Effects of retrorsine on mouse hepatocyte proliferation after liver injury

AIM： To study the effect of retrorsine on mouse hepatocyte proliferation. METHODS： Mice and rats were treated respectively with two injections of retrorsine （as retrosine-treated group） or saline （as non-treated group） at 2 wk intervals. They received a single injection of carbon tetrachloride （CCh） 4 wk later. On d 0, 1, 2, 3, 4, 6, 15 after CCh administration, the animals were killed and their livers were excised. Hematoxylin and eosin （HE） staining and Ki-67 antibody immunohistochemical analysis of liver samples were used to evaluate the pathological changes and hepatocyte proliferation. RESULTS： In rats treated with retrorsine and CCl4, the liver displayed obvious megalocytosis, proliferation of mild bile duct, small hepatocyte-forming nodule, which were not found in liver samples from non-treated group. However, in mice treated with retrorsine combined with CCh, the liver displayed hepatocyte degeneration and necrosis in perivenous areas. There was no obvious difference between retrorsine-treated group and nontreated group. Ki-67 immunohistochemical analysis showed that in rats treated with retrorsine, the positive hepatocytes mainly found in small hepatocyte nodules, were obviously less than those in non-treated group. The mice treated with retrorsine showed that the number of Ki-67 positive hepatocytes was very high and more than that in non-treated group. CONCLUSION： Retrorsine has no effect on mouse hepatocyte proliferation.

Disrupted NF-κB activation after partial hepatectomy does not impair hepatocyte proliferation in rats

AIM： To analyze the effects of NF-kB inhibition by antioxidant pyrrolidine dithiocarbamate （PDTC） or TNF inhibitor pentoxifylline （PTX） on liver regeneration after partial hepatectomy （PH）. METHODS： Saline, PDTC or PTX were injected 1 h before PH and rats were killed at 0.5 and 24 h after PH. Several control groups were used for comparison （injection control groups）. RESULTS： Compared to saline injected controls, NF-kB activation was absent 0.5 h after PH in rats treated with PDTC or PTX. At 24 h after PH, DNA synthesis and PCNA expression were identical in treated and control rats and thus occurred irrespectively of the status of NF-kB activation at 0.5 h. Signal transducer and activator of transcription 3 （Stat3） acUvatJon was observed already 0.5 h after PH in saline, PDTC or PTX group and was similar to Stat3 activation in response to injection without PH. CONCLUSION： These data strongly suggest that （1） NF-kB p65/p50 DNA binding produced in response to PH is not a signal necessary to initiate the liver regeneration, （2） star3 activation is a stress response unrelated to the activation of NF-kB. In conclusion, NF-kB activation is not critically required for the process of liver regeneration after PH.

Identification of Human Hepatocyte Proliferation Related Gene C2orf69

Objective To construct the prokaryotic expression vector p ET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained.Methods Gene fragment of C2orf69 was amplified by PCR and then prokaryotic expression plasmid pE T-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identified by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and specificity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay(ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully amplified, results of gene sequencing were consistent with the sequence in Gen Bank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors

The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor(EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGFinduced receptor down-regulation. Furthermore, the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma sMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.

Hepatotropic growth factors protect hepatocytes during inflammation by upregulation of antioxidative systems

AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-containing cytokine mixture of interleukin-1β,tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors,e.g.hepatocyte growth factor,epidermal growth factor and/or transforming growth factor-α.Cells were analyzed for glutathione levels.Culture supernatants were assayed for produc-tion of reactive oxygen intermediates(ROIs) as well as NO2-,NO3-and S-nitrosothiols.To determine cellular damage,release of aspartate aminotransferase(AST) into the culture medium was analyzed.Activation of nuclear factor(NF)-κB was measured by electrophoretic mobility shift assay.RESULTS:Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation.AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture,independent of growth-factor co-stimulation.However,pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione.Application of growth factors did not result in increased NF-κB activation.Pretreatment with growth factors further increased formation of NO2-,NO3-and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture.Thus,we propose that,together with an increase in glutathione increased NO2-,NO3-formation might shift their metabolism towards non-toxic products.CONCLUSION:Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.

Hepatoprotective effects of baicalein against CCl 4-induced acute liver injury in mice

AIM:To investigate the hepatoprotective effect of baicalein against carbon tetrachloride(CCl 4)-induced liver damage in mice.METHODS:Mice were orally administered with baicalein after CCl 4 injection,and therapeutic baicalein was given twice a day for 4 d.The anti-inflammation effects of baicalein were assessed directly by hepatic histology and serum alanine aminotranferease and aspartate aminotransferase measurement.Proliferating cell nuclear antigen was used to evaluate the effect of baicalein in promoting hepatocyte proliferation.Serum interleukin(IL)-6,IL-1β and tumor necrosis factor-α(TNF-α) levels were measured by enzyme-linked immunosorbent assay and liver IL-6,TNF-α,transforming growth factor-α(TGF-α),hepatocyte growth factor(HGF) and epidermal growth factor(EGF) genes expression were determined by quantitative real-time polymerase chain reaction.RESULTS:CCl4-induced acute liver failure model offers a survival benefit in baicalein-treated mice.The data indicated that the mRNA levels of IL-6 and TNF-α significantly increased within 12 h after CCl 4 treatment in baicalein administration groups,but at 24,48 and 72 h,the expression of IL-6 and TNF-α was kept at lower levels compared with the control.The expression of TGF-α,HGF and EGF was enhanced dramatically in baicalein administration group at 12,24,48 and 72 h.Furthermore,we found that baicalein significantly elevated the serum level of TNF-α and IL-6 at the early phase,which indicated that baicalein could facilitate the initiating events in liver regeneration.CONCLUSION:Baicalein may be a therapeutic candidate for acute liver injury.Baicalein accelerates liver regeneration by regulating TNF-α and IL-6 mediated pathways.

Protective effect of recombinant human IL-1Ra on CCl_4-induced acute liver injury in mice

AIM: To evaluate the effects of positive regulation of recombinant human interleukin 1 receptor antagonist (rhIL-1Ra) on hepatic tissue recovery in acute liver injury in mice induced by carbon tetrachloride (CCl 4 ). METHODS: Acute liver damage was induced by injecting 8-wk-old mice with CCl 4 1 mL/kg (1:3 dilution in corn oil) intraperitoneally (ip). Survival after liver failure was assessed by injecting 8-wk-old mice with a lethal dose of CCl 4 2.6 mL/kg (1:1 dilution in corn oil) ip. Mice were subcutaneously injected with 1 mg/kg recombinant human IL-1Ra twice a day after CCl 4 treatment for 5 d. Serum alanine amino transferase (ALT) and aspartate aminotransferase (AST) levels were determined with a commercial assay kit. Serum IL-1β, IL-1Ra levels were measured by enzyme-linked immunosorbent assay kit. Quantitative real-time polymerase chain reaction was used to determine liver IL-1β, IL-1Ra and IL-6 expression during CCl 4-induced acute liver injury. Liver sections were stained with hematoxylin-eosin. A histology-injury grading system was used to evaluate the degree of necrosis after acute liver injury. Proliferating cell nuclear antigen (PCNA) staining was used to evaluate the role of rhIL-1Ra in promoting hepatocyte proliferation. RESULTS: Quantitative analysis showed a higher level of IL-6 mRNA expression and reduced serum AST and ALT levels in the livers of the rhIL-1Ra-treated group at the early phase of CCl 4-induced acute liver injury. Histological examination indicated a decrease in centrilobular necrotic areas in mice treated with rhIL-1Ra, and a novel role of rhIL-1Ra in promoting hepatocyte proliferation was also supported by an increase of PCNA staining. All these results, accompanied by a strong survival benefit in rhIL-1Ra-treated vs PBS-treated groups, demonstrated that rhIL-1Ra administration ameliorated the histological damage and accelerated the regeneration and recovery process of the liver. CONCLUSION: rhIL-1Ra could be further developed as a novel therapeutic agent for the treatment of acute liver injury because of its ability to reduce hepatocellular damage and facilitate liver regeneration.

Dimensions of hepatocellular carcinoma phenotypic diversity

Hepatocellular carcinoma(HCC) is the 3^(rd) leading cause of cancer-related death worldwide. More than 80% of HCCs arise within chronic liver disease resulting from viral hepatitis, alcohol, hemochromatosis, obesity and metabolic syndrome or genotoxins. Projections based on Western lifestyle and its metabolic consequences anticipate a further increase in incidence, despite recent breakthroughs in the management of viral hepatitis. HCCs display high heterogeneity of molecular phenotypes, which challenges clinical management. However, emerging molecular classifications of HCCs have not yet formed a unified corpus translatable to the clinical practice. Thus, patient management is currently based upon tumor number, size, vascular invasion, performance status and functional liver reserve. Nonetheless, an impressive body of molecular evidence emerged within the last 20 years and is becoming increasingly available to medical practitioners and researchers in the form of repositories. Therefore, the aim this work is to review molecular data underlying HCC classifications and to organize this corpus into the major dimensions explaining HCC phenotypic diversity. Major efforts have been recently made worldwide toward a unifying "clinically-friendly" molecular landscape. As a result, a consensus emerges on three major dimensions explaining the HCC heterogeneity. In the first dimension, tumor cell proliferation and differentiation enabled allocation of HCCs to two major classes presenting profoundly different clinical aggressiveness. In the second dimension, HCC microenvironment and tumor immunity underlie recent therapeutic breakthroughs prolonging patients' survival. In the third dimension,metabolic reprogramming, with the recent emergence of subclass-specific metabolic profiles, may lead to adaptive and combined therapeutic approaches. Therefore, here we review recent molecular evidence, their impact on tumor histopathological features and clinical behavior and highlight the remaining challenges to translate our cognitive corpus into patient diagnosis and allocation to therapeutic options.

YAP regulates the liver size during the fasting-refeeding transition in mice

Liver is the central hub regulating energy metabolism during feeding-fasting transition.Evidence suggests that fasting and refeeding induce dynamic changes in liver size,but the underlying mechanisms remain unclear.Yes-associated protein(YAP)is a key regulator of organ size.This study aims to explore the role of YAP in fasting-and refeeding-induced changes in liver size.Here,fasting significantly reduced liver size,which was recovered to the normal level after refeeding.Moreover,hepatocyte size was decreased and hepatocyte proliferation was inhibited after fasting.Conversely,refeeding promoted hepatocyte enlargement and proliferation compared to fasted state.Mechanistically,fasting or refeeding regulated the expression of YAP and its downstream targets,as well as the proliferation-related protein cyclin D1(CCND1).Furthermore,fasting significantly reduced the liver size in AAV-control mice,which was mitigated in AAV Yap(5SA)mice.Yap overexpression also prevented the effect of fasting on hepatocyte size and proliferation.Besides,the recovery of liver size after refeeding was delayed in AAV Yap shRNA mice.Yap knockdown attenuated refeeding-induced hepatocyte enlargement and proliferation.In summary,this study demonstrated that YAP plays an important role in dynamic changes of liver size during fasting-refeeding transition,which provides new evidence for YAP in regulating liver size under energy stress.