Role of Ras-related Nuclear Protein/Polypyrimidine Tract Binding Protein in Facilitating the Replication of Hepatitis C Virus.

BACKGROUND AND AIMS: Ras-related nuclear (RAN) protein is a small GTP-binding protein that is indispensable for the translocation of RNA and proteins through the nuclear pore complex. Recent studies have indicated that RAN plays an important role in virus infection. However, the role of RAN in hepatitis C virus (HCV) infection is unclear. The objective of this study was to investigate the role and underlying mechanisms of RAN in HCV infection. METHODS: Huh7.5.1 cells were infected with the JC1-Luc virus for 24 h and then were incubated with complete medium for an additional 48 h. HCV infection and RAN expression were determined using luciferase assay, quantitative reverse transcription-PCR and western blotting. Small interfering RNA was used to silence RAN. Western blotting and immunofluorescence were used to evaluate the cytoplasmic translocation of polypyrimidine tract-binding (PTB), and coimmunoprecipitation was used to examine the interaction between RAN and PTB. RESULTS: HCV infection significantly induced RAN expression and cytoplasmic redistribution of PTB. Knockdown of RAN dramatically inhibited HCV infection and the cytoplasmic accumulation of PTB. Colocalization of RAN and PTB was determined by immunofluorescence, and a direct interaction of RAN with PTB was demonstrated by coimmunoprecipitation. CONCLUSIONS: PTB in the host cytoplasm is directly associated with HCV replication. These findings demonstrate that the involvement of RAN in HCV infection is mediated by influencing the cytoplasmic translocation of PTB.

[Effect of alisol A 24-acetate on proliferation of aorta smooth muscle cells in rats induced by ox-LDL].

Alisol A 24-acetate, a triterpenoid extracted from Alisma orientale, has shown anti-atherosclerotic actions and many studies have proved that oxidized low density lipoprotein (Ox-LDL) could promote proliferation of aorta smooth muscle cells (VSMCs) which are closely related to atherosclerosis (AS). The purpose of this study was to evaluate the effect of alisol A 24-acetate on the proliferation of VSMCs isolated from the thoracic aorta of rats induced by ox-LDL. VSMCs were induced by ox-LDL(50 mg·L⁻¹) to establish the proliferation model and intervened by alisol A 24-acetate (5, 10, 20 mg·L⁻¹) for 12, 24 and 48 h. Then the proliferation of VSMCs was detected by MTT assay; protein expression levels of VSMCs PCNA, cyclinD1, cyclinE, p21, p27 and VSMCs PCNA, p21and p27 mRNA expression levels were detected by Western blot and Real-time polymerase chain reaction (RT-PCR) respectively. The results showed that ox-LDL could induce the proliferation of VSMCs (P<0.05), increase the protein expression levels of PCNA, cyclinD1 and cyclinE in the VSMCs (P<0.05) and inhibit the protein and mRNA expression levels of p21 and p27 (P<0.05). As compared with the model group, alisol A 24-acetate inhibited the proliferation of VSMCs in rats induced by ox-LDL and inhibited the protein expression of VSMCs PCNA, cyclinD1, cyclinE and enhanced the protein and mRNA p21 and p27 expression levels (P<0.05). The effect was more obvious with the increase of concentration of alisol A 24-acetate. These data indicate that alisol A 24-acetate can inhibit the proliferation of VSMCs induced by ox-LDL and the mechanism may be associated with inhibiting expression of cyclin protein, including cyclinD1, cyclinE, p21, p27 and so on.

MicroRNA-141 Targets Sirt1 and Inhibits Autophagy to Reduce HBV Replication.

BACKGROUND/AIMS: About 400 million individuals are chronically infected with hepatitis B virus, at high risk of developing liver cirrhosis and hepatocellular carcinoma. Recent studies have demonstrated an interaction between hepatitis B virus replication and autophagy activity of hepatocytes. In the present study, we aimed to investigate the role of miR-141 in regulating autophagy and hepatitis B virus replication. METHODS: The expression of HBV-DNA, miR-141 and Sirt1 mRNA was determined by quantitative real-time PCR analysis. The expression of HBsAg and HBeAg was determined by ELISA. Western blotting was performed to detect protein expression. The LC3 puncta was determined by immunofluorescence. To test whether miR-141 directly regulate the expression level of Sirt1 mRNA, dual-luciferase reporter gene assay was performed. RESULTS: In vitro studies showed that miR-141 mimic inhibited the autophagic response, hepatitis B virus and the expression of Sirt1 in hepatocytes. And transfection with miR-141 inhibitor enhanced autophagic response and Sirt1 expression. The autophagy induced by overexpression of Sirt1 was inhibited by miR-141 mimic. In addition, miR-141 mimic also decreased the expression of Sirt1 mRNA. Sirt1 was predicted as a potential miR-141 target by bioinformatic analysis of its 3'-UTR, and confirmed by luciferase reporter assays which analyzing the interaction of miR-141 with the wild- type or the mutated Sirt1 3'-UTR. CONCLUSION: We have therefore demonstrated a role of miR-141 in regulating autophagy-mediated hepatitis B virus inhibition by targeting Sirt1, and may provide potential targets for drug development.

Resveratrol enhances HBV replication through activating Sirt1-PGC-1α-PPARα pathway.

The population of hepatitis B combined with a number of metabolic disorders is increasing significantly. Resveratrol (RSV) has been used as a preclinical drug for the treatment of the metabolic disorders. However, the impact of RSV on HBV replication remains unknown. In this study, the HBV-expressing hepatocelluar carcinoma cell line and mouse model created by hydrodynamic injection of viral DNA were used. We found that RSV activates Sirt1, which in turn deacetylates PGC-1α and subsequently increases the transcriptional activity of PPARα, leading to the enhanced HBV transcription and replication in vitro and in vivo. In addition, we found that this pathway is also required for fasting-induced HBV transcription. Taken together, this study identifies that RSV enhances HBV transcription and replication especially acting on the core promoter, which depends on Sirt1-PGC-1α-PPARα pathway. We conclude that RSV may exacerbate the progression of hepatitis B and that patients with hepatitis B infection should be cautious taking RSV as a dietary supplement.

MEAN inhibits hepatitis C virus replication by interfering with a polypyrimidine tract-binding protein.

MEAN (6-methoxyethylamino-numonafide) is a small molecule compound, and here, we report that it effectively inhibits hepatitis C virus (HCV) infection in an HCV cell culture system using a JC1-Luc chimeric virus, with a 50% effective concentration (EC50) of 2.36 ± 0.29 µM. Drug combination usage analyses demonstrated that MEAN was synergistic with interferon α, ITX5061 and ribavirin. In addition, MEAN effectively inhibits N415D mutant virus and G451R mutant viral infections. Mechanistic studies show that the treatment of HCV-infected hepatocytes with MEAN inhibits HCV replication but not translation. Furthermore, treatment with MEAN significantly reduces polypyrimidine tract-binding protein (PTB) levels and blocks the cytoplasmic redistribution of PTB upon infection. In the host cytoplasm, PTB is directly associated with HCV replication, and the inhibition of HCV replication by MEAN can result in the sequestration of PTB in treated nuclei. Taken together, these results indicate that MEAN is a potential therapeutic candidate for HCV infection, and the targeting of the nucleo-cytoplasmic translocation of the host PTB protein could be a novel strategy to interrupt HCV replication.

Isoliquiritigenin Inhibits Interferon-γ-Inducible Genes Expression in Hepatocytes through Down-Regulating Activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK and PI3K/Akt Signaling Pathways.

BACKGROUND & AIMS: The high expression levels of interferon-γ (IFN-γ)-inducible genes correlate positively with liver diseases. The present study aimed to explore the effect of isoliquiritigenin (ISL) on the expression of genes induced by IFN-γ in vitro, and to elucidate the underlying molecular mechanisms. METHODS: HepG2 and L02 cells were divided into control, ISL, IFN-γ, and IFN-γ plus ISL groups. The cytotoxicity of compounds to cells was evaluated by Cell Counting Kit 8 (CCK8) assay; the expression levels of chemokine (C-X-C motif) ligand 9 (CXCL9), CXCL10, CXCL11, and interleukin-6 (IL-6) in cells and supernatant were measured by quantitative real time polymerase chain reaction (qRT-PCR) and ELISA, respectively. Moreover, western blot was used to examine the phosphorylated levels of janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1), nuclear factor (NF)-κB, interferon regulatory factor 3 (IRF3)/myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Protein Kinase B (Akt) in HepG2 and L02 cells exposed to ISL, IFN-γ and IFN-γ plus ISL. RESULTS: The results showed that IFN-γ treatment induced the expression of CXCL9, CXCL10, CXCL11, and IL-6 in HepG2 and LO2 cells, which could be significantly and dose-dependently inhibited by ISL treatment (P < 0.05 or P < 0.01), but the inhibitory effect of ISL on IL-6 expression was not so good as on CXCL9, CXCL10, and CXCL11 expression. Furthermore, ISL treatment dose-dependently inhibited the activation of JAK1/STAT1, IRF3/MyD88, extracellular signal-regulated kinase (ERK)/MAPK, c-Jun N-terminal kinase (JNK)/MAPK, and PI3K/Akt signaling pathways (P < 0.05), but had no effect on the activation of JAK2/STAT1, NF-κB and p38/MAPK signaling pathways. CONCLUSION: We demonstrate that ISL inhibits IFN-γ-induced inflammation in hepatocytes via influencing the activation of JAK1/STAT1, IRF3/MyD88, ERK/MAPK, JNK/MAPK, and PI3K/Akt signaling pathways.

Lipocalin 2 Upregulation Protects Hepatocytes from IL1-ß-Induced Stress.

BACKGROUND: Lipocalin 2 (LCN2), a protein primarily produced by hepatocytes, is highly upregulated under various conditions that induce cellular stress, such as intoxication, infection or inflammation. However, the precise biological functions and underlying mechanisms of LCN2 in hepatocytes remains unknown. METHODS: Hepatocyte stress was successfully induced by treating Huh7 cells with interleukin-1ß (IL-1ß). Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α) and LCN2 levels were measured in IL-1ß treated Huh7 cells and supernatant. Additionally, microarray analysis was conducted to identify genes differentially expressed in LCN2-silenced and control Huh7 cells. RESULTS: TNF-α, IL-6 and LCN2 were significantly elevated in Huh7 cells after IL-1ß) treatment. In LCN2-silenced Huh7 cells, expression of IL-6 and TNF-α was significantly increased when compared with the expression levels of control Huh7 cells. Furthermore, differentially expressed genes were observed between the LCN2-silenced and control cells. Microarray analysis indicated that LCN2 acted by influencing genes involved in protein metabolism, stress response, cell cycle and proliferation. CONCLUSIONS: Our results suggest that LCN2 upregulation protects hepatocytes from IL-1ß-induced stress. Additionally, our microarray analysis of LCN2-silenced and control cells provides a better understanding of the mechanisms that may be influenced by LCN2 induction.

The p53/miR-34a/SIRT1 Positive Feedback Loop in Quercetin-Induced Apoptosis.

BACKGROUND: The anti-tumor effects of quercetin have been reported, but the underlying molecular mechanisms remain to be elucidated. The aim of present study was to explore the role of miRNA in the anticancer effects of quercetin. METHODS: The differential miRNAs expression between the HepG2 and Huh7 cells treated by quercetin were detected by microarray. The xCELLigence, Flow cytometry, RT-PCR and Western blot were used to analyze the cell proliferation, cell apoptosis, cell cycle arrest, anti-tumor genes, and protein expression. RESULTS: miR-34a was up-regulated in HepG2 cells treated by quercetin exhibiting wild-type p53. When inhibiting the miR-34a, the sensitivity of the cells to quercetin decreased and the expression of the SIRT1 was up-regulated, but the acetylation of p53 and the expression of some genes related to p53 down-regulated. CONCLUSION: miR-34a plays an important role in the anti-tumor effects of querctin in HCC, miR-34a may be a tiemolecule between the p53 and SIRT1 and is composed of a p53/miR-34a/SIRT1 signal feedback loop, which could enhance apoptosis signal and significantly promote cell apoptosis.

Emodin protects against concanavalin A-induced hepatitis in mice through inhibiting activation of the p38 MAPK-NF-κB signaling pathway.

BACKGROUND/AIMS: To investigate the effects of emodin on concanavalin A (Con A)-induced hepatitis in mice and to elucidate its underlying molecular mechanisms. METHODS: A fulminant hepatitis model was established successfully by the intravenous administration of Con A (20 mg/kg) to male Balb/c mice. Emodin was administered to the mice by gavage before and after Con A injection. The levels of pro-inflammatory cytokines and chemokines, numbers of CD4(+) and F4/80(+) cells infiltrated into the liver, and amounts of phosphorylated p38 MAPK and NF-κB in mouse livers and RAW264.7 and EL4 cells were measured. RESULTS: Pretreatment with emodin significantly protected the animals from T cell-mediated hepatitis, as shown by the decreased elevations of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as reduced hepatic necrosis. In addition, emodin pretreatment markedly reduced the intrahepatic expression of pro-inflammatory cytokines and chemokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1ß, IL-6, IL-12, inducible nitric oxide synthase (iNOS), integrin alpha M (ITGAM), chemokine (C-C motif) ligand 2 (CCL2), macrophage inflammatory protein 2 (MIP-2) and chemokine (CXC motif) receptor 2 (CXCR2). Furthermore, emodin pretreatment dramatically suppressed the numbers of CD4(+) and F4/80(+) cells infiltrating into the liver as well as the activation of p38 MAPK and NF-κB in Con A-treated mouse livers and RAW264.7 and EL4 cells. CONCLUSION: The results indicate that emodin pretreatment protects against Con A-induced liver injury in mice; these beneficial effects may occur partially through inhibition of both the infiltration of CD4(+) and F4/80(+) cells and the activation of the p38 MAPK-NF-κB pathway in CD4(+) T cells and macrophages.

Interferon-associated retinopathy risk in patients with diabetes and hypertensive hepatitis C.

AIM: To investigate the association of hypertension and diabetes mellitus (DM) with interferon-associated retinopathy (IAR) risk in chronic hepatitis C (CHC). METHODS: Two investigators independently searched PubMed and Embase for eligible articles published prior to December 2013; additional studies were identified by reviewing the bibliographies. Only case-control or cohort studies that evaluated the association between hypertension and/or DM and IAR incidence in CHC patients were included. IAR was characterized by the presence of cotton-wool spots and/or retinal hemorrhage, and was defined as the primary efficacy measure. Pooled relative risks (RRs) with 95% confidence intervals (CIs) were estimated using data extracted from papers based on random-effects models. RESULTS: Eight eligible studies were included in the present meta-analysis. The outcomes showed that patients with CHC and hypertension were at higher risk of IAR (48/189 vs 96/455, RR = 1.90; 95%CI: 1.15-3.15, P < 0.05). Patients with DM receiving interferon (IFN)-based therapy for CHC infection may be at higher risk for IAR (18/72 vs 60/256, RR = 1.56, 95%CI: 1.11-2.20, P < 0.05); however, the outcome was not stable. There was no significant difference in IAR risk between genotype-1-infected patients and non-genotype-1-infected patients (RR = 1.09, 95%CI: 0.64-1.87, P > 0.05). Comparable incidences of IAR were also found between patients treated with pegylated interferon (PIFN) α-2a and those treated with PIFN α-2b (RR = 0.84, 95%CI: 0.56-1.24, P > 0.05) and between patients treated with IFN α and those treated with PIFN α (RR = 1.04, 95%CI: 0.72-1.50, P > 0.05). CONCLUSION: Patients with hypertension have a higher risk of retinopathy when receiving IFN-based therapy for CHC.

Therapeutic vaccines against hepatitis C virus.

Hepatitis C virus (HCV) is a blood-borne pathogen which has chronically infected about 130-210 million people worldwide. Current standard-of-care (SoC) therapy is an inadequate and expensive treatment with more side effects. Two direct-acting antiviral agents (DAAs) (telaprevir and boceprevir) in combination with SoC therapy have been used in patients infected with HCV genotype 1. Although these drugs result in a shortening of therapy, they also have additional side effects and are expensive. In their stead, several second-generation DAAs are being investigated. What important is that all-oral, interferon (IFN)- and ribavirin-free regimens for the treatment of HCV-infected patients are now being investigated, and will be applied in the next year. Preventive measures against HCV, including vaccine development, are also now in progress. However, no therapeutic vaccine against HCV has been produced to date. An effective vaccine should induce robust and broadly cross-reactive CD4(+), CD8(+)T-cell and neutralising antibody (NAb) responses. Current data indicate that vaccines can usually not completely prevent HCV infection but rather prevent the progression of HCV infection to chronic and persistent infection, which may be a realistic goal. This review discusses the important roles of NAbs and CD8(+)T-cells in the development of therapeutic vaccines, and summarizes some important epitopes of HCV recognized by CD8(+)T-cells and some prospective therapeutic vaccine approaches.

Identification of serum biomarkers of hepatocarcinoma through liquid chromatography/mass spectrometry-based metabonomic method.

Late diagnosis of hepatocarcinoma (HCC) is one of the most primary factors for the poor survival of patients. Thereby, identification of sensitive and specific biomarkers for HCC early diagnosis is of great importance in biological medicine to date. In the present study, serum metabolites of the HCC patients and healthy controls were investigated using the improved liquid chromatography-mass spectrometry (LC/MS). A wavelet-based method was utilized to find and align peaks of LC-MS. The characteristic peaks were selected by performing a two-sample t test statistics (p value <0.05). Clustering analysis based on principal component analysis showed a clear separation between HCC patients and healthy individuals. The serum metabolite, namely 1-methyladenosine, was identified as the characteristic metabolite for HCC. Moreover, receiver-operator curves were calculated with 1-methyladenosine and/or alpha fetal protein (AFP). The higher area under curve value was achieved in 1-methyladenosine group than AFP group (0.802 vs. 0.592), and the diagnostic model combining 1-methyladenosine with AFP exhibited significant improved sensitivity, which could identify those patients who missed the diagnosis of HCC by determining serum AFP alone. Overall, these results suggested that LC/MS-based metabonomic study is a potent and promising strategy for identifying novel biomarkers of HCC.

Involvement of REST corepressor 3 in prognosis of human hepatitis B.

AIM: To examine the potential correlation between serum REST corepressor 3 (RCOR3) level and the outcome of patients with hepatitis B. METHODS: Concanavalin A (ConA)-induced mouse hepatitis model was used. The mRNA level of RCOR3 in mouse liver was measured using GeneChip array and real-time PCR. One hundred seventy-seven patients with hepatitis B and 34 healthy individuals were categorized into six groups including mild chronic hepatitis, moderate chronic hepatitis B, severe hepatitis B (SHB), cirrhosis, hepatocellular carcinoma (HCC) and healthy control. Serum levels of human RCOR3 were measured using ELISA. RESULTS: In the mouse hepatitis model, the mRNA level of RCOR3 in liver was reduced early after exposure to ConA, then increased after 6 h of exposure. There was no significant difference in the serum RCOR3 level between the mild chronic hepatitis B and the control groups. The serum RCOR3 level was significantly increased in the moderate chronic hepatitis B group, but significantly reduced in SHB, cirrhosis and HCC groups, as compared with the control group. Moreover, the serum RCOR3 levels in SHB, cirrhosis and liver cancer patients were significantly lower than those in the patients with moderate chronic hepatitis B and with mild chronic hepatitis B. Rank correlation analysis revealed a significant correlation between serum RCOR3 level and total bilirubin (r=-0.305, P<0.01). There was no significant correlation between RCOR3 on one hand, and alanine transaminase (r=0.014, P>0.05) or aspartate transaminase (r=-0.079, P>0.05) on the other hand. CONCLUSION: Serum RCOR3 level may reflect the degree of liver damage, which might be a potential biomarker for the outcome of patients with hepatitis B.