The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats*

Background: Hepatopoietin Cn (HPPCn) is a member of the leucine‐rich acidic nuclear protein family (LANP), and studies of partially hepatectomized (PH) mice show that levels of HPPCn mRNA increase following liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Aim: The aim of the study was to evaluate the protective effect of rhHPPCn on liver injury and fibrosis induced by carbon tetrachloride (CCl4) injection. Methods: Wistar rats weighing 200 g were given a single and repeated intraperitoneal injections of CCl4. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in rat serum were measured using biochemical assay. Hepatic hydroxyproline (Hyp) level was determined in the hydrolysates of liver samples. Immunostaining and Masson's trichrome staining were conducted to evaluate hepatocyte proliferation and fibrosis. Results: The results showed that exogenous rhHPPCn could alleviate hepatocyte necrosis and protect the liver from the development of fibrotic lesions by proliferation stimulation. Additionally, HPPCn could reduce ALT/AST levels in rat serum following single and repeated CCl4 injection. Conclusion: It was suggested that HPPCn could protect hepatocytes from injury induced by CCl4 as a proliferation stimulator.     

Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat

BACKGROUND/AIMS: In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl4) administration in the rat. METHODS: Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl4 (1ml/kg, twice per week) and dalteparin (50IU/kg, daily) for 7 weeks. RESULTS: Dalteparin did not prevent acute CCl4-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24h after simultaneous administration of CCl4 and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12h after injection of CCl4 were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl4 treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl4-induction of smooth muscle alpha-actin expression, alpha1(I)procollagen and transforming growth factor (TGF)-beta1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2'deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner. CONCLUSIONS: Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl4 treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.     

Significance and therapeutic potential of endothelial progenitor cell transplantation in a cirrhotic liver rat model

BACKGROUND & AIMS: We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells. METHODS: Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks. RESULTS: At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates. CONCLUSIONS: We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.     

肌细胞增强因子2A与肝星状细胞活化关系的体内实验研究

背景：转录调控在肝星状细胞（HSC）活化过程中起重要作用,研究显示转录因子肌细胞增强因子2（MEF2）参与了HSC的活化过程。目的：探讨肝纤维化形成过程中MEF2家族成员MEF2A与HSC活化的关系。方法：实验开始时处死6只大鼠作为0周对照组,64只大鼠随机分为正常对照组和肝纤维化模型组。模型组大鼠皮下注射60％CCL（0．3ml／100g,每周2次）复制肝纤维化模型;正常对照组大鼠与模型组于相同条件下饲养,不予任何处理。造模开始后3、6、9、12周分批处死大鼠,取肝组织,实时荧光定量聚合酶链反应（PCR）检测MEF2AmRNA表达．蛋白质印迹法检测MEF2A蛋白和HSC活化标记物α-平滑肌肌动蛋白（α-SMA）表达,VanGieson染色定量分析肝内胶原含量。结果：正常肝组织中仅有少量MEF2AmRNA和蛋白表达;随着肝纤维化的形成,肝组织MEF2AmRNA和蛋白表达量逐渐增加（P〈0．05）,MEF2A与Q—SMA蛋白表达呈正相关（r=0．88,P〈0．05）。肝内胶原含量随肝纤维化的形成呈递增趋势（P〈0．01）。结论：MEF2A参与了CCl4。诱导的大鼠肝纤维化形成过程中HSC的活化和增殖。     

体外四氯化碳损伤肝细胞对肝星状细胞活化的影响及其丹酚酸A的干预效果

目的 体外观察肝细胞四氯化碳过氧化损伤后肝星状细胞（ＨＳＣ）活化的影响,并观察丹参水溶性成分丹酚酸Ａ的干预效果,以研究脂质过氧化损伤与肝纤维化的关系和丹酚酸Ａ的作用机理。方法 肝细胞分离后分组,分别添加不同浓度的丹酚酸Ａ以及对照药维生素Ｅ,同时以四氯化碳熏蒸２４ｈ并测定各组细胞上清液ＡＬＴ、ＡＳＴ活性后,换液培养２４ｈ并测定ＭＤＡ含量,再以此上清液作为“肝细胞条件培养液”作用于ＨＳＣ４８ｈ,观察Ｈ     

Increased susceptibility to liver fibrosis with age is correlated with an altered inflammatory response

It has been suggested that increasing age is correlated with an acceleration of the progression of liver fibrosis induced by various agents, such as hepatitis C virus or chronic alcohol consumption. However, the cellular and molecular changes underlying this predisposition are not entirely understood. In the context of an aging population, it becomes challenging to decipher the mechanisms responsible for this higher susceptibility of older individuals to this acquired liver disorder. To address this issue, we induced liver fibrosis by carbon tetrachloride (CCl(4)) chronic administration to 8-week- and 15-month-old mice. We confirmed that susceptibility to fibrosis development increased with age and showed that aging did not affect fibrosis resolution capacity. We then focused on the impairment of hepatocyte proliferation, oxidative stress, and inflammation as potential mechanisms accelerating the development of fibrosis in the elderly. We detected no inhibition of hepatocyte proliferation after CCl(4) injury in 15-month-old mice, whereas it was inhibited after a partial hepatectomy. Finally, we observed that, in a context in which liver oxidative stress was not differentially increased in both experimental groups, there was a higher recruitment of inflammatory cells, including mostly macrophages and lymphocytes, oriented toward a T helper 2 (T(H)2) response in older mice. Our data show that in conditions of equivalent levels of oxidative stress and maintained hepatocyte proliferative capacity, an increased inflammatory reaction mainly composed of CD4(+) lymphocytes and macrophages expressing T(H)2 cytokines is the main factor involved in the higher susceptibility to fibrosis with increasing age.     

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice

BACKGROUND/AIMS: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied. METHODS: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis. RESULTS: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice. CONCLUSIONS: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.     

Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration.

An enzyme-linked immunosorbent assay was used to measure the level of hepatocyte growth factor in rat plasma at various times after two-thirds partial hepatectomy or CCl4 administration. An initial 17-fold rise and 13-fold rise in the level of hepatocyte growth factor was observed 2 hr after partial hepatectomy and CCl4 treatment, respectively, well before the onset of DNA synthesis in the liver. The peaks of DNA synthesis in remnant livers and livers exposed to CCl4 occurred at 24 hr and 48 hr, respectively, as determined by 5-bromo-2'-deoxyuridine labeling and [3H]thymidine uptake by the liver. A later peak level (17-fold above control) of hepatocyte growth factor at 24 hr after CCl4 treatment coincided with strong immunostaining of damaged or necrotic hepatocytes around central veins with an antibody to hepatocyte growth factor. This suggests a later intrahepatic origin of the signals for liver regeneration after hepatotoxic injury subsequent to the early extrahepatic production of hepatocyte growth factor at 2 hr after CCl4 administration. The absence of staining in the liver remnants in partially hepatectomized rats implies that the increase in hepatocyte growth factor seen in the plasma is caused by production at extrahepatic site(s). Possible sources include the pancreas, brain, thyroid and salivary glands, and Brunner's glands of the duodenum. Norepinephrine also increases in plasma as early as 2 hr after hepatectomy. In vitro, [3H]thymidine incorporation into hepatocyte DNA in the presence of hepatocyte growth factor is greater if 10(-5) mol/L norepinephrine is also present in the media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice

Oncostatin M (OSM) is a member of the IL-6 family of cytokines. Mice deficient in the OSM receptor (OSMR(-/-)) showed impaired liver regeneration with persistent parenchymal necrosis after carbon tetrachloride (CCl(4)) exposure. The recovery of liver mass from partial hepatectomy was also significantly delayed in OSMR(-/-) mice. In contrast to wildtype mice, CCl(4) administration only marginally induced expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 genes in OSMR(-/-) mice, correlating with the increased gelatinase activity of matrix metalloproteinase (MMP)-9 and matrix degradation in injured livers. The activation of STAT3 and expression of immediate early genes and cyclins were decreased in OSMR(-/-) liver, indicating that OSM signaling is required for hepatocyte proliferation and tissue remodeling during liver regeneration. We also found that CCl(4) administration in IL-6(-/-) mice failed to induce OSM expression and that OSM administration in IL-6(-/-) mice after CCl(4) injection induced the expression of cyclin D1 and proliferating cell nuclear antigen, suggesting that OSM is a key mediator of IL-6 in liver regeneration. Consistent with these results, administration of OSM ameliorated liver injury in wildtype mice by preventing hepatocyte apoptosis as well as tissue destruction. In conclusion, OSM and its signaling pathway may provide a useful therapeutic target for liver regeneration.     

High,but not low,dietary retinoids aggravate manifestation of rat liver fibrosis

BACKGROUND: Although there is strong implication that retinoids regulate Ito cell proliferation and collagen synthesis, results from in vivo studies on the relationship between vitamin A and liver fibrosis are conflicting. The present study focuses on the role of vitamin A in carbon tetrachloride (CCl4)-induced fibrosis by chronic feeding of rats with either a vitamin A-supplemented or -depleted diet. METHODS AND RESULTS: In animals with high dietary hepatic retinoid levels, liver fibrosis was more pronounced and was associated with an increased CCl4-toxicity resulting in high mortality (73%). Enhanced liver fibrosis was confirmed by in vivo fluorescence microscopic determination of both collagen deposits (7.4 +/- 1.1 vs 3.9 +/- 0.3% in high vitamin A diet-fed and standard diet-fed fibrotic animals, respectively; P < 0.05) and rarefication of sinusoids (1.5 +/- 0.2 vs 2.4 +/- 0.2 sinusoids/200 microm in high vitamin A diet-fed and standard diet-fed fibrotic animals, respectively; P < 0.05). It was further associated with decreased bile flow and increased parenchymal cell damage. CCl4 reduced hepatic retinoid levels in high vitamin A diet-fed animals, but restored hepatic retinoid levels in animals fed with a vitamin A-deficient diet, implying major interference of vitamin A metabolism with hepatotoxic agents such as CCl4. Low vitamin A feeding did not modulate liver fibrogenesis and caused no mortality. CONCLUSIONS: These results show that the vitamin A status of the liver plays an important role in liver fibrogenesis. While dietary vitamin A shortage does not promote liver fibrogenesis, high levels of vitamin A have the potential to increase systemic and hepatic toxicity of CCl4. Thus, the narrow therapeutic window for nutritional vitamin A substitution must take into account that liver fibrotic patients may display enhanced susceptibility to the adverse effects of vitamin A.     

Dietary iron overload inhibits carbon tetrachloride-induced promotion in chemical hepatocarcinogenesis: effects on cell proliferation, apoptosis, and antioxidation

BACKGROUND/AIMS: The aim of this study was to investigate if feeding with carbonyl iron would facilitate the development of preneoplastic lesions initiated by diethylnitrosamine (DEN) and promoted by CCl4-induced liver cirrhosis. METHODS: Male Wistar rats were fed a diet with 1.25%-2.5% carbonyl iron for 23 weeks and received intragastric injections of CCl4 (1.0 or 2.0 ml/kg per week) for 13 weeks, followed by one i.p. injection of DEN (200 mg/kg), after which CCl4 was administered for 8 additional weeks. Animals were killed 48 h after the first CCl4 injection to evaluate liver necrosis, 8 weeks later to evaluate fibrosis, and 9 weeks after DEN to determine formation of glutathione S-transferase 7,7 (GST-7,7) positive foci. RESULTS: Treatment with iron counteracted the increased serum alanine aminotransferase levels and liver necrosis following CCl4 administration. Hepatic levels of reduced Q9 and alpha-tocopherol were elevated in rats treated with CCl4 and decreased in rats treated with iron compared to the controls. Fibrogenesis was not altered by iron treatment. Nine weeks after DEN initiation, the number and volume density of GST-7,7-positive foci in rats treated with CCl4 were significantly increased as compared with controls, but co-treatment with iron inhibited this increase. Apoptotic index was increased in iron-loaded livers, and labelling index (the fraction of S-phase hepatocytes) was decreased by co-treatment with iron in livers exposed to CCl4. CONCLUSION: Carbonyl iron depleted hepatic levels of antioxidants, it decreased CCl4-induced necrosis and cell proliferation, it enhanced apoptosis and did not facilitate fibrogenesis. These effects together may explain the suppression of CCl4-induced promotion after DEN initiation exerted by carbonyl iron in the present study.     

Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice

Liver fibrogenesis is associated with the transition of quiescent hepatocytes and hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl(4) . To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive CcnE1(-/-) and CcnE2(-/-) knock-out mice with CCl(4) to induce liver fibrosis. Interestingly, CcnE1(-/-) mice were protected against CCl(4) -mediated liver fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2(-/-) mice showed accelerated fibrogenesis after CCl(4) treatment. We isolated primary HSCs from WT, CcnE1(-/-) , and CcnE2(-/-) mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1(-/-) HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis. (HEPATOLOGY 2012;56:1140-1149).     

Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells

Rodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34(+) or highly purified CD34(+)CD38(-)CD7(-) human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl(4)) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl(4)-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl(4) injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl(4)-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl(4)-mediated liver injury. Our studies provide evidence that human "hematopoietic" stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.