (r = –0.69) in the CCl₄-induced fibrotic liver. In the process of fibrosis, hepatic retinol levels were elevated in association with a decrease in retinyl palmitate. In particular in the early stage of fibrosis, lipocytes increased remarkably in number in fibrotic areas in spite of a decrease in total hepatic vitamin A. The present study suggests that an increase in hepatic retinol as well as a decrease in retinyl palmitate may facilitate the process of hepatic fibrosis produced by lipocytes.     

罗汉果甜苷对四氯化碳诱导的肝纤维化大鼠的保护作用

目的研究罗汉果甜苷对大鼠肝纤维化的保护作用。方法除正常组外,其余各组采用四氯化碳(CCL4)诱导肝纤维化模型,于造模第7周起,给药组分别灌胃给予相应的受试药,疗程6周。实验结束后,观察罗汉果甜苷对大鼠血清ALT、AST活性,HA、PⅢP、CⅣ含量,肝组织SOD活性及MDA含量的影响,观察罗汉果甜苷对肝组织转化生长因子β1(TGF-β1)表达的影响及对肝组织病理变化的影响。结果罗汉果甜苷各剂量组能明显降低肝纤维化大鼠ALT、AST、MDA、CⅣ、PⅢNP含量,升高SOD水平,减轻肝组织病理损伤程度,抑制肝组织中TGF-β1表达。结论罗汉果甜苷对CCL4诱导的肝纤维化大鼠有很好的保护作用。     

熊去氧胆酸对大鼠免疫性肝纤维化的干预作用研究

研究熊去氧胆酸对大鼠免疫性肝纤维化的干预作用,探讨其抗肝纤维化的机制。64只SD大鼠随机分为4组。32只大鼠腹腔内注射猪血清诱导建立肝纤维化模型;其中药物干预组16只予以熊去氧胆酸（UDCA）灌胃,余16只为模型对照组。另32只大鼠中16只给予UDCA为药物对照组,其余16只大鼠为空白对照组。于2、4、6、8周末分批处死动物,观察肝组织纤维化程度并进行免疫组化染色。发现正常肝组织不表达或微弱表达血小板衍生生长因子（PDGF-B）、组织基质金属蛋白酶抑制剂-1（TIMP-1）,纤维化的肝组织PDGF-B、TIMP-1表达随肝纤维化分期的升高而增强,两者的表达呈正相关。药物干预组较模型组病理改变轻,而且肝组织PDGF-B和TIMP-1的表达下降。UDCA对大鼠免疫性肝纤维化有明显改善作用,其机制可能是抑制PDGF-B分泌及其介导的与TIMP-1增强有关的肝纤维化过程。     

Hypervitaminosis A-induced liver fibrosis: stellate cell activation and daily dose consumption.

Hypervitaminosis A-related liver toxicity may be severe and may even lead to cirrhosis. In the normal liver, vitamin A is stored in hepatic stellate cells (HSC), which are prone to becoming activated and acquiring a myofibroblast-like phenotype, producing large amounts of extracellular matrix. AIMS: In order to assess the relationship between vitamin A intake, HSC activation and fibrosis, we studied nine liver biopsies from patients belonging to a well-characterized series of 41 patients with vitamin A hepatotoxicity. METHODS: Fibrosis was underlined by Sirius-red staining, whereas activated HSC were immunohistochemically identified using an antibody against alpha smooth muscle actin. The volume density (Vv) of sinusoidal and total fibrosis and of sinusoidal and total activated HSC was quantified by the point-counting method. RESULTS: Morphology ranged from HSC hypertrophy and hyperplasia as the sole features to severe architectural distortion. There was a significant positive correlation between Vv of perisinusoidal fibrosis and the daily consumption of vitamin A (P=0.004). CONCLUSION: The close correlation between the severity of perisinusoidal fibrosis and the daily dose of the retinol intake suggests the existence of a dose-effect relationship.     

Liver ischemia/reperfusion induces an increase of microvascular leukocyte flux,but not heterogeneity of leukocyte trafficking

Abstract: Leukocytic response plays a major role in the manifestation of hepatic ischemia/reperfusion (I/R) injury. To clarify whether post-ischemic hepatic leukocyte accumulation is based on increased leukocyte flux to the hepatic tissue due to systemic inflammation or chemoattractant activities or whether it represents solely a local tissue response without changing overall leukocyte flux and trafficking characteristics through the microvasculature, we studied acinar and sinusoidal leukocyte flux and distribution in rat livers in vivo both under normal (sham, n=8) and post-ischemic (60′ ischemia/75′ reperfusion) conditions (I/R, n=8), using fluorescence epi-illumination microscopy (rhodamine-6G). Hepatic ischemia/reperfusion significantly (p<0.05) increased acinar leukocyte flux (58.4±20.9 cells/min vs 36.4±12.8 cells/min in sham controls); however, it did not exhibit increased heterogeneity of acinar leukocyte distribution, as indicated by the unchanged coefficient of variance (CV) of 0.36±0.16 (sham controls: 0.31 ±0.14). In parallel, analysis of individual sinusoidal leukocyte flux demonstrated significantly (p<0.05) higher values (8.9±3.7 cells/min) after ischemia/reperfusion when compared with sham controls (5.7±1.9 cells/min), which, however, was not associated with increased heterogeneity of sinusoidal leukocyte trafficking (CV: 0.85±0.15 vs 0.85±0.16 in sham controls) and manifestation of preferential pathways. Analysis of blood cell count did not demonstrate an overall increase of total blood leukoycte count; however, an increased (p<0.01) fraction of polymorphonuclear leukocytes (65.2±11.2%) and stab cells (9.5±7.9%) during post-ischemic reperfusion when compared with sham controls (8.8±3.5% and 0.2±0.4%) was demonstrated. Thus, the increase of hepatic leukocyte flux after ischemia/reperfusion may be the result of both the manifestation of a systemic inflammatory response and the increase of local chemoattractant activities, such as the production and release of the cytokine-induced neutrophil chemoattractant of the IL-8 family.     

Ursodeoxycholic acid treatment improves hepatocyte ultrastructure in rat liver fibrosis

AIM： To examine the ultrastructural changes after ursodeoxycholic acid （UDCA） treatment in hepatocytes from experimentally induced fibrotic livers.METHODS： Liver fibrosis was induced in male Sprague-Dawley rats with CCl4 for 12 wk, and the rats were divided into two groups. Group I was treated with saline and group Ⅱ with UDCA （25 mg/kg per day） for 4 wk. All the rats were killed at wk 16. Mitochondria, nuclei, rough endoplasmic reticulum （RER） and smooth endoplasmic reticulum （SER） of hepatocytes were evaluated according to a scoring system.RESULTS： Mitochondria, nuclei, RER and SER injury scores in group Ⅱ were significantly lower than those in groupⅠ（P 〈 0.001）. CONCLUSION： UDCA alleviates hepatocyte organelle injury in CCl4-induced liver fibrosis.     

复方汉防己冲剂抗肝纤维化的实验研究

我们应用复方汉防己冲剂对Wistar大鼠CCl4造成肝纤维化动物模型进行疗效研究，现将研究结果报道如下。[第一段]     

四氯化碳肝纤维化大鼠肝组织差异蛋白质组的动态变化

目的 探索肝纤维化形成过程中肝组织蛋白质组的变化,部分解析肝纤维化发生、发展的病理生理机制. 方法 采用四氯化碳(CCl4)诱导大鼠肝纤维化模型,应用双向凝胶电泳技术分离大鼠肝组织总蛋白质,经考马斯亮蓝染色、图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间串联质谱及数据库查询鉴定差异蛋白质点.采用Western blot、免疫组织化学方法对细胞角蛋白(CK)8、CK18进行蛋白质表达水平的验证.计量资料采用单因素方差分析中的LSD法或非参数检验中的H检验进行统计学分析. 结果 大鼠造模后,随时间的推移,肝纤维化评分逐渐增加(3周组＜6周组＜9周组),建立了正常大鼠和大鼠CCl4肝纤维化形成过程3、6、9周肝组织双向凝胶电泳图谱,质谱鉴定了44种差异蛋白质;采用Western blot、免疫组织化学方法对CK8、CK18的验证结果与双向凝胶电泳结果基本一致.正常组及3、6、9周模型组CK8/CK18蛋白相对表达量分别为:0.113±0.005/0.170±0.030、0.473±0.046/0.530±0.070、0.682±0.087/0.780±0.080、0.837±0.096/1.390±0.130,各组间差异均有统计学意义(F值分别为196.085/74.088、13.870/16.115、75.800/75.900,P值均＜0.01). 结论 CCl4大鼠肝纤维化形成与发展过程中差异表达蛋白质的功能主要涉及细胞生长、发育与分化,细胞增殖与凋亡,血管新生或重构,氧化应激,物质代谢及转运,信号转导等.     

Inhibitory effect of acyclic retinoid (polyprenoic acid) on hepatic fibrosis in CCl4-treated rats

A study was conducted to examine the inhibitory effect of acyclic retinoid (polyprenoic acid) on the development of hepatic fibrosis induced by CC1₄ in rats. Oral administration of the compound brought about a significant reduction in both serum and tissue levels of immunoreactive prolyl hydroxylase, a key enzyme of collagen formation. The result indicated that the rate of collagen synthesis in the liver was decreased which was consistent with histological findings. Acyclic retinoid also decreased both AST and ALT activities in serum, demonstrating the reduction in hepatic parenchymal damage. This cytoprotective effect on parenchymal cells may be related, at least in part, to inhibition of hepatic fibrosis. No significant side effects were observed, despite a long-term administration of the acyclic retinoid. The present findings suggest the potential scope of therapy of hepatic fibrosis by retinoid.     

Significance of septal fibrosis for disturbance of hepatic circulation

Abstract: To examine the significance of fibrous septa of the liver for hepatic circulatory disturbance, haemodynamic changes were investigated in rats with septal fibrosis induced with horse serum injections. The fibrotic liver showed thin fibrous bands originating in the vicinity of the peripheral branches of the hepatic vein connected with each other and partly with the portal triads, without conspicuous periportal, pericentral or perisinusoidal fibrosis. There was no evidence of hepatic cell enlargement, disarrangement of hepatic cell plates or narrowing of the sinusoids in the fibrotic livers. Portal vascular resistance, bile production and hepatic oxygen consumption, which were measured by an isolated liver perfusion method, were much the same in the normal and the fibrotic livers. Moreover, there was no significant difference in in vivo blood pressures of the portal vein, the terminal portal venule, the terminal hepatic venule and the inferior vena cava between the normal and the fibrotic rats. These data suggest that septal fibrosis in itself does not disturb hepatic circulation.     

Increased liver echogenicity at ultrasound examination reflects degree of steatosis but not of fibrosis in asymptomatic patients with mild/moderate abnormalities of liver transaminases

AIMS: To investigate whether hyperechogenicity of liver can reliably be interpreted as liver steatosis and if any concomitant or isolated fibrosis can be disclosed. PATIENTS AND METHODS: A series of 165 patients with no signs or symptoms of liver disease referred because of slightly to moderately raised aminotransferases (alanine aminotransferase and/or aspartate aminotransferase 0.7-5.0 microkat/l) for more than 6 months were prospectively investigated with a comprehensive laboratory profile, ultrasound examination of liver and percutaneous liver biopsy Fibrosis was assessed quantitatively and according to Metavir. Steatosis was graded as none, mild, moderate or severe. RESULTS: Of 98 (59.4%) patients with raised echogenicity, 85 (86.7%) had liver steatosis of at least moderate degree, 9 patients with same degree of steatosis had normal echogenicity and 13 patients with no or only mild steatosis had a hyperechogenic liver (sensitivity 0.90, specificity 0.82, positive predictive value 0.87, negative predictive value 0.87). About the same relations were found regardless of body mass index and degree of fibrosis. With increased echogenicity together with high attenuation (n = 591 and reduced portal vessel wall distinction (n = 79), positive predictive value increased to 0.93 and 0.94, respectively. Quantitatively assessed fibrosis (mean +/- SD) was 3.2 +/- 4.6% of biopsy area with normal and 2.3 +/- 1.8% with raised echogenicity (ns). Echogenicity was normal in 5 out of 9 patients with septal fibrosis and in 4 out of 6 patients with cirrhosis. Any structural, non-homogenous findings at ultrasound were not associated with architectural fibrotic changes and none had nodular contours of liver surface. CONCLUSIONS: Assessment of liver echogenicity is of value for detection or exclusion of moderate to pronounced fatty infiltration (correct classification 86.6%) but cannot be relied upon in diagnosing fibrosis, not even cirrhosis in asymptomatic patients with mild to moderately elevated liver transaminases.     

绞股蓝总皂苷对四氯化碳诱导大鼠肝纤维化的防治作用

目的:观察绞股蓝总皂苷对四氯化碳( CCl4)诱导的大鼠肝纤维化的防治作用.方法:采用CCl4诱导的大鼠肝纤维化模型,分为正常组(Z,n=6)、模型组(M,n=8)、绞股蓝总皂苷组(J,n=8)、秋水仙碱(Q,n=8).造模6周末开始给药(股蓝总皂苷200mg/kg体重、秋水仙碱0.1mg/kg体重),给药3周.观察:①大鼠体重、肝脾比值的变化;②血清丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、谷氨酰转肽酶(GGT)活性、白蛋白( Alb)、总胆红素(TBil)含量、肝组织羟脯氨酸(Hyp)含量;③肝组织超氧化物歧化酶(SOD)活性及丙二醛(MDA)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)含量;④肝组织病理及胶原沉积情况.结果:①M组大鼠血清ALT、AST、GGT、TBil显著升高,Alb显著降低;J和Q组大鼠血清ALT、AST、GGT、TBil显著下降,Alb显著升高;②M组大鼠肝组织Hyp含量显著升高,J组及Q组大鼠肝组织Hyp含量显著下降;③肝组织HE染色显示:M组大鼠肝细胞脂肪变性,大量纤维结缔组织增生,假小叶形成.J组及Q组大鼠肝细胞脂肪变性减轻,纤维增生减少,少见完整假小叶结构.天狼星红染色显示:M组大鼠肝窦周胶原沉积明显,形成较厚汇管区和中央静脉间的纤维间隔,J组和Q组大鼠肝脏汇管区胶原纤维染较M组明显减轻;④M组大鼠肝组织SOD活性及GSH含量明显降低,MDA含量显著升高.J组大鼠肝组织SOD活性显著提高.结论:绞股蓝总皂苷具有显著抗CCl4诱导的大鼠肝纤维化及氧化损伤的作用.     

血瘀型肝纤维化大鼠模型的建立与评价

目的：复制血瘀型肝纤维化大鼠病证结合动物模型。方法：采用复合多因素的造模方法，在DMN（二甲基亚硝胺）制作大鼠肝纤维化模型的同时，多次静脉培予去甲肾上腺素和小牛血清白蛋白综合追模，对模型大鼠进行血瘀证的体征观察，采用多部位微循环显微镜行肠系膜微循环检测，并进行肝脏病理组织学观察。结果：模型（血瘀组）大鼠出现明显的血瘀表现，血瘀证候积分明显高于DMN组；血瘀组大鼠肠系膜微血管均出现典型微循环障碍。肠系膜血管管径血瘀组较DMN组增粗显著（P〈0．01）；血瘀组红细胞聚集明显。血囊流态以粒流或粒缓流为主。肝脏病理显示血瘀组纤维化改变程度较DMN组更严重，部分肝窦中可见明显扩张瘀血。结论：病证结合动物模型既具备肝纤维化的病理特征。又符合中医血瘀证的证候表现。     

Kupffer cells are mandatory for adequate liver regeneration by mediating hyperperfusion via modulation of vasoactive proteins

OBJECTIVE: Physiological liver regeneration requires adequate microvascular perfusion after partial hepatectomy. Although Kupffer cells (KCs) are known to play a key role in modulating hepatocyte proliferation, their impact on regulating hepatic microcirculation during liver regeneration has so far been disregarded. With respect to their expression and modulation of vasoactive mediators, KCs may provide important signals that regulate hepatic perfusion during liver regeneration. METHODS: Intravital fluorescence microscopy, immunohistochemistry, Western blot analysis, and RT-PCR were used to analyze livers of KC-depleted mice (liposome-encapsulated clodronate application) and KC-competent mice at days 3, 5, and 8 after 68% hepatectomy. RESULTS: Selective and long-lasting KC elimination limited the resection-associated hyperperfusion, as evidenced by an only 1.7- to 2-fold increase of sinusoidal volumetric blood flow and shear stress in contrast to a 3.5- to 5-fold increase in KC-competent mice. In accordance to that livers of KC-depleted mice showed an altered pattern of vasoregulatory gene expression. KC-depleted mice failed to show resection-induced increase of HO-1 and eNOS protein expression, but revealed a reduction of hepatic eNOS and HO-1 protein levels to 22 and 12% of the corresponding values in KC-competent mice. In addition, the eNOS inhibitory protein caveolin-1 was increased in KC-depleted animals prior to as well as after resection. Furthermore, resection-associated accumulation of ET-1 mRNA was absent in KC-depleted livers. Finally, liver mass restoration was impaired, with a regain of only 79% weight within 8 days after resection in KC-depleted mice. CONCLUSION: The present study documents a remarkable change of the vasoactive mediator profile upon KC-depletion and liver resection, limiting intrahepatic hyperperfusion. Therefore, KC-dependent molecular mechanisms seem to be mandatory in regulating vasotonus during the process of liver regeneration and therewith maintaining intrahepatic shear stress as a trigger of hepatic proliferation.     

高血糖对四氯化碳中毒大鼠肝纤维化进展的影响

本研究通过链脲佐菌素(STZ)和(或)四氯化碳(CCI4) 制备糖尿病和肝纤维化模型,探讨高血糖对CCI4中毒大鼠肝纤维化进展的影响。一、材料与方法 1．动物模型:雄性SD大鼠24只,体重245-275g,购自上海西普尔-必凯实验动物中心,随机分为模型组(n=18)和     

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM To observe the inhibition of antisenseoligonucleotides(asON)phosphorthioate to thetissue inhibitors metalloproteinase-1(TIMP-1)gene and protein expression in the liver tissue ofimmunologically induced hepatic fibrosis rats.The possibility of reversing hepatic fibrosisthrough gene therapy was observed.METHODS Human serum albumin(HSA)wasused to attack rats,as hepatic fibrosis model,inwhich asONs were used to block the gene andprotein expressing TIMP-1.According to theanalysis of modulator,structure protein,codingseries of TIMP-1 genome,we designed fourdifferent asONs.These asONs were injected intothe hepatic fibrosis models through coccygealvein.The results was observed by RT-PCR formeasuring TIMP-1 mRNA expression,immunohistochemistry and in situ hybridizationfor collagen Ⅰ,Ⅲ,special staining of collagenfiber,and electron microscopic examination.RESULTS Hepatic fibrosis could last within 363days in our modified model.The expressinglevel of TiMP-1 was high during hepatic fibrosisprocess.It has been proved by theimmunohistochemical and the electronmicroscopic examination that the asON phosphorthioate of TIMP-1 could exactly expressin vivo,The effect of colchicine wasdemonstrated to inhibit the expressing level ofmRNA and the content of collagen Ⅰ,Ⅲ in theliver of experimental hepatic fibrosis rats,However,the electron microscopy research andthe pathologic grading of hepatic fibrosisshowed that there was no significant differencebetween the treatment group and the modelgroup(P>0.05).CONCLUSION The experimental rat model ofhepatic fibrosis is one of the preferable modelsto estimate the curative effect of anti-hepaticfibrosis drugs.The asON phosphorthioate ofTIMP-1 could block the gene and proteinexpression of TIMP-1 in the liver of experimentalhepatic fibrosis rats at the mRNA level.It ispossible to reverse hepatic fibrosis,and it isexpected to study a new drug of anti-hepaticfibrosis on the genetic level.Colchicine has verylimited therapeutic effect on hepatic fibrosis,furthermore,its toxicity and side effects areobvious.     

茴三硫对大鼠酒精及四氯化碳致肝纤维化的防治作用

目的：复制大鼠酒精及四氯化碳致肝纤维化动物模型，观察茴三硫对其起防治作用的剂量大小。方法：健康雄性Wistar大鼠60只，随机分为6组，实验持续4周。A组(空白对照组)：生理盐水灌胃，每天1ml／100g体重。B组(模型组)：白酒灌胃，每天1mg／100g体重，同时CCl4腹腔内每次注射0．025mg／100g体重，每周2次。C、D、E组(茴三硫小、中、大剂量组)：在B组基础上预先灌胃茴三硫每天0．5、1．0、2．0mg／100g体重。F组(阳性对照组)：在B组基础上凯西莱预先灌胃，每天10mg／100g体重。第2周末、第4周末分别取血检测ALT、AST、ALP，GSH-Px、MDA等项目。实验结束处死动物，取肝组织行病理检验。结果：酒精及四氯化碳可引起大鼠肝功能损害、脂质过氧化加重，组织学出现肝纤维化表现。给予茴三硫可明显减轻这些改变。结论：茴三硫对大鼠酒精及四氯化碳致肝纤维化有明显防治作用。     

不同剂量γ-干扰素治疗大鼠肝纤维化疗效研究

目的 观察不同剂量重组人γ-干扰素（IFN-γ）对不同原因引起的大鼠肝纤维化的治疗效果。方法 用四氯化碳（CCl4）和二甲基亚硝胺（DMN）分别诱导形成大鼠纤维化模型,采用3种不同剂量IFN-γ对2种模型肝纤维化大鼠进行治疗,并与正常对照组、秋水仙碱治疗组和模型对照组相比较,观察各组肝功能、血清透明质酸（HA）水平、肝组织羟脯氨酸（Hyp）含量、病理组织学、免疫组化α-SMA的变化。结果 2种模型组中,3组IFN-γ治疗肝纤维化程度均明显小于秋水仙碱治疗组和模型对照组,部分肝功能指标、肝Hyp含量、血清HA水平较秋水仙碱治疗组和模型对照组亦有显著降低。3组IFN-γ治疗组之间,随剂量增大存在明显量效关系。结论 IFN-γ对不同原因引起的大鼠纤维化有较好疗效,IFN-γ的治疗效果与剂量有关。