Yuewen Gong

Antioxidative function of L‐FABP in L‐FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L‐FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L‐FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L‐FABP mRNA level. Full‐length L‐FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA‐FABP). Chang cells were stably transfected with pc‐DNA‐FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 μmol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L‐FABP was documented by regular reverse transcription polymerase chain reaction (RT‐PCR), real‐time RT‐PCR, and Western blot. The pcDNA‐FABP–transfected cells expressed full‐length L‐FABP mRNA, which was absent from vector‐transfected control cells. Western blot showed expression of 14‐kd L‐FABP protein in pcDNA‐FABP–transfected cells, but not in vector‐transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L‐FABP expression. Lower LDH release was observed in the higher L‐FABP–expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L‐FABP gene. The L‐FABP–expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L‐FABP has a significant role in oxidative stress. (HEPATOLOGY 2005;42:871–879.)

Use of proliferating cell nuclear antigen as a marker of liver regeneration after partial hepatectomy in rats

In order to document and compare proliferating cell nuclear antigen (PCNA) mRNA and protein levels with more traditional parameters of hepatic regenerative activity in a rat model, adult male Sprague-Dawley rats (4 to 6 per group) were killed at various times up to 96 hours after 70% partial hepatectomy. At each time interval, tissue PCNA mRNA abundance and protein levels were documented (by Northern and Western blot analysis, respectively) and compared with the results of PCNA immunostaining and 3H-thymidine incorporation into hepatic DNA. Tissue PCNA protein levels were also documented in additional groups of rats 12, 24, 36, and 48 hours after sham or 30% partial hepatectomy. PCNA mRNA expression after partial hepatectomy was variable: almost undetectable at 24 hours, levels returned to baseline at 36 hours, then fell again to low levels at 96 hours. PCNA protein levels remained stable for 36 hours, increased to fourfold above baseline (p < 0.01) at 48 hours, then remained elevated for the duration of the 96-hour study. Changes in PCNA by immunostaining were similar but tended to occur somewhat earlier (significant increases being detectable at 24 hours), whereas 3H-thymidine incorporation detected the earliest increases in DNA synthesis at 12 hours and peaked at 36 hours. Peak PCNA protein levels correlated with the extent (0%, 30%, or 70%) of hepatic resection. The results indicate that PCNA protein level as determined by Western blot analysis, but not PCNA mRNA expression, correlates with PCNA immunostaining and 3H-thymidine incorporation in the regenerating liver. These findings support the use of PCNA protein determinations as an additional quantitative measure of hepatic regenerative activity after partial hepatectomy in rats.

Increased Renal Aldose Reductase Activity, Immunoreactivity, and mRNA in Streptozocin-Induced Diabetic Rats

Increased accumulation of renal sorbitol has been documented in the diabetic rat, and it has been suggested that this accumulation may be important in the pathogenesis of diabetic nephropathy. It is not clear whether sorbitol accumulation results from increases in substrate, activity of the aldose reductase (AR) protein molecule, or activity due to an increase in the amount of enzyme present. In this study, we have quantitated renal AR activity, immunoreactivity, and mRNA in rats 3 mo after induction of diabetes with streptozocin (STZ-D, 65 mg/kg body wt). Renal AR activity was significantly increased in diabetic rats compared with age-matched nondiabetic controls (0.95 ± 0.05 vs. 0.51 ± 0.03 U · mg−1 · h−1, respectively, P < .0005). Western blot analysis demonstrated that the antiserums recognized a single 40,000-Mr, protein species in renal homogenates from both diabetic and nondiabetic rats. When quantitated in an immunodot assay, AR immunoreactivity was significantly increased in diabetic rats compared with nondiabetic controls (0.57 ± 0.03 vs. 0.33 ± 0.02 U, respectively, P < .0005). Hybridization of Northern blots with a synthetic 36-nucleotide oligomer and an AR cDNA identified a 1.4-kilobase pair transcript; the abundance of the transcript was significantly increased in poly(A)+RNA from the kidneys of diabetic compared with nondiabetic rats (P < .005). This study demonstrates that renal AR activity is increased in the STZ-D rats and suggests that the increased AR activity can be in part explained by enhanced AR gene expression.

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma.

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity.

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABPs antioxidant activity is through inactivation of the free radicals by L-FABPs methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Meta-analysis of prophylactic corticosteroid use in post-ERCP pancreatitis

BackgroundAcute pancreatitis is a common complication of endoscopic retrograde cholangiopancreatography and benefit of pharmacological treatment is unclear. Although prophylactic use of corticosteroid for reduction of pancreatic injury after ERCP has been evaluated, discrepancy about beneficial effect of corticosteroid on pancreatic injury still exists. The aim of current study is to evaluate effectiveness and safety of corticosteroid in prophylaxis of post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP).MethodsWe employed the method recommended by the Cochrane Collaboration to perform a meta-analysis of seven randomized controlled trials (RCTs) of corticosteroid in prevention of post-ERCP pancreatitis (PEP) around the world.ResultsMost of the seven RCTs were of high quality. When the RCTs were analyzed, odds ratios (OR) for corticosteroid were 1.13 [95% CI (0.89~1.44), p = 0.32] for PEP, 1.61 [95% CI (0.74~3.52), p = 0.23] for severe PEP, 0.92 [95% CI (0.57~1.48), p = 0.73] for post-ERCP hyperamylasemia respectively. The results indicated that there were no beneficial effects of corticosteroid on acute pancreatitis and hyperamylasemia. No evidence of publication bias was found.ConclusionCorticosteroids cannot prevent pancreatic injury after ERCP. Therefore, their use in the prophylaxis of PEP is not recommended.

Serum immunoglobulins predict the extent of hepatic fibrosis in patients with chronic hepatitis C virus infection

Summary.  Recently, we documented that immunoglobulins stimulate the proliferative activity of rat hepatic stellate cells in vitro. The aim of the present study was to determine whether there is any association between serum immunoglobulin levels and hepatic fibrosis in patients with chronic hepatitis C virus (HCV) infection. Charts from 116 patients with biochemical, serologic, virologic and histologic evidence of chronic hepatitis C infection and serum immunoglobulin levels (IgA, IgG, IgM and total) were reviewed. The mean (±SD) age of the study population was 46 ± 11 years and 67 (58%) were male. There were significant correlations between serum IgA (r = 0.39, P = 0.00001), IgG (r = 0.49, P = 0.000002) and total (r = 0.51, P = 0.000003) immunoglobulin levels and the stage of hepatic fibrosis. When serum immunoglobulin levels were included into logistic regression analysis with variables known to be associated with advanced disease (male gender, age >40 years at onset of infection, duration of infection beyond 20 years and concurrent alcohol abuse) only IgA, IgG and total immunoglobulin levels (P < 0.05, <0.05 and <0.005, respectively) emerged as independent predictors of hepatic fibrosis. Our data indicate a strong association between serum immunoglobulin levels (IgA, IgG and total) and hepatic fibrosis in patients with HCV infection. This finding supports the need to further investigate whether immunoglobulins independently promote disease progression in patients with chronic HCV infection.

Randomized trial of autologous bone marrow mesenchymal stem cells transplantation for hepatitis B virus cirrhosis: regulation of Treg/Th17 cells.

Liver cirrhosis is one of the major consequences of hepatitis B virus (HBV) infection, and transplantation of autologous bone marrow mesenchymal stem cells (ABMSCs) is one of promising therapies for patients with HBV‐related liver cirrhosis (HBV‐LC). However, the mechanism is unclear. The aim of the current study was to explore the role of Treg/Th17 cells in ABMSCs transplantation in patients with HBV‐LC.

Bone Morphogenetic Protein-4 Induced Rat Hepatic Progenitor Cell (WB-F344 Cell) Differentiation Toward Hepatocyte Lineage

Hepatic progenitor cells are local stem cells in the liver and they can be differentiated into either hepatocytes or cholangiocytes depending on different stimulations. These stimulations include extracellular growth factors and intracellular transcription factors. Bone morphogenetic protein 4 (BMP4) is a member of transforming growth factor beta (TGF‐β) superfamily and was first identified as growth factor to induce ectopic bone formation from skeletal muscle. Role of BMP4 in the liver is still unclear especially its role in hepatic progenitor cells (HPCs) differentiation. BMP4 was used to stimulate rat HPCs (WB‐F344 cells) and differentiation of WB‐F344 cells was investigated by reverse transcriptase polymerase chain reaction (RT‐PCR) and Western blot analysis. Both adenovirus delivered BMP4 and recombinant BMP4 were able to induce expression of hepatocyte markers such as albumin, TAT‐1, and G6Pase but not cholangiocyte markers such as β4‐integrin and CK19. BMP4 induced differentiation of WB‐F344 cells toward hepatocytes was mediated by increase in phosphorylation of Smad1 and ERK1/2. Moreover, BMP4 also stimulated expression of transcription factor—C/EBP‐α, which involved in differentiation of WB‐F344 cells toward hepatocytes. BMP4 is able to stimulate WB‐F344 cells differentiation toward hepatocyte lineage. J. Cell. Physiol. 220: 72–81, 2009.

Decreased hepatocyte membrane potential differences and GABAa‐β3 expression in human hepatocellular carcinoma

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and γ‐aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real‐time reverse‐transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (−19.8 ± 1.3 versus −25.9 ± 3.2 mV, respectively [P < 0.05]), and GABAA‐β3 expression was down‐regulated (GABAA‐β3 mRNA and protein expression in HCC; 5,693 ± 1,385 and 0.29 ± 0.11 versus 11,046 ± 4,979 copies/100 mg RNA and 0.62 ± 0.16 optical density in adjacent tumor tissues, respectively [P = 0.002 and P < 0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down‐regulation, human malignant hepatocytes deficient in GABAA‐β3 receptor expression (Huh‐7 cells) were transfected with GABAA‐β3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n = 16‐17 group). Tumors developed after a mean (± SD) of 51 ± 6 days (range: 41‐60 days) in 7/16 (44%) mice injected with vector‐transfected cells and 70 ± 12 days (range: 59‐86 days) in 4/17 (24%) mice injected with GABAA‐β3 cDNA‐transfected cells (P < 0.005). Conclusion: The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA‐β3 receptor expression is down‐regulated in human HCC, and (3) restoration of GABAA‐β3 receptor expression results in attenuated in vivo tumor growth in nude mice. (HEPATOLOGY 2007;45:735–745.)