The extent of liver injury determines hepatocyte fate toward senescence or cancer.

It is well known that induction of hepatocyte senescence could inhibit the development of hepatocellular carcinoma (HCC). Until now, it is still unclear how the degree of liver injury dictates hepatocyte senescence and carcinogenesis. In this study, we investigated whether the severity of injury determines cell fate decisions between hepatocyte senescence and carcinogenesis. After testing of different degrees of liver injury, we found that hepatocyte senescence is strongly induced in the setting of severe acute liver injury. Longer-term, moderate liver injury, on the contrary did not result into hepatocyte senescence, but led to a significant incidence of HCC instead. In addition, carcinogenesis was significantly reduced by the induction of severe acute injury after chronic moderate liver injury. Meanwhile, immune surveillance, especially the activations of macrophages, was activated after re-induction of senescence by severe acute liver injury. We conclude that severe acute liver injury leads to hepatocyte senescence along with activating immune surveillance and a low incidence of HCC, whereas chronic moderate injury allows hepatocytes to proliferate rather than to enter into senescence, and correlates with a high incidence of HCC. This study improves our understanding in hepatocyte cell fate decisions and suggests a potential clinical strategy to induce senescence to treat HCC.

The effect of carvedilol and propranolol on portal hypertension in patients with cirrhosis: a meta-analysis.

PURPOSE: Several randomized controlled clinical trials have been conducted to investigate the role of carvedilol and propranolol on the effect of portal pressure in patients with cirrhosis, leading to controversial results. Current meta-analysis was performed to compare the efficacy of the two drugs on portal pressure. PATIENTS AND METHODS: Two-hundred and ninety eligible patients were recruited. Published studies were selected based on PubMed, the Cochrane Library, Chinese Journal Full-text Database, and Wanfang Database. The outcome measurements included the mean difference (MD) in the percentage of hepatic vein pressure gradient reduction (%HVPG reduction), the risk ratio (RR) of nonresponders in hemodynamic assessment, and the percentage of mean arterial pressure reduction (%MAP reduction). Subgroup analysis was performed. RESULTS: Seven trials were identified (including five acute and three long-term drug administration randomized controlled trials). A summary of pooled MD between the %HVPG reduction is as follows: overall -8.62 (confidence interval [CI] -11.76, -5.48, P<0.00001), acute -10.05 (CI -14.24, -5.86, P<0.00001), and long term -6.80 (CI -11.53, -2.07, P=0.005), while summary of pooled RR of hemodynamic nonresponders with carvedilol was as follows: overall 0.64 (CI 0.51, 0.81, P=0.0002), acute 0.63 (CI 0.47, 0.85, P=0.002), and long term 0.67 (CI 0.47, 0.97, P=0.03). Both of the outcome measurements favored carvedilol. Significant heterogeneity (P<0.1, I (2)=92%) existed between the two treatment groups in %MAP reduction. No considerable difference could be observed in the %MAP reduction through the poor overlapping CI boundaries. CONCLUSION: Carvedilol has a greater portal hypertensive effect than propranolol. Further comparative trials of the two drugs are required to identify the effect of MAP reduction.

GDC-0980-induced apoptosis is enhanced by autophagy inhibition in human pancreatic cancer cells.

Pancreatic cancer remains fatal to the fast majority of affected patients. Activation of phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway plays an important role in pancreatic cancer progression and chemo-resistance. In the present study, we examined the activity of GDC-0980, a novel class I PI3K/mTOR kinase inhibitor, against pancreatic cancer cells in vitro. GDC-0980 inhibited AKT-mTOR activation and pancreatic cancer cell (PANC-1 and Capan-1 lines) survival. In both cancer cell lines, GDC-0980 simultaneously activated apoptosis and autophagy, the latter was detected by p62 degradation, Beclin-1 upregulation and light chain 3B (LC3B) conversion from a cytosolic (LC3B-I) to a membrane-bound (LC3B-II) form. Autophagy inhibitors including 3-methyladenine, hydroxychloroquine, NH4Cl and bafilomycin A1 enhanced apoptosis and cytotoxicity by GDC-0980, such an effect was reversed by caspase inhibitors (z-VAD-FMK and z-ITED-FMK). Furthermore, knockdown of LC3B or Beclin-1 through siRNA increased GDC-0980-induced anti-pancreatic cancer cell activity. Thus, inhibition of autophagy sensitizes GDC-0980-induced anti-pancreatic cancer activity, suggesting a novel therapeutic strategy for GDC-0980 sensitization.

miR-186 regulates glycolysis through Glut1 during the formation of cancer-associated fibroblasts.

Emerging evidence has suggested that glycolysis is enhanced in cancer-associated fibroblasts (CAF), and miR-186 is downregulated during the CAF formation. However, it is not clear whether miR-186 is involved in the regulation of glycolysis and what the role of miR-186 plays during the CAF formation. In this study, quantitative PCR analysises show miR-186 is downregulated during the CAF formation. Moreover, miR-186 targets the 3' UTR of Glut1, and its overexpression results in the degradation of Glut1 mRNA, which eventually reduces the level of Glut1 protein. On the other hand, knockdown of miR-186 increased the expression of Glut1. Both time course and dose response experiments also demonstrated that the protein and mRNA levels of Glut1 increase during CAF formation, according to Western blot and quantitative PCR analyses, respectively. Most importantly, besides the regulation on cell cycle progression, miR-186 regulates glucose uptake and lactate production which is mediated by Glut1. These observations suggest that miR-186 plays important roles in glycolysis regulation as well as cell cycle checkpoint activation.

Hexokinase 2 regulates G1/S checkpoint through CDK2 in cancer-associated fibroblasts.

Hexokinase 2 (HK2), a pivotal glycolytic enzyme, is often overexpressed in tumor cells and contributes to glycolysis. Emerging evidence has suggested that glycolysis is also enhanced in cancer-associated fibroblasts (CAF). However, it is not clear whether HK2 is involved in enhanced glycolysis in CAFs or what role HK2 plays in the CAFs. In this study, both time course experiments and dose response experiments demonstrated that the protein and mRNA levels of HK2 increase in CAF cells, according to western blot and quantitative PCR analyses, respectively. Additionally, miR-182 targets the 3' UTR of HK2, and its overexpression results in the degradation of HK2 mRNA, which eventually reduces the level of HK2 protein. On the other hand, knockdown of miR-182 increased the expression of HK2. Most importantly, HK2 regulated the protein level and T14 phosphorylation of CDK2, and knockdown of HK2 resulted in a G1 phase cell cycle arrest. These observations suggest that HK2 plays important roles in glycolysis regulation and in cell cycle checkpoint activation.

Wnt-induced secreted protein 1/CCN4 in liver fibrosis both in vitro and in vivo.

BACKGROUND: Wnt-induced secreted protein-1 (WISP-1/CCN4) is a member of the CCN family growth factors, and its role in liver fibrosis is largely unknown. METHODS: For in vitro, hepatic stellate cells (HSCs) were isolated from Sprague-Dawley rats. Expression of WISP-1 during progressive activation of cultured rat HSCs was analyzed by qRT-PCR. The effects of TNF-a and TGF-beta1 on WISP-1 expression were analyzed in stellate cell lines HSC-T6 and LX-2. The effect of exogenous WISP-1 protein on LX-2 proliferation was examined. For in vivo, expressions of WISP-1 in fibrotic liver of a carbon tetrachloride (CCl4)-induced fibrosis rat model were analyzed by qRT-PCR and immunohistochemistry. RESULTS: In vitro, WISP-1 was increasingly expressed during progressive activation of cultured rat HSCs. WISP-1 was significantly induced in HSC-T6 cells by TNF-a and in LX-2 cells by TGF-beta1. Recombinant WISP-1 protein promoted LX-2 proliferation in a dose-dependent manner. In vivo, both mRNA and protein expression levels of WISP-1 were increased significantly in experimental hepatic fibrosis model. CONCLUSIONS: Our results showed the upregulation of WISP-1 in both in vitro and in vivo liver fibrosis models, and WISP-1 stimulated the proliferation of HSCs in vitro. These results may be helpful to elucidate the exact role of WISP-1 in liver fibrogenesis.

Focal peliosis hepatis in a colon cancer patient resembling metastatic liver tumor.

Peliosis hepatis (PH) is a rare benign condition characterized by the presence of multiple, randomly distributed, blood filled cystic areas of variable size within the liver parenchyma. PH is difficult to recognize and may be mistaken for neoplasm, metastases or multiple abscesses. A 75-year-old female with a previous history of colon cancer was admitted when a liver mass in the right liver lobe was found 11 mo after surgery during the follow-up period. Computed tomography and magnetic resonance imaging scan of the abdomen were performed. The initial possible diagnosis was metastatic hepatocellular carcinoma. The patient underwent excision of the hepatic segment where the nodule was located. The pathological diagnosis of the surgical specimen was PH. PH should be considered in the differential diagnosis of new liver lesions in patients whose clinical settings do not clearly favor metastasization. Clinicians and radiologists must recognize these lesions to minimize the probability of misdiagnosis and inappropriate treatment.

Evaluation of in vivo antioxidant and immunity enhancing activities of sodium aescinate injection liquid.

Oxidative stress is involved in the development and progression of disease. Because sodium aescinate has been reported to have immunity enhancing and antioxidative effects, we investigated its activity by employing a hepatocellular carcinoma (HCC) mouse model. Sixty BALB/c mice were randomly divided into four groups, including a 1.4 mg/kg treated group (n = 15), a 2.8 mg/kg treated group (n = 15), an untreated hepatocellular carcinoma control group (n = 15) and a normal control group (n = 15). After H22 cells were cultured for one week, we collected 2 × 106 cells and injected them subcutaneously as 0.2 mL cell suspensions in sterile saline into the right shoulder region of every mouse. The animals were monitored for changes in activity, physical condition and body weight during the experiment. The next day after injection of H22 cells, animals in these test groups received one intraperitoneal injection of drug or physiological saline for 13 days. Results showed that in the sodium aescinate injection liquid (SAIL)-treated HCC mice, serum interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), Gamma-glutamyltransferase (γ-GT), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels were significantly decreased compared with normal control mice. In addition, treatment with sodium aescinate injection liquid significantly decreased blood and liver malondialdehyde (MDA) levels, increased glutathione (GSH) levels, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)] activities in a dose-dependent manner. We conclude that sodium aescinate injection liquid can decrease oxidative injury and enhance immunity functions in HCC mice.

Effect of oxymatrine on hepatic gene expression profile in experimental liver fibrosis of rats.

OBJECTIVE: To investigate the effects of oxymatrine on hepatic gene expression profile in a rat model of liver fibrosis. METHODS: Forty healthy male SD rats were randomly divided into three groups, a normal group (n=8), a model group (n=16), and an oxymatrine treatment group (n=16). Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)). The rats in the treatment group received oxymatrine via celiac injection at a dosage of 40 mg/kg once a day at the same time. The rats in the model and normal groups received saline at the same dosage via celiac injection. Serum levels of aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (AKP), hyaluronic acid (HA), and laminin (LN) were assayed. The deposition of collagen was observed with HE and Masson staining. Effect of oxymatrine on hepatic gene expression profile was detected by oligonucleotide microarray analysis with Affymetrix gene chip rat U230A. Quantitative real-time polymerase chain reaction (QRT-PCR) was carried out to confirm the expression changes of six genes. RESULTS: Oxymatrine significantly improved liver function, lowered serum levels of HA and LN, and decreased the degree of liver fibrosis, compared with the model group (P<0.05). A total of 754 differentially expressed genes were identified by gene chip between the model group and the normal group, among which 438 genes increased and 316 genes decreased over two folds. Compared with the model group, 86 genes were downregulated markedly in the oxymatrine group (P<0.05), including collagen I and other genes related to extracellular material (ECM), integrin signal transduction genes, early growth response factor genes, and proinflammatory genes; 28 genes were upregulated significantly (P<0.05), including cytochrome P450 (CYP450) superfamily genes, glycolipids metabolism and biological transformation related genes. Six genes were confirmed with QRT-PCR, consistent with the result from microarray. CONCLUSION: Oxymatrine could affect the expression of many functional genes and may be useful in the prevention and treatment of liver fibrosis.

Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays.

AIM: To identify differentially expressed genes in quiescent and activated hepatic stellate cells (HSCs) and explore their functions. METHODS: HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. Total RNA and mRNA of quiescent HSCs, and culture-activated HSCs were extracted, quantified and reversely transcripted into cDNA. The global gene expression profile was analyzed by microarray with Affymetrix rat genechip. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Microarray data were validated by quantitative real-time polymerase chain reaction (qRT-PCR). The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi. The expression of Wnt5a in fibrotic liver of a carbon tetrachloride (CCl(4))-induced fibrosis rat model was also analyzed with Western blotting. RESULTS: Of the 28 700 genes represented on this chip, 2566 genes displayed at least a 2-fold increase or decrease in expression at a P < 0.01 level with a false discovery rate. Of these, 1396 genes were upregulated, while 1170 genes were downregulated in culture-activated HSCs. These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms. The most enriched GO terms included response to wounding, wound healing, regulation of cell growth, vasculature development and actin cytoskeleton organization. KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs. Wnt5a was significantly increased in culture-activated HSCs as compared with quiescent HSCs. qRT-PCR validated the microarray data. Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation, downregulated expressions of type I collagen and transforming growth factor-ß1. Wnt5a was upregulated in the fibrotic liver of a CCl(4)-induced fibrosis rat model. CONCLUSION: Wnt5a is involved in the activation of HSCs, and it may serve as a novel therapeutic target in the treatment of liver fibrosis.

Simultaneous determination of l-citrulline and l-arginine in plasma by high performance liquid chromatography.

OBJECTIVE: To develop a method for simultaneously determining l-citrulline and L-arginine levels in plasma using RP-HPLC with ultraviolet detection. DESIGN AND METHODS: Plasma samples were deproteinized by trichloroacetic acid and heat. Phenyl-isothiocyanate (PITC) solution was used as derivatization reagent and a gradient elution was carried out. RESULTS: The linearity for L-citrulline and L-arginine ranged from 0 to at least 1000 micromol/L. R(2) values were above 0.9999 for both. LODs for L-citrulline and L-arginine were 0.0201 micromol/L and 0.0476 micromol/L, respectively, while LOQs were 0.240 micromol/L and 0.448 micromol/L, respectively. Intra- and inter-day CVs were less than 3.40% and 7.2%, respectively. The average recovery was from 86.22% to 118.9%. L-citrulline and L-arginine concentrations in healthy controls were 60.77+/-9.18 micromol/L and 58.19+/-16.43 micromol/L, respectively. CONCLUSION: This approach offers a reliable, efficient analytical platform for the simultaneous determination of citrulline and arginine levels in plasma.

Combined MELD and blood lipid level in evaluating the prognosis of decompensated cirrhosis.

AIM: To evaluate the prognostic value of the combined model for end-stage liver disease (MELD) and blood lipid level in patients with decompensated cirrhosis. METHODS: A total of 198 patients with decompensated cirrhosis were enrolled into the study. The values of triglyceride (TG), cholesterol (TC), high density lipoproteins (HDL) and low density lipoprotein (LDL) of each patient on the first day of admission were retrieved from the medical records, and MELD was calculated. All the patients were followed up for 1 year. The relationship between the change of blood lipid level and the value of MELD score was studied by analysis of variance. The prognostic factors were screened by multivariate Cox proportional hazard model. Draw Kaplan-Meier survival curves were drawn. RESULTS: Forty-five patients died within 3 mo and 83 patients died within 1 year. The levels of TG, TC, HDL and LDL of the death group were all lower than those of the survivors. The serum TG, TC, HDL and LDL levels were lowered with the increase of the MELD score. Multivariate Cox proportional hazard model showed that MELD >or= 18 and TC or= 18 combined with TC or= 18 and TC

Comparison of four models for end-stage liver disease in evaluating the prognosis of cirrhosis.

AIM: To investigate the prognostic value of the model for end-stage liver disease (MELD) and three new MELD-based models combination with serum sodium in decompensated cirrhosis patients-the MELD with the incorporation of serum sodium (MELD-Na), the integrated MELD (iMELD), and the MELD to sodium (MESO) index. METHODS: A total of 166 patients with decompensated cirrhosis were enrolled into the study. MELD, MELD-Na, iMELD and MESO scores were calculated for each patient following the original formula on the first day of admission. All patients were followed up at least 1 year. The predictive prognosis related with the four models was determined by the area under the receiver operating characteristic curve (AUC) of the four parameters. Kaplan-Meier survival curves were made using the cut-offs identified by means of receiver operating characteristic (ROC). RESULTS: Out of 166 patients, 38 patients with significantly higher MELD-Na (28.84 +/- 2.43 vs 14.72 +/- 0.60), iMELD (49.04 +/- 1.72 vs 35.52 +/- 0.67), MESO scores (1.59 +/- 0.82 vs 0.99 +/- 0.42) compared to the survivors died within 3 mo (P < 0.001). Of 166 patients, 75 with markedly higher MELD-Na (23.01 +/- 1.51 vs 13.78 +/- 0.69), iMELD (44.06 +/- 1.19 vs 34.12 +/- 0.69), MESO scores (1.37 +/- 0.70 vs 0.93 +/- 0.40) than the survivors died within 1 year (P < 0.001). At 3 mo of enrollment, the iMELD had the highest AUC (0.841), and was followed by the MELD-Na (0.766), MESO (0.723), all larger than MELD (0.773); At 1 year, the iMELD still had the highest AUC (0.783), the difference between the iMELD and MELD was statistically significant (P < 0.05). Survival curves showed that the three new models were all clearly discriminated the patients who survived or died in short-term as well as intermediate-term (P < 0.001). CONCLUSION: Three new models, changed with serum sodium (MELD-Na, iMELD, MESO) can exactly predict the prognosis of patients with decompensated cirrhosis for short and intermediate period, and may enhance the prognostic accuracy of MELD. The iMELD is better prognostic model for outcome prediction in patients with decompensated cirrhosis.

Protective effect of Jiechangning decoction in treating experimental ulcerative colitis in guinea pigs.

OBJECTIVE: To study the therapeutic effects and mechanism of Jiechangning (JCN) decoction on carrageenan induced experimental ulcerative colitis (UC). METHODS: After sensitizing guinea pigs with carrageenan, we established UC animal models by free drinking water containing 2% acid degraded carrageenan (ADC). JCN decoction was orally administered once a day for 2 weeks after carrageenan treatment. Salicylazosulfapyridine (SASP) and normal saline were given to the other two groups as control. The levels of colon lipid peroxide (LPO), acid phosphatase (ACP) activity and tumor necrosis factor-alpha (TNF-alpha) were measured; colitis activity score (CAS) was carried out for assessment of the degree of tissue inflammation and injury; the colonic pathological changes were examined simultaneously with hematoxylin and eosin (HE) and toluidine blue staining used to evaluate the therapeutic effects of JCN decoction and SASP. RESULTS: Experimental colitis models resembling human UC were successfully induced. The levels of tissue LPO, ACP activity and the content of tissue TNF-alpha were markedly increased in the model group as compared with the normal control group (P < 0.01) and were positively correlated with CAS. JCN decoction could reverse these changes like SASP. HE staining showed that JCN decoction and SASP could reduce CAS and the degree of tissue injury, toluidine blue staining revealed that mucosa and submucosa red metachromasia pellets in JCN group and SASP group were markedly fewer than those in the model group. CONCLUSION: JCN decoction is effective in treating experimental UC, which provides theoretical basis for its clinical application.