Integration of Metabolomics and Transcriptomics to Reveal the Metabolic Characteristics of Exercise-Improved Bone Mass.

(1) Background: Exercise is effective in promoting and maintaining bone mass. The aim of this study was to detect the exercise-induced metabolic changes in bone tissue of zebrafish. (2) Methods: Thirty-eight zebrafish (Danio rerio, six months old) were analyzed. The exercise group (n = 19) received 8 weeks of counter-current swimming training. The control group (n = 19) was not subjected to exercise. Mineralization was quantified, and alkaline phosphatase (Alp) and anti-tartrate acid phosphatase (Trap) activities were estimated (n = 12). The metabolomics (n = 12) and transcriptomics (n = 14) data of bone tissue were used for the integration analyses. (3) Results: The results showed that the exercise training improved the bone mineralization of zebrafish, e.g., the exercise group (5.74 × 104 ± 7.63 × 103) had a higher mean optical density than the control group (5.26 × 104 ± 8.56 × 103, p = 0.046) for the caudal vertebrae. The amount of mineralized matrix in scales of the exercised zebrafish was also higher (0.156 ± 0.012 vs. 0.102 ± 0.003, p = 0.005). Both histological staining and biochemical analysis revealed increased Alp activity (0.81 ± 0.26 vs. 0.76 ± 0.01, p = 0.002) and decreased Trap activity (1.34 ± 0.01 vs. 1.36 ± 0.01, p = 0.005) in the exercise group. A total of 103 different metabolites (DMs, VIP ≥ 1, fold change (FC) ≥ 1.20 or ≤0.83, p < 0.050) were identified. Alanine, aspartate and glutamate metabolism, ß-alanine metabolism, pyrimidine metabolism, and pantothenate and CoA biosynthesis were the significantly enriched metabolic pathways (p < 0.050). A total of 35 genes (q ≤ 0.050 (BH), |Log2FC| ≥ 0.5) were coenriched with the 103 DMs in the four identified pathways. Protein-protein interaction network analysis of the 35 genes showed that entpd3, entpd1, and cmpk2 were the core genes. (4) Conclusions: The results of this study suggest that alanine, aspartate and glutamate metabolism, ß-alanine metabolism, pyrimidine metabolism, and pantothenate and CoA biosynthesis contributed to exercise-induced improvements in bone mass.

Associations between Dietary Antioxidant Vitamin Intake and the Changes in Bone Mass in Chinese Adolescents: A 2.5-Year Longitudinal Study.

(1) Background: Optimal bone mass accumulation during adolescence is crucial for maximising peak bone mass during adulthood. Dietary antioxidant vitamins may contribute to bone mass accumulation. This 2.5-year-long longitudinal study aimed to evaluate the relationships between dietary vitamin A, C, and E intakes and the annual changes in bone parameters among Chinese adolescents. (2) Method: Subjects aged 10-18 years (n = 1418) were recruited from a secondary school in Jiangmen, China. Dietary vitamin A, C, and E intakes were assessed using 24 h dietary records over 3 consecutive days. The Sahara Clinical Bone Sonometer was used to measure the broadband ultrasound attenuation (BUA) and the speed of sound (SOS). Their annual changes were then calculated (i.e., BUA%/year, SOS%/year). The associations were detected after adjusting for the baseline bone phenotype; age; sex; weight; height; pubertal stage; physical activity; and dietary intakes of vitamin D, calcium and energy. (3) Results: A curvilinear relationship was found between the dietary intake of vitamin C and BUA%/year (p = 0.026); further analyses in the subgroups revealed that this relationship was observed in male adolescents (p = 0.012). A positive association was observed only in boys with a dietary vitamin C intake of ≥159.01 mg/day (ß = 0.395, p = 0.036). Moreover, a linear positive association was shown between the dietary intake of vitamin E and BUA%/year in female adolescents (ß = 0.082, p = 0.033). (4) Conclusion: Our findings indicated that dietary vitamin C intake has a threshold effect on bone mass gain in male adolescents and that dietary vitamin E intake could be a positive predictor of bone mass gain in female adolescents.

A two photon fluorescent probe for highly selective detection and endogenous imaging of hydrogen sulfide.

Hydrogen sulfide (H2S), one of redox-active sulfur species, is known as a signaling molecule and an antioxidant in biological tissues to maintain cellular functions. The development of selective and sensitive H2S detection is important to understand the role of H2S in vivo. Herein, a new two-photon probe NNE was developed to detect hydrogen sulfide using 6-acetyl-N-methyl-2-naphthylamine with an attachment of 7-nitrobenzo-oxadiazole. The probe NNE exhibits high selectivity towards hydrogen sulfide over other anions. Nucleophilic substitution of H2S leads to a turn-on response with 28-fold enhancement in quantum yield (from 0.004 to 0.117). NNE shows a high sensitivity towards hydrogen sulfide with an extremely low detection limit at 6.8 nM. Furthermore, the probe NNE exhibits two-photon excited fluorescence, making it a suitable probe for monitoring H2S distribution in live cells and tissues without background fluorescence interference.

Association between Body Composition and Bone Mineral Density in Children and Adolescents: A Systematic Review and Meta-Analysis.

BACKGROUND: Bone mass acquisition during growth is a major determinant of the risk of developing osteoporosis later in life. Body composition is an anthropometric determinant of bone mineral density (BMD) and significantly influences its development during childhood and adolescence. OBJECTIVE: This study aimed to systematically examine the association between body composition and bone mineral density in children and adolescents. METHODS: Observational studies addressing this association were identified from PubMed (MEDLINE), Embase, Scopus and the Cochrane Library (up to January 2021). The study populations consisted of healthy children and adolescents. The DerSimonian and Laird method was used to compute pooled estimates of effect size and the respective 95% confidence intervals for upper limbs, femoral neck (FN), lumbar spine (LS) and total body, respectively. Subgroup analyses were further performed based on age, sex and ethnicity. RESULTS: Thirty-one published studies were eligible for inclusion in this systematic review and meta-analysis, including three longitudinal studies. The combined population from all the studies amounted to 21,393 (11,205 males and 10,188 females). The pooled estimates of the correlation coefficients for lean mass (LM) and BMD ranged from 0.53 to 0.74 (p < 0.050), and the pooled regression coefficients ranged from 0.23 to 0.79 for FN, LS and total body (p < 0.050). For fat mass (FM), the pooled correlation coefficients ranged from 0.10 to 0.50 (p < 0.050) and the pooled regression coefficient was only significant for FN BMD with a weak strength (pooled ß = 0.07, p < 0.050). The pooled regression coefficients for body fat percentage (BF%) were between -0.54 and -0.04 (p < 0.050). The subgroup analysis revealed a stronger association in Asians than in Caucasians for LM and in males compared to females for BF% (p < 0.050). CONCLUSIONS: This systematic review and meta-analysis supports a positive association between LM and BMD. BF% appears to have a deleterious effect on bone acquisition in children and adolescents.

Analysis of the Association between Fat Mass Distribution and Bone Mass in Chinese Male Adolescents at Different Stages of Puberty.

BACKGROUND: Bone mineral acquisition during adolescence is crucial for maximizing peak bone mass. Fat mass (FM) and bone mass are closely related. This study investigated the association of FM distribution with bone mass in Chinese male adolescents. METHOD: A total of 693 male adolescents aged 10-18 years were recruited from a secondary school in Jiangmen, China. Their bone mass and body composition were measured by quantitative ultrasound and bioelectrical impedance analysis, respectively. The associations of the measures of fat distribution with bone parameters, i.e., broadband ultrasound attenuation, speed of sound (SOS), and stiffness index (SI), were analyzed using multiple linear regression. Age, height, body mass index, stage of puberty, physical activity, sedentary behavior, dietary energy intake, and dietary calcium and vitamin D intake were adjusted in the model. Further subgroup analyses of prepubertal and pubertal participants were conducted. RESULTS: The measures of fat distribution showed negative associations with SOS and SI in total subjects (p < 0.010). In prepubertal boys, the measures of fat distribution were only associated with SOS (ß = -0.377 to -0.393, p < 0.050). In pubertal boys, the measures of fat distribution had associations with all bone parameters (ß = -0.205 to -0.584, p < 0.050). The strongest association was between trunk FM and SOS (ß = -0.584, p < 0.001). CONCLUSION: This study supported that the measures of fat distribution were negatively associated with bone parameters in Chinese male adolescents. Trunk FM had the strongest association with bone parameter. These associations appear to be stronger in pubertal boys than in prepubertal boys.

Low molecular weight fucoidan inhibits hepatocarcinogenesis and nonalcoholic fatty liver disease in zebrafish via ASGR/STAT3/HNF4A signaling.

BACKGROUND: Hepatocellular carcinoma ranks fourth in cancer-related mortality currently lacks effective therapeutics. Fucoidan is sulfated polysaccharide that is mainly found in brown seaweeds. In this study, we investigated the effects and mechanisms of low molecular weight fucoidan (i.e. oligo-fucoidan [OF]) preventing hepatocarcinogenesis. METHODS: We used [HBx,src], [HBx,src,p53-/+ ], and [CD36] transgenic zebrafish liver cancer model treated with OF, and performed molecular and histopathological analysis. Transcriptomic and pathways analysis was performed. RESULTS: Decreased expression of lipogenic enzymes, fibrosis markers, and cell cycle/proliferation markers by OF in [HBx,src] and [HBx,src,p53-/+ ] transgenic fish. Liver fibrosis was decreased as revealed by Sirius Red staining, and the liver cancer formation was eventually reduced by feeding OF. OF was also found to be capable of reducing lipid accumulation and cancer formation in non-B non-C Hepatocellular carcinoma (HCC) model in CD36 transgenic zebrafish. Whole-genome expression analysis showed that 661 genes were up-regulated, and 451 genes were downregulated by feeding OF. Upregulated genes were mostly found in protein transporter activity, and downregulated genes were enriched with response to extracellular stimulus and metal binding in gene ontology analysis. The driver gene was HNF4A revealed by NetworkAnalyst from OF differential regulated genes at various insults. OF is able to bind the asialoglycoprotein receptor (ASGR) in hepatoma cells, and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both hepatoma cells and [HBx,src,p53-/+ ] transgenic fish liver cancer model. Using chromatin-immunoprecipitation, we found pSTAT3 could associate with the P1 promoter of HNF4A. Knockdown of either ASGR or HNF4A reversed OF mediated anti-cancer cell proliferation. CONCLUSIONS: Taken together, we provide evidence that OF exhibits the anti-HCC, anti-steatosis, and anti-fibrosis effect for liver in zebrafish models, and the anti-cancer potential of OF attributed to the binding to ASGR and activation of STAT3/HNF4A signaling. OF might be potentially valuable for the management of HCC.

Transcriptomically Revealed Oligo-Fucoidan Enhances the Immune System and Protects Hepatocytes via the ASGPR/STAT3/HNF4A Axis.

Oligo-fucoidan, a sulfated polysaccharide extracted from brown seaweed, exhibits anti-inflammatory and anti-tumor effects. However, the knowledge concerning the detailed mechanism of oligo-fucoidan on liver cells is obscure. In this study, we investigate the effect of oligo-fucoidan in normal hepatocytes by transcriptomic analysis. Using an oligo-fucoidan oral gavage in wild-type adult zebrafish, we find that oligo-fucoidan pretreatment enhances the immune system and anti-viral genes in hepatocytes. Oligo-fucoidan pretreatment also decreases the expression of lipogenic enzymes and liver fibrosis genes. Using pathway analysis, we identify hepatocyte nuclear factor 4 alpha (HNF4A) to be the potential driver gene. We further investigate whether hepatocyte nuclear factor 4 alpha (HNF4A) could be induced by oligo-fucoidan and the underlying mechanism. Therefore, a normal hepatocyte clone 9 cell as an in vitro model was used. We demonstrate that oligo-fucoidan increases cell viability, Cyp3a4 activity, and Hnf4a expression in clone 9 cells. We further demonstrate that oligo-fucoidan might bind to asialoglycoprotein receptors (ASGPR) in normal hepatocytes through both in vitro and in vivo competition assays. This binding, consequently activating the signal transducer and activator of transcription 3 (STAT3), increases the expression of the P1 isoform of HNF4A. According to our data, we suggest that oligo-fucoidan not only enhances the gene expression associated with anti-viral ability and immunity, but also increases P1-HNF4A levels through ASGPR/STAT3 axis, resulting in protecting hepatocytes.

Low Molecular Weight Fucoidan Prevents Radiation-Induced Fibrosis and Secondary Tumors in a Zebrafish Model.

Radiotherapy often causes unwanted side effects such as radiation-induced fibrosis and second malignancies. Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has many biological effects including anti-inflammation and anti-tumor. In the present study, we investigated the radioprotective effect of Oligo-Fucoidan (OF) using a zebrafish animal model. Adult zebrafish of wild-type and transgenic fish with hepatocellular carcinoma were orally fed with Oligo-Fucoidan before irradiation. Quantitative PCR, Sirius red stain, hematoxylin, and eosin stain were used for molecular and pathological analysis. Whole genomic microarrays were used to discover the global program of gene expression after Oligo-Fucoidan treatment and identified distinct classes of up- and downregulated genes/pathways during this process. Using Oligo-Fucoidan oral gavage in adult wild-type zebrafish, we found Oligo-Fucoidan pretreatment decreased irradiation-induced fibrosis in hepatocyte. Using hepatitis B virus X antigen (HBx), Src and HBx, Src, p53-/+ transgenic zebrafish liver cancer model, we found that Oligo-Fucoidan pretreatment before irradiation could lower the expression of lipogenic factors and enzymes, fibrosis, and cell cycle/proliferation markers, which eventually reduced formation of liver cancer compared to irradiation alone. Gene ontology analysis revealed that Oligo-Fucoidan pretreatment increased the expression of genes involved in oxidoreductase activity in zebrafish irradiation. Oligo-Fucoidan also decreased the expression of genes involved in transferase activity in wild-type fish without irradiation (WT), nuclear outer membrane-endoplasmic reticulum membrane network, and non-homologous end-joining (NHEJ) in hepatocellular carcinoma (HCC) transgenic fish. Rescue of those genes can prevent liver cancer formation. Conclusions: Our results provide evidence for the ability of Oligo-Fucoidan to prevent radiation-induced fibrosis and second malignancies in zebrafish.

Omics-based Investigation of Diet-induced Obesity Synergized with HBx, Src, and p53 Mutation Accelerating Hepatocarcinogenesis in Zebrafish Model.

The primary type of liver cancer, hepatocellular carcinoma (HCC), has been associated with nonalcoholic steatohepatitis, diabetes, and obesity. Previous studies have identified some genetic risk factors, such as hepatitis B virus X antigens, overexpression of SRC oncogene, and mutation of the p53 tumor suppressor gene; however, the synergism between diet and genetic risk factors is still unclear. To investigate the synergism between diet and genetic risk factors in hepatocarcinogenesis, we used zebrafish with four genetic backgrounds and overfeeding or high-fat-diet-induced obesity with an omics-based expression of genes and histopathological changes. The results show that overfeeding and high-fat diet can induce obesity and nonalcoholic steatohepatitis in wild-type fish. In HBx, Src (p53-) triple transgenic zebrafish, diet-induced obesity accelerated HCC formation at five months of age and increased the cancer incidence threefold. We developed a global omics data analysis method to investigate genes, pathways, and biological systems based on microarray and next-generation sequencing (NGS, RNA-seq) omics data of zebrafish with four diet and genetic risk factors. The results show that two Kyoto Encyclopedia of Genes and Genomes (KEGG) systems, metabolism and genetic information processing, as well as the pathways of fatty acid metabolism, steroid biosynthesis, and ribosome biogenesis, are activated during hepatocarcinogenesis. This study provides a systematic view of the synergism between genetic and diet factors in the dynamic liver cancer formation process, and indicate that overfeeding or a high-fat diet and the risk genes have a synergistic effect in causing liver cancer by affecting fatty acid metabolism and ribosome biogenesis.

A Novel AURKA Mutant-Induced Early-Onset Severe Hepatocarcinogenesis Greater than Wild-Type via Activating Different Pathways in Zebrafish.

Aurora A kinase (AURKA) is an important regulator in mitotic progression and is overexpressed frequently in human cancers, including hepatocellular carcinoma (HCC). Many AURKA mutations were identified in cancer patients. Overexpressing wild-type Aurka developed a low incidence of hepatic tumors after long latency in mice. However, none of the AURKA mutant animal models have ever been described. The mechanism of mutant AURKA-mediated hepatocarcinogenesis is still unclear. A novel AURKA mutation with a.a.352 Valine to Isoleucine (V352I) was identified from clinical specimens. By using liver-specific transgenic fish overexpressing both the mutant and wild-type AURKA, the AURKA(V352I)-induced hepatocarcinogenesis was earlier and much more severe than wild-type AURKA. Although an increase of the expression of lipogenic enzyme and lipogenic factor was observed in both AURKA(V352I) and AURKA(WT) transgenic fish, AURKA(V352I) has a greater probability to promote fibrosis at 3 months compared to AURKA(WT). Furthermore, the expression levels of cell cycle/proliferation markers were higher in the AURKA(V352I) mutant than AURKA(WT) in transgenic fish, implying that the AURKA(V352I) mutant may accelerate HCC progression. Moreover, we found that the AURKA(V352I) mutant activates AKT signaling and increases nuclear ß-catenin, but AURKA(WT) only activates membrane form ß-catenin, which may account for the differences. In this study, we provide a new insight, that the AURKA(V352I) mutation contributes to early onset hepatocarcinogenesis, possibly through activation of different pathways than AURKA(WT). This transgenic fish may serve as a drug-screening platform for potential precision medicine therapeutics.

Up-regulation of golgi α-mannosidase IA and down-regulation of golgi α-mannosidase IC activates unfolded protein response during hepatocarcinogenesis.

α-1,2 mannosidases, key enzymes in N-glycosylation, are required for the formation of mature glycoproteins in eukaryotes. Aberrant regulation of α-1,2 mannosidases can result in cancer, although the underlying mechanisms are unclear. Here, we report the distinct roles of α-1,2 mannosidase subtypes (MAN1A, MAN1B, ERMAN1, MAN1C) in the formation of hepatocellular carcinoma (HCC). Clinicopathological analyses revealed that the clinical stage, tumor size, α-fetoprotein level, and invasion status were positively correlated with the expression levels of MAN1A1, MAN1B1, and MAN1A2. In contrast, the expression of MAN1C1 was decreased as early as stage I of HCC. Survival analyses showed that high MAN1A1, MAN1A2, and MAN1B1 expression levels combined with low MAN1C1 expression levels were significantly correlated with shorter overall survival rates. Functionally, the overexpression of MAN1A1 promoted proliferation, migration, and transformation as well as in vivo migration in zebrafish. Conversely, overexpression of MAN1C1 reduced the migration ability both in vitro and in vivo, decreased the colony formation ability, and shortened the S phase of the cell cycle. Furthermore, the expression of genes involved in cell cycle/proliferation and migration was increased in MAN1A1-overexpressing cells but decreased in MAN1C1-overexpressing cells. MAN1A1 activated the expression of key regulators of the unfolded protein response (UPR), while treatment with endoplasmic reticulum stress inhibitors blocked the expression of MAN1A1-activated genes. Using the MAN1A1 liver-specific overexpression zebrafish model, we observed steatosis and inflammation at earlier stages and HCC formation at a later stage accompanied by the increased expression of the UPR modulator binding immunoglobulin protein (BiP). These data suggest that the up-regulation of MAN1A1 activates the UPR and might initiate metastasis. Conclusion: MAN1A1 represents a novel oncogene while MAN1C1 plays a role in tumor suppression in hepatocarcinogenesis. (Hepatology Communications 2017;1:230-247).

Frequency of asthma education in primary care in the years 2007-2010.

OBJECTIVE: Recent research suggests that health disparities persist among asthmatic patients and receipt of asthma education, though recent guidelines have highlighted the importance of receiving asthma education. The purpose of this study was to identify trends in the receipt of asthma education as well as to identify disparities in asthma education using the most recently available data in National Ambulatory Medical Care Survey, 2007-2010. METHODS: Weighted chi-square tests were conducted to identify associations between asthma education and variables of interest. A weighted multivariate logistic regression model was subsequently constructed to jointly assess the association of factors of interest on receipt of asthma education. Submission to the Campbell University Institutional Review Board resulted in expedited approval. RESULTS: The percentage of patients who receive asthma education remains quite low. After adjusting for all variables of interest: no statistically significant difference in receipt of asthma education between year groups (2007-2008, 2009-2010) was found (odds ratio [OR] 0.84, 95% confidence interval [CI] 0.52-1.34); patients seen by pediatricians (vs. internal medicine physicians) and Hispanic or Latino patients (vs. non-Hispanic or Latino patients) were more likely to receive asthma education (OR 2.72, 95% CI 1.11-6.66 and OR 2.33, 95% CI 1.18-4.60, respectively); and patients not prescribed a controller medication were less likely to receive asthma education than those who were (OR 0.56, 95% CI 0.37-0.82). CONCLUSIONS: Combined with previously published results, it appears the provision of asthma education continues to be low, despite proven benefits. Additionally, some patient and physician characteristics may be associated with the delivery of asthma education.

Overexpression of endothelin 1 triggers hepatocarcinogenesis in zebrafish and promotes cell proliferation and migration through the AKT pathway.

Hepatocarcinogenesis commonly involves the gradual progression from hepatitis to fibrosis and cirrhosis, and ultimately to hepatocellular carcinoma (HCC). Endothelin 1 (Edn1) has been identified as a gene that is significantly up-regulated in HBx-induced HCC in mice. In this study, we further investigated the role of edn1 in hepatocarcinogenesis using a transgenic zebrafish model and a cell culture system. Liver-specific edn1 expression caused steatosis, fibrosis, glycogen accumulation, bile duct dilation, hyperplasia, and HCC in zebrafish. Overexpression of EDN1 in 293T cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with up-regulation of several cell cycle/proliferation- and migration-specific genes. Furthermore, expression of the unfolded protein response (UPR) pathway-related mediators, such as spliced XBP1, ATF6, IRE1, and PERK, was also up-regulated at both the RNA and protein levels. In the presence of an EDN1 inhibitor or an AKT inhibitor, these increases were diminished and the EDN1-induced migration ability also was disappeared, suggesting that the EDN1 effects act through activation of the AKT pathway to enhance the UPR and subsequently activate the expression of downstream genes. Additionally, p-AKT is enhanced in the edn1 transgenic fish compared to the GFP-mCherry control. The micro RNA miR-1 was found to inhibit the expression of EDN1. We also observed an inverse correlation between EDN1 and miR-1 expression in HCC patients. In conclusion, our data suggest that EDN1 plays an important role in HCC progression by activating the PI3K/AKT pathway and is regulated by miR-1.

Liver-specific expressions of HBx and src in the p53 mutant trigger hepatocarcinogenesis in zebrafish.

Hepatocarcinogenesis is a multistep process that starts from fatty liver and transitions to fibrosis and, finally, into cancer. Many etiological factors, including hepatitis B virus X antigen (HBx) and p53 mutations, have been implicated in hepatocarcinogenesis. However, potential synergistic effects between these two factors and the underlying mechanisms by which they promote hepatocarcinogenesis are still unclear. In this report, we show that the synergistic action of HBx and p53 mutation triggers progressive hepatocellular carcinoma (HCC) formation via src activation in zebrafish. Liver-specific expression of HBx in wild-type zebrafish caused steatosis, fibrosis and glycogen accumulation. However, the induction of tumorigenesis by HBx was only observed in p53 mutant fish and occurred in association with the up-regulation and activation of the src tyrosine kinase pathway. Furthermore, the overexpression of src in p53 mutant zebrafish also caused hyperplasia, HCC, and sarcomatoid HCC, which were accompanied by increased levels of the signaling proteins p-erk, p-akt, myc, jnk1 and vegf. Increased expression levels of lipogenic factors and the genes involved in lipid metabolism and glycogen storage were detected during the early stages of hepatocarcinogenesis in the HBx and src transgenic zebrafish. The up-regulation of genes involved in cell cycle regulation, tumor progression and other molecular hallmarks of human liver cancer were found at later stages in both HBx and src transgenic, p53 mutant zebrafish. Together, our study demonstrates that HBx and src overexpression induced hepatocarcinogenesis in p53 mutant zebrafish. This phenomenon mimics human HCC formation and provides potential in vivo platforms for drug screening for therapies for human liver cancer.

Hepatitis B virus X antigen and aflatoxin B1 synergistically cause hepatitis, steatosis and liver hyperplasia in transgenic zebrafish.

Aflatoxin B1 (AFB1) and the hepatitis B virus X antigen (HBx) are linked to the formation of liver diseases and hepatocellular carcinoma (HCC). The aim of this study was to investigate the synergistic effects between HBx and AFB1 in causing liver disorders using a transgenic zebrafish animal model. Histopathology, Periodic acid-Schiff (PAS) staining, Sirius red staining, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) were used to examine the livers of the HBx transgenic fish injected with AFB1. We found that HBx and AFB1 synergistically promoted steatosis as indicated by histopathological examinations and the increased expression of lipogenic factors, enzymes, and genes related to lipid metabolism. Moreover, treatment of AFB1 in HBx transgenic fish accelerated the development of liver hyperplasia and enhanced the expression of cell cycle related genes. PCNA was co-localized with active caspase 3 protein expression in HBx zebrafish liver samples and human HBV positive HCC samples by double fluorescence immunostaining. Finally, we found that in human patients with liver disease, significant glycogen accumulated in the inflammation, cirrhosis stage, and all cases of hepatocellular and cholangiocellular carcinoma showed a moderate cytoplasmic accumulation of glycogen. Our data demonstrated a synergistic effect of AFB1 and HBx on the regulation of lipid metabolism related genes and cell cycle/division-related genes which might contribute to enhanced steatosis and hyperplasia at 5.75months.

Apoptosis signal-regulating kinase 1 is mediated in TNF-α-induced CCL2 expression in human synovial fibroblasts.

Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with a critical role in osteoarthritis (OA), was primarily produced by monocytes/macrophages and plays a crucial role in the inflammatory response. Here, we investigated the intracellular signaling pathways involved in TNF-α-induced monocyte chemoattractant protein 1 (MCP-1)/CCL2 expression in human synovial fibroblast cells. Stimulation of synovial fibroblasts (OASF) with TNF-α induced concentration- and time-dependent increases in CCL2 expression. TNF-α-mediated CCL2 production was attenuated by TNFR1 monoclonal antibody (Ab). Pretreatment with an apoptosis signal-regulating kinase 1 (ASK1) inhibitor (thioredoxin), JNK inhibitor (SP600125), p38 inhibitor (SB203580), or AP-1 inhibitor (curcumin or tanshinone IIA) also blocked the potentiating action of TNF-α. Stimulation of cells with TNF-α enhanced ASK1, JNK, and p38 activation. Treatment of OASF with TNF-α also increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the CCL2 promoter. TNF-α-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element were inhibited by TNFR1 Ab, thioredoxin, SP600125, and SB203580. Our results suggest that the interaction between TNF-α and TNFR1 increases CCL2 expression in human synovial fibroblasts via the ASK1, JNK/p38, c-Jun, and AP-1 signaling pathway.

Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model.

Hepatitis B virus X antigen plays an important role in the development of human hepatocellular carcinoma (HCC). The key regulators controlling the temporal downstream gene expression for HCC progression remains unknown. In this study, we took advantage of systems biology approach and analyzed the microarray data of the HBx transgenic mouse as a screening process to identify the differentially expressed genes and applied the software Pathway Studio to identify potential pathways and regulators involved in HCC. Using subnetwork enrichment analysis, we identified five common regulator genes: EDN1, BMP7, BMP4, SPIB and SRC. Upregulation of the common regulators was validated in the other independent HBx transgenic mouse lines. Furthermore, we verified the correlation of their RNA expression levels by using the human HCC samples, and their protein levels by using the human liver disease tissue arrays. EDN1, bone morphogenetic protein (BMP) 4 and BMP7 were upregulated in cirrhosis, BMP4, BMP7 and SRC were further upregulated in hepatocellular or cholangiocellular carcinoma samples. The trend of increasing expression of the common regulators correlates well with the progression of human liver cancer. Overexpression of the common regulators increases the cell viability, promotes migration and invasiveness and enhances the colony formation ability in Hep3B cells. Our approach allows us to identify the critical genes in hepatocarcinogenesis in an HBx-induced mouse model. The validation of the gene expressions in the liver cancer of human patients and their cellular function assays suggests that the identified common regulators may serve as useful molecular targets for the early-stage diagnosis or therapy for HCC.

Liver development and cancer formation in zebrafish.

Liver is the largest organ in the human body, and it regulates many physiological processes. Many studies on liver development in different model organisms have demonstrated that the mechanism of hepatogenesis is conserved in vertebrates. The identification of the genes and regulatory pathways involved in liver formation provides a basis for the diagnosis of liver diseases and therapeutic interventions. Hepatocellular carcinoma is the third leading cause of mortality worldwide. In the last decade, genetic alterations, which include the gain and loss of DNA, as well as mutations and epigenomic changes, have been identified as important factors in liver cancer. Many genetic pathways are dysregulated during carcinogenesis. Here, we review the gene regulatory networks that underlie liver organogenesis and the dysregulation of these pathways in liver cancer. The genes and pathways involved in hepatogenesis and liver cancer are largely conserved between zebrafish and humans, making this an ideal model organism for the study of this disease. A better understanding of liver development may aid in the development of new diagnostic and therapeutic approaches to liver cancer.

CTGF inhibits cell motility and COX-2 expression in oral cancer cells.

Oral squamous cell carcinoma (SCC) has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity and COX-2 expression in human oral cells is mostly unknown. Here we found that CTGF reduced the migration and expression of COX-2 in human oral cancer cells. αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin) and Akt inhibitor reversed the CTGF-inhibited the migration and COX-2 down-regulation of oral cancer cells. CTGF stimulation decreased the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt. In addition, c-Jun siRNA also antagonized the CTGF-inhibited migration and COX-2 expression. Moreover, CTGF decreased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Taken together, our results indicated that CTGF inhibits the migration of oral cancer cells by decreasing COX-2 expression through the αvß5 integrin receptor, FAK, PI3K, Akt, c-Jun and AP-1 signal transduction pathways.