Hepatoprotective drugs for prevention of liver injury resulting from anti-tuberculosis treatment: A meta-analysis of cohort studies.

Background: The incidence of liver injury caused by anti-tuberculous (TB) drugs is very high. However, owing to a lack of sufficient evidence, preventive use of hepatoprotective drugs is not yet recommended. Therefore, we aimed to assess the protective effect of hepatoprotective drugs for anti-TB drug-induced liver injury. Methods: We conducted a literature search in China Biology Medicine disc, China National Knowledge Infrastructure, WanFang, Chinese Scientific and Technological Journal, PubMed, Cochrane Library, Web of Science, and Embase. We performed meta-analysis using R 4.0 and Review Manager 5.3 software. Results: A total of 18 studies involving 3589 patients from 2 groups were included. Use of hepatoprotective drugs contributed to a lower incidence of liver injury as compared with conventional anti-TB treatment alone (relative risk [RR] = 0.39, 95% confidence interval [CI]: 0.28-0.53, p < 0.001). In subgroup analysis, significant protective effects were noted for mild liver injury (RR = 0.30, 95% CI 0.15-0.58), moderate (or severe) liver injury (RR = 0.35, 95% CI 0.19-0.65), and liver injury within 2-4 weeks (RR = 0.37, 95% CI 0.19-0.71). We also found a statistically significant difference in the incidence of drug withdrawal (RR = 0.58, 95% CI 0.34-0.97, p = 0.040). Conclusions: Our results demonstrate that hepatoprotective drugs are effective in preventing liver injury in patients receiving anti-TB treatment, to some extent.

Identifying potential biomarkers of nonalcoholic fatty liver disease via genome-wide analysis of copy number variation.

BACKGROUND: The prevalence of Non-alcoholic fatty liver disease (NAFLD) is increasing and emerging as a global health burden. In addition to environmental factors, numerous studies have shown that genetic factors play an important role in the development of NAFLD. Copy number variation (CNV) as a genetic variation plays an important role in the evaluation of disease susceptibility and genetic differences. The aim of the present study was to assess the contribution of CNV to the evaluation of NAFLD in a Chinese population. METHODS: Genome-wide analysis of CNV was performed using high-density comparative genomic hybridisation microarrays (ACGH). To validate the CNV regions, TaqMan real-time quantitative PCR (qPCR) was utilized. RESULTS: A total of 441 CNVs were identified, including 381 autosomal CNVs and 60 sex chromosome CNVs. By merging overlapping CNVs, a genomic CNV map of NAFLD patients was constructed. A total of 338 autosomal CNVRs were identified, including 275 CNVRs with consistent trends (197 losses and 78 gains) and 63 CNVRs with inconsistent trends. The length of the 338 CNVRs ranged from 5.7 kb to 2.23 Mb, with an average size of 117.44 kb. These CNVRs spanned 39.70 Mb of the genome and accounted for ~ 1.32% of the genome sequence. Through Gene Ontology and genetic pathway analysis, we found evidence that CNVs involving nine genes may be associated with the pathogenesis of NAFLD progression. One of the genes (NLRP4 gene) was selected and verified by quantitative PCR (qPCR) method with large sample size. We found the copy number deletion of NLRP4 was related to the risk of NAFLD. CONCLUSIONS: This study indicate the copy number variation is associated with NAFLD. The copy number deletion of NLRP4 was related to the risk of NAFLD. These results could prove valuable for predicting patients at risk of developing NAFLD.

[Temporal and Spatial Characteristics of Total Phosphorus in the Middle and Lower Yangtze River Mainstem Under New Water and Sediment Conditions].

After the completion of the Three Gorges Reservoir and the upstream reservoir group of the Yangtze River, new water and sediment conditions appeared in the middle and lower reaches of the Yangtze River, and its influence on the phosphorus concentrations in water has attracted much attention. Therefore, the spatial and temporal distributions of total phosphorus (TP) concentrations in the middle and lower reaches of the mainstem under the new water and sediment conditions were studied. The results show that:① after the construction of the Three Gorges Reservoir, the concentrations of TCP (samples were allowed to settle for 30 min) in the middle and lower reaches of the Yangtze River fluctuates between 0.10 and 0.15 mg·L-1, and generally increased during 2004-2010 and then decreased during 2014-2019, and increases along the flow direction. The concentrations of dissolved total phosphorus (TDP) have slowly increased with time. ② The settleable solids influence the phosphorus content to varying degrees. The median values of TCP/TP ratio in Nanjinguan, Hankou, and 23 km below Wusongkou, are 0.900, 0.720, and 0.609, respectively, which decreases successively from upstream to downstream. The proportion of TPP (total particulate phosphorus)/TP shows an increasing trend along the flow direction. The median values of TPP/TP ratios in Nanjinguan, Hankou, and 23 km below Wusongkou were 0.439, 0.567, and 0.738, respectively. ③ According to the "Environmental quality standard for surface water GB 3838-2002", the water quality was assessed using TCP concentrations, and the assessment results showed that the water quality of the middle and lower reaches of the Yangtze River was generally good. However, considering the influence of settleable solids, the water quality categories assessed based on TP concentrations would be worse, especially near estuaries. ④ In the middle and lower reaches of Yangtze River, there is little difference in the phosphorus concentration of different monitoring sites in the upper section of main stream; however, the difference is obvious near the estuary. ⑤ The concentration of TCP in the coastal waters of the urban river section of the middle and lower reaches of the Yangtze River is significantly higher than that of the main channel, and there are obvious coastal pollution zones in the coastal waters of the urban river section.

The role of hepatitis B infection in anti-tuberculosis drug-induced liver injury: a meta-analysis of cohort studies.

Drug-induced liver injury (DILI) is a common adverse drug reaction leading to the interruption of tuberculosis (TB) therapy. We aimed to identify whether the hepatitis B virus (HBV) infection would increase the risk of DILI during first-line TB treatment. A meta-analysis of cohort studies searched in PubMed, Web of Science and China National Knowledge Infrastructure was conducted. Effect sizes were reported as risk ratios (RRs) and 95% confidence intervals (CIs) and calculated by R software. Sixteen studies with 3960 TB patients were eligible for analysis. The risk of DILI appeared to be higher in TB patients co-infected with HBV (RR 2.66; 95% CI 2.13-3.32) than those without HBV infection. Moreover, patients with positive hepatitis B e antigen (HBeAg) were more likely to develop DILI (RR 3.42; 95% CI 1.95-5.98) compared to those with negative HBeAg (RR 2.30; 95% CI 1.66-3.18). Co-infection with HBV was not associated with a higher rate of anti-TB DILI in latent TB patients (RR 4.48; 95% CI 0.80-24.99). The effect of HBV infection on aggravating anti-TB DILI was independent of study participants, whether they were newly diagnosed with TB or not. Besides, TB and HBV co-infection patients had a longer duration of recovery from DILI compared to non-co-infected patients (SMD 2.26; 95% CI 1.87-2.66). To conclude, the results demonstrate that HBV infection would increase the risk of DILI during TB therapy, especially in patients with positive HBeAg, and close liver function monitoring is needed for TB and HBV co-infection patients.

Microcystin-LR promotes necroptosis in primary mouse hepatocytes by overproducing reactive oxygen species.

Microcystin-LR (MC-LR) is a type of cyclic heptapeptide toxin produced by cyanobacteria during bloom events. MC-LR-induced cell death is critically involved in its potent specific hepatotoxicity. Many studies have demonstrated that prototypical apoptosis as a form of programmed cell death after MC-LR is associated with liver injury. However, whether another form of programmed cell death exists and the underlying mechanism have not been reported. Here, we demonstrate that MC-LR can induce necroptosis via ROS overactivation in primary mouse hepatocytes. Various potential pathways of programmed cell death induced by MC-LR were evaluated by annexin V/PI dual staining for flow cytometric analysis, image-based PI staining analysis and western blot analysis. Cell viability was determined by the CCK8 assay. Rupture of the plasma membrane was indicated by lactate dehydrogenase release. ROS was evaluated with the carboxy-H2DCFDA fluorescent probe. It was found that in MC-LR-treated cells, as the plasma membrane was damaged, annexin V/PI-stained double-positive cells were significantly induced and PI-stained nuclei were more diffuse. Western blot analysis showed that MC-LR treatment significantly upregulated the expression of necroptotic and apoptotic proteins. Mechanistically, MC-LR induced ROS overproduction by dysregulating the expression and activity of the pro-oxidants SOD1, MAOA, and NOX4 and the antioxidant GPX1. These results indicate the presence of a novel mechanism for MC-LR-mediated liver injury and present a novel target in the treatment of MC-LR-exposed patients.

Hepatitis B Virus X Protein Increases 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine (8-Oxodg) Level via Repressing MTH1/ MTH2 Expression in Hepatocytes.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection markedly increases the risk of development of hepatocellular carcinoma (HCC). Among the seven viral proteins that HBV encodes, HBV X protein (HBx) appears to have the most oncogenic potential. The mitochondria-associated HBx can induce oxidative stress in hepatocytes, leading to the production of abundant reactive oxygen species (ROS). High levels of ROS usually induce oxidative DNA damage and 8-hydroxy-2-deoxyguanosine (8-OHdG), also known as 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), which is one of the major products of DNA oxidation and an important biomarker for oxidative stress and carcinogenesis. Cells have evolved a mechanism to prevent oxidized nucleotides from their incorporation into DNA through nucleotide pool sanitization enzymes of MTH1 (NUDT1), MTH2 (NUDT15), MTH3 (NUDT18) and NUDT5. However, little is known as to whether HBx can regulate the expression of those enzymes and modulate the formation and accumulation of 8-oxodG in hepatocytes. METHODS: The level of 8-oxodG was assessed by ELISA in stable HBV-producing hepatoma cell lines, an HBV infectious mouse model, HBV and HBx transgenic mice and HBV-infected patients versus their respective controls. Expression of MTH1, MTH2, MTH3 and NUDT5 was determined by a real-time quantitative PCR and western blot analysis. Transcriptional regulation of MTH1 and MTH2 expression by HBx and the effect of HBx on MTH1 and MTH2 promoter hypermethylation were examined using a luciferase reporter assay and bisulfite sequencing analysis. RESULTS: In comparison with controls, significantly higher levels of 8-oxodG were detected in the genome and culture supernatant of stable HBV-producing HepG2.2.15 cells, in the sera and liver tissues of HBV infectious mice and HBV or HBx transgenic mice, and in the sera of HBV-infected patients. Expression of HBx in hepatocytes significantly increased 8-oxodG level and reduced the expression of MTH1 and MTH2 at both mRNA and protein levels. It was also demonstrated that HBx markedly attenuated the MTH1 or MTH2 promoter activities through hypermethylation. Furthermore, enhancement of 8-oxodG production by HBx was reversible by overexpression of MTH1 and MTH2. CONCLUSION: Our data show that HBx expression results in the accumulation of 8-oxodG in hepatocytes through inhibiting the expression of MTH1 and MTH2. This may implicate that HBx may act as a tumor promoter through facilitating the mutational potential of 8-oxodG thus connecting a possible link between HBV infection and liver carcinogenesis.

Multiple MicroRNAs Ameliorate Hepatocyte Steatosis and Injury by Suppressing FABP1 Expression.

BACKGROUND/AIMS: Liver fatty acid-binding protein (FABP1) is a key regulator of hepatic lipid metabolism. MicroRNAs (miRNAs) are thought to be involved in nonalcoholic fatty liver disease (NAFLD), and the underlying mechanism is largely unclear. We investigated whether miRNAs influence hepatocyte steatosis by regulating the FABP1 gene. METHODS: Candidate FABP1-targeting miRNAs were evaluated using luciferase reporter assay. FABP1 expression was measured using western blotting and quantitative reverse transcription-PCR. Intracellular lipid accumulation was measured based on Oil Red O staining and intracellular triglyceride content. Hepatocyte injury was evaluated based on culture supernatant levels of alanine aminotransferase, aspartate aminotransferase, and intracellular adenosine triphosphate, and mitochondrial membrane potential. RESULTS: Dicer1 knockdown significantly elevated FABP1 expression. In total, 68 miRNAs potentially targeting FABP1 were selected; of these, miR-3941, miR-4517, and miR-4672 directly targeted the FABP1 3' untranslated region. Mimics of the three miRNAs substantially repressed FABP1 expression at translational level and led to HepG2 cell resistance to steatosis and cell injury induced by free fatty acids mixture, which rescue of FABP1 overexpression reversed. CONCLUSION: Our findings identify a novel mechanism by which miRNAs protect against hepatocyte steatosis and injury by downregulating FABP1 expression.

Deubiquitylation of hepatitis B virus X protein (HBx) by ubiquitin-specific peptidase 15 (USP15) increases HBx stability and its transactivation activity.

Hepatitis B virus X protein (HBx) plays important roles in viral replication and the development of hepatocellular carcinoma. HBx is a rapid turnover protein and ubiquitin-proteasome pathway has been suggested to influence HBx stability as treatment with proteasome inhibitors increases the levels of HBx protein and causes accumulation of the polyubiquitinated forms of HBx. Deubiquitinases (DUBs) are known to act by removing ubiquitin moieties from proteins and thereby reverse their stability and/or activity. However, no information is available regarding the involvement of DUBs in regulation of ubiquitylation-dependent proteasomal degradation of HBx protein. This study identified the deubiquitylating enzyme USP15 as a critical regulator of HBx protein level. USP15 was found to directly interact with HBx via binding to the HBx region between amino acid residues 51 and 80. USP15 increased HBx protein levels in a dose-dependent manner and siRNA-mediated knockdown of endogenous USP15 reduced HBx protein levels. Increased HBx stability and steady-state level by USP15 were attributable to reduced HBx ubiquitination and proteasomal degradation. Importantly, the transcriptional transactivation function of HBx is enhanced by overexpression of USP15. These results suggest that USP15 plays an essential role in stabilizing HBx and subsequently affects the biological function of HBx.

[Temporal and Spatial Variation Characteristics of the Heavy Metals Content in the Surface Sediment and the Potential Ecological Risk Trends in the Three Gorges Reservoir Area].

With the construction progress of the Three Gorges Project, the hydrological situation of Three Gorges Reservoir changes greatly, which causes the changes of suspended solids precipitation conditions and surface sediment traits. This research analyzed the temporal and spatial variation of the heavy metal pollution in the surface sediment and the potential ecological risk trends during the years from 2000 to 2015 in the trunk stream of the Yangtze River from Jiangjin to the Three Gorges Dam area and some major tributaries, such as Jialing River, Yulin River, Wujiang River, Xiaojiang River, Xiangxi River. The results showed that the average content ranges of heavy metals (including Cu, Pb, Mn, As, Hg etc.) in surface sediments at the main stream monitoring sections during the study period were 46.5-85.7 mg·kg-1(Cu), 43.8-65.1 mg·kg-1(Pb), 784.2-910.6 mg·kg-1(Mn), 8.44-11.91 mg·kg-1(As), 0.193-0.236 mg·kg-1(Hg) respectively; The average content ranges of the heavy metals in surface sediments at the main stream monitoring sections during the study period were 16.5-85.6 mg·kg-1(Cu), 25.8-74.8 mg·kg-1(Pb), 573.7-996.3 mg·kg-1 (Mn), 6.96-13.31 mg·kg-1 (As), 0.160-0.232 mg·kg-1 (Hg) respectively. The results also showed that there were obvious differences of the heavy metals content in some areas between the left and right bank of the trunk stream of the Yangtze River in the Three Gorges Reservoir area. The results also showed that the concentration variation trends of the heavy metals were different in the surface sediments, such as Cu, Pb, Mn, As and Hg. The content of Hg was the most influenced element by the water period effect. The concentration variation trends of these elements were different at different monitoring sections during the construction and water harvesting progress of the Three Gorges project. The contents of Cu, Pb, Mn and As in sediment from the Three Gorges Reservoir area showed strong positive correlations between each other. But there was a low correlation between the content of As and those of other elements. There was no significant correlation between the content of Hg and those of other heavy metal elements. According to the geoaccumulation index analytical method, the contents of the heavy metals in surface sediments in the Three Gorges Reservoir area were generally at low enrichment level. But the pollution caused by Hg should be paid attention. The change of potential ecological risk index was small in the main stream and the major tributaries of the Yangtze River (except Wujiang River) in most of the time. It should also be noted that the change of potential ecological risk index fluctuated obviously at a high level before the year of 2008, and then the change fluctuations trends descended with time and tended to be stable.

Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein.

UNLABELLED: Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3ß-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3ß and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3ß, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. IMPORTANCE: Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV-induced pathogenesis of hepatic steatosis still remains to be elucidated. In this study, we found that expression of liver fatty acid binding protein (FABP1) was dramatically increased in the sera of HBV-infected patients and in both sera and liver tissues of HBV-transgenic mice. Forced expression of HBx led to FABP1 upregulation, whereas knockdown of FABP1 remarkably diminished lipid accumulation in both in vitro and in vivo models. It is possible that HBx promotes hepatic lipid accumulation through upregulating FABP1 in the development of HBV-induced nonalcoholic fatty liver disease. Therefore, inhibition of FABP1 might have therapeutic value in steatosis-associated chronic HBV infection.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

Liver fatty acid-binding protein (L-FABP), also known as fatty acid-binding protein 1 (FABP1), is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD) and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs) of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182) from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032).Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05). The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01). In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (ß = -0.320, P = 0.003), while serum TG levels were positively associated with serum FABP1 levels (ß = 0.487, P = 0.014). Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

Transcriptional regulation of the apolipoprotein F (ApoF) gene by ETS and C/EBPα in hepatoma cells.

Apolipoprotein F (ApoF) inhibits cholesteryl ester transfer protein (CETP) activity and plays an important role in lipid metabolism. In the present study, the full-length human ApoF promoter was cloned, and the molecular mechanism of the regulation of ApoF was investigated. The ApoF promoter displayed higher activities in hepatoma cell lines, and the -198 nt to +79 nt promoter region contained the maximum promoter activity. In the promoter region of -198 nt to -2 nt there were four putative binding sites for transcription factors ETS-1/ETS-2 (named EBS-1 to EBS-4) and one for C/EBP. Mutation of EBS-2, EBS4 and the C/EBP binding site abolished the promoter activity, and ETS-1/ETS-2 and C/EBPα could interact with corresponding binding sites. In addition, overexpression of ETS-1/2 or C/EBPα enhanced, while dominant-negative mutants of ETS-1/2 and knockdown of C/EBPα decreased, ApoF promoter activities. ETS-1 and C/EBPα associated physically, and acted synergistically to activate ApoF transcription. These results demonstrated combined activation of the ApoF promoter by liver-enriched and ubiquitous transcription factors. Direct interactions between C/EBPα and ETS-1 were important for high liver-specific expression of ApoF.

Hepatitis B virus core protein inhibits Fas-mediated apoptosis of hepatoma cells via regulation of mFas/FasL and sFas expression.

Hepatitis B virus core protein (HBc) has been implicated in hepatocarcinogenesis through several mechanisms. Resistance of hepatitis B virus (HBV)-infected hepatocytes to apoptosis is considered one of the major contributors to the progression of chronic hepatitis to cirrhosis and ultimately to hepatocellular carcinoma. The Fas receptor/ligand (Fas/FasL) system plays a prominent role in hepatocyte death during HBV infection. Here we report that HBc mediates resistance of hepatoma cells to agonistic anti-Fas antibody (CH11)-induced apoptosis. When HBc was introduced into human hepatoma cells, the cells became resistant to CH11 cytotoxicity in a p53-dependent manner. HBc significantly down-regulated the expression of p53, total Fas, and membrane-bound Fas at the mRNA and protein levels and reduced FasL mRNA expression. In contrast, HBc up-regulated the expression of soluble forms of Fas by increasing Fas alternative mRNA splicing. Mechanistically, HBc-mediated Fas alternative mRNA splicing was associated with up-regulation of polypyrimidine tract-binding protein 1 and down-regulation of Fas-activated serine/threonine kinase. These results indicated that HBc may prevent hepatocytes from Fas-induced apoptosis by the dual effects of reducing the expression of the proapoptotic form of Fas and enhancing the expression of the antiapoptotic form of the receptor, which may contribute to the survival and persistence of infected hepatocytes during chronic infection.

Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity.

BACKGROUND: The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. METHODS: The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. RESULTS: HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. CONCLUSIONS: Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity.

Epigenetic silencing of NAD(P)H:quinone oxidoreductase 1 by hepatitis B virus X protein increases mitochondrial injury and cellular susceptibility to oxidative stress in hepatoma cells.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II enzyme that participates in the detoxification of dopamine-derived quinone molecules and reactive oxygen species. Our prior work using a proteomic approach found that NQO1 protein levels were significantly decreased in stable hepatitis B virus (HBV)-producing hepatoma cells relative to the empty-vector-transfected controls. However, the mechanism and biological significance of the NQO1 suppression remain elusive. In this study we demonstrate that HBV X protein (HBx) induces epigenetic silencing of NQO1 in hepatoma cells through promoter hypermethylation via recruitment of DNA methyltransferase DNMT3A to the promoter region of the NQO1 gene. In HBV-related hepatocellular carcinoma (HCC) specimens, HBx expression was correlated negatively to NQO1 transcripts but positively to NQO1 promoter hypermethylation. Downregulation of NQO1 by HBx reduced intracellular glutathione levels, impaired mitochondrial function, and increased susceptibility of hepatoma cells to oxidative stress-induced cell injury. These results suggest a novel mechanism for HBV-mediated pathogenesis of chronic liver diseases, including HCC.

The roles of HBV infection and host factors in ultrasound-diagnosed fatty liver: a case-control study.

BACKGROUND/AIMS: The present study is to evaluate the roles of HBV infection, host factors and their interactions in the development of ultrasound-diagnosed fatty liver in Fujian province of China, a highly HBV endemic area. METHODOLOGY: From June 2007 to May 2008, 527 ultrasound-diagnosed fatty liver patients and 1042 controls were ultrasonically diagnosed in this hospital-based case-control study. Their demographic, anthropometric, biochemical, behavioral factors and HBV markers were compared, and factors associated with fatty liver were determined by multivariate logistic regression analysis. RESULTS: Multivariate ORs and 95% confidence intervals (Cls) of fatty liver were 3.96 (2.10-7.48) for HBsAg positivity, 2.97 (1.78-4.94) for HBsAg negativity with antibodies positivity (either anti-HBe or anti-HBc or both), 1.66 (1.10-2.51) for low alcohol consumption, 7.09 (4.28- 11.75) for obesity, 3.19 (1.75-5.81) for reduced high density lipoprotein cholesterol (HDL-C), 2.17 (1.00-4.72) for elevated fasting plasma glucose (FPG), 2.71 (1.72-4.27) for hypertriglyceridemia, 9.19 (4.32-19.57) for hypertension, and 2.89 (1.86-4.48) for hyperuricemia. Furthermore, synergistic interactions on the additive model were observed between HBV infection and obesity, hypertriglyceridemia, reduced HDL-C, and hyperuricemia with regard to the risk of fatty liver. CONCLUSIONS: Ultrasound-diagnosed fatty liver in Fujian province of China is closely associated with HBV infection, low alcohol consumption, obesity, and other features of the metabolic syndrome. In addition, HBV infection was synergistic with obesity, hypertriglyceridemia, reduced HDL-C, and hyperuricemia as far as the risk of fatty liver concerned.

Genetic variants in PNPLA3 and risk of non-alcoholic fatty liver disease in a Han Chinese population.

We investigated the possible association between genetic variants in the Patatin like phospholipase-3 (PNPLA3) gene and nonalcoholic fatty liver disease (NAFLD) in a Han Chinese population. We evaluated twelve tagging single-nucleotide polymorphisms (tSNPs) of the PNPLA3 gene in a frequency matched case-control study from Fuzhou city of China (553 cases, 553 controls). In the multivariate logistic regression analysis, the rs738409 GG or GC, and rs139051 TT genotypes were found to be associated with increased risk of NAFLD, and a significant trend of increased risk with increasing numbers of risk genotype was observed in the cumulative effect analysis of these single nucleotide polymorphisms. Furthermore, haplotype association analysis showed that, compared with the most common haplotype, the CAAGAATGCGTG and CGAAGGTGTCCG haplotypes conferred a statistically significant increased risk for NAFLD, while the CGGGAACCCGCG haplotype decreased the risk of NAFLD. Moreover, rs738409 C>G appeared to have a multiplicative joint effect with tea drinking (P<0.005) and an additive joint effect with obesity (Interaction contrast ratio (ICR) = 2.31, 95% CI: 0.7-8.86), hypertriglyceridemia (ICR = 3.07, 95% CI: 0.98-5.09) or hypertension (ICR = 1.74, 95% CI: 0.52-3.12). Our data suggests that PNPLA3 genetic polymorphisms might influence the susceptibility to NAFLD development independently or jointly in Han Chinese.

Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion.

Hepatitis B spliced protein (HBSP) is involved in the pathogenicity and/or persistence of hepatitis B virus (HBV). Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma (HCC). However, whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown. This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility. Conversely, small interfering RNA (siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion. By glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and a mammalian two-hybrid assay, HBSP was found to directly interact with cathepsin B (CTSB). Similar to HBSP knockdown, knocking down CTSB also reduced cell migration and invasion. Furthermore, the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), and overexpression of HBSP significantly enhanced tumor-induced vascularization of endothelial cells. In contrast, knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis. Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase (MAPK) and Akt signaling pathway, as evidenced by increases in phosphorylation of p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt. Taken together, these findings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes, increased tumor-induced angiogenesis, and activation of the MAPK/Akt signaling, thereby leading to the aggressiveness of hepatoma cells.

Two genetic variants in FABP1 and susceptibility to non-alcohol fatty liver disease in a Chinese population.

Liver fatty acid-binding protein (FABP1) serves as a key regulator of hepatic lipid metabolism, and polymorphisms within the FABP1 gene have been associated with several metabolic traits. To investigate the association between FABP1 polymorphisms and the risk of non-alcohol fatty liver disease (NAFLD) in a Chinese population, the genotypes and haplotypes of FABP1 (rs2241883 T/C and rs1545224G/A) were determined in 553 patients with NAFLD and 553 healthy controls. The results showed that individuals with at least one copy of the rs2241883 C allele (TC or CC genotype) had an elevated risk for developing NAFLD (odds ratio [OR]=1.32, 95% CI: 1.01-1.71), and individuals with at least one copy of the rs1545224 A allele (GA or AA genotype) also had a significantly increased risk for NAFLD (OR=1.52, 95% CI: 1.14-2.02). Cumulative effect analysis of the two SNPs revealed that individuals with two risk genotypes were at significantly higher risk of NAFLD than those without risk genotype, and a significant trend of increased risk with increasing numbers of risk genotype was observed. Stratification analysis showed that the rs2241883 C allele carriers had higher level of LDL-C and the rs1545224 A allele carriers had higher level of FPG than those without this allele. In addition, haplotype analysis revealed that the one composed of the rs1545224 A and rs2241883 C variants was significantly associated with an increased risk for NAFLD (OR=1.34; 95% CI=1.05-1.40) compared to the GT haplotype. Taken together, the present study suggests that genetic variations within FABP1 influence susceptibility to NAFLD independently or jointly.

Hepatitis B virus X protein blocks filamentous actin bundles by interaction with eukaryotic translation elongat ion factor 1 alpha 1.

Hepatitis B virus (HBV)-encoded X protein (HBx protein) is a multi-functional regulatory protein. It functions by protein-protein interaction and plays a pivotal role in the pathogenesis of HBV-related diseases. However, the partners in hepatocytes interacting with HBx protein are far from understood fully. In this study, immunoprecipitation was employed to screen for binding partners for the HBx protein from huh-7 hepatoma cells infected with recombinant adenovirus expressing HBx protein, and five cellular proteins including eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), were identified. The interaction between HBx protein and eEF1A1 was confirmed further using a GST pull-down assay and co-immunoprecipitation, respectively. In Huh-7 hepatoma cells, the HBx protein inhibits dimer formation of eEF1A1, hence blocks filamentous actin bundling. These findings provide new insights into the molecular mechanisms involved in the functions of the HBx protein.