CB2R agonist GW405833 alleviates acute liver failure in mice via inhibiting HIF-1α-mediated reprogramming of glycometabolism and macrophage proliferation.

The inflammatory responses involving infiltration and activation of liver macrophages play a vital role in acute liver failure (ALF). In the liver of ALF mice, cannabinoid receptor 2 (CB2R) is significantly upregulated on macrophages, while CB2R agonist GW405833 (GW) could protect against cell death in acute liver damage. In this study, we investigated the molecular mechanisms underlying the protective effects of GW against ALF in vivo and in vitro from a perspective of macrophage glycometabolism. Mice were pretreated with GW (10 mg/kg, i.p.), then were injected with D-GalN (750 mg/kg, i.p.) and LPS (10 mg/kg, i.p.) to induce ALF. We verified the protective effects of GW pretreatment in ALF mice. Furthermore, GW pretreatment significantly reduced liver macrophage infiltration and M1 polarization, and inhibited the release of inflammatory factors TNF-α and IL-1ß in ALF mice. These protective effects were eliminated by CB2R antagonist SR144528 or in CB2R-/- ALF mice. We used LPS-stimulated RAW264.7 cells as an in vitro M1 macrophage-centered model of inflammatory response, and demonstrated that pretreatment with GW (10 µM) significantly reduced glucose metabolism by inhibiting glycolysis, which inhibited LPS-induced macrophage proliferation and inflammatory cytokines release. We verified these results in a stable CB2R-/- RAW264.7 cell line. Moreover, we found that GW significantly inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Using a stable HIF-1α-/- RAW264.7 cell line, we confirmed that GW reduced the release of inflammatory cytokines from macrophages and inhibited glycolysis by downregulating HIF-1α expression. In conclusion, activation of CB2Rs inhibits the proliferation of hepatic macrophages and release of inflammatory factors in ALF mice through downregulating HIF-1α to inhibit glycolysis.

Melittin ameliorates inflammation in mouse acute liver failure via inhibition of PKM2-mediated Warburg effect.

Acute liver failure (ALF) is a fatal clinical syndrome with no special drug. Recent evidence shows that modulation of macrophage to inhibit inflammation may be a promising strategy for ALF treatment. In this study we investigated the potential therapeutic effects of melittin, a major peptide component of bee venom both in mice model of ALF and in LPS-stimulated macrophages in vitro, and elucidated the underlying mechanisms. ALF was induced in mice by intraperitoneal injection of D-galactosamine/LPS. Then the mice were treated with melittin (2, 4, and 8 mg/kg, ip). We showed that melittin treatment markedly improved mortality, attenuated severe symptoms and signs, and alleviated hepatic inflammation in D-galactosamine/LPS-induced ALF mice with the optimal dose being 4 mg/kg. In addition, melittin within the effective doses did not cause significant in vivo toxicity. In LPS-stimulated RAW264.7 macrophages, melittin (0.7 µM) exerted anti-oxidation and anti-inflammation effects. We showed that LPS stimulation promoted aerobic glycolysis of macrophages through increasing glycolytic rate, upregulated the levels of Warburg effect-related enzymes and metabolites including lactate, LDHA, LDH, and GLUT-1, and activated Akt/mTOR/PKM2/HIF-1α signaling. Melittin treatment suppressed M2 isoform of pyruvate kinase (PKM2), thus disrupted the Warburg effect to alleviate inflammation. Molecular docking analysis confirmed that melittin targeted PKM2. In LPS-stimulated RAW264.7 macrophages, knockdown of PKM2 caused similar anti-inflammation effects as melittin did. In D-galactosamine/LPS-induced ALF mice, melittin treatment markedly decreased the expression levels of PKM2 and HIF-1α in liver. This work demonstrates that melittin inhibits macrophage activation-mediated inflammation via inhibition of aerobic glycolysis by targeting PKM2, which highlights a novel strategy of using melittin for ALF treatment.

Cargo-laden erythrocyte ghosts target liver mediated by macrophages.

Liver-targeted cargo delivery possesses great potential for the treatment of liver disease. It is urgent to find an efficient and biocompatible liver targeted delivery system. This study focused on the liver targeting properties of erythrocyte ghosts and its possible mechanism. Herein, we optimized conditions to fabricate human and mouse erythrocyte ghosts with sufficient room capable of incorporating various model substances. Erythrocyte ghosts are biocompatible cargo carriers because it is derived from autologous red blood cells (RBCs), and the cell size, zeta potential, and biconcave-disk shape of the ghosts were consistent with those of RBCs. An in vivo imaging system and positron emission tomography/computed tomography imaging showed that the ghosts were captured mainly in the liver by intravenous injection of fluorescence or 18F-fluorodeoxyglucose (FDG)-labelled ghosts into mice. In contrast, the main concentration of naked octreotide was trapped in the lungs while naked 18F-FDG was trapped in the heart. However, the concentration of cargo-loaded ghosts decreased significantly in the liver in macrophage-depleted mice. Accordingly, in vitro experiments showed that higher phosphatidylserine exposure was observed in the ghosts (38.9 %) compared to normal erythrocytes (0.69 %), and the phagocytic activity of the macrophage RAW 264.7. on the ghosts was significantly higher than that of normal erythrocytes (p < 0.001). Together they indicate that erythrocyte ghosts show liver targeting properties, and possibly owing to macrophage phagocytosis. This promising and effective therapeutic delivery system may provide therapeutic benefits for liver disease.

Efficacy and safety of direct acting antiviral regimens for hepatitis C virus and human immunodeficiency virus co-infection: systematic review and network meta-analysis.

BACKGROUND AND AIM: Various all-oral direct-acting antiviral (DAA) regimens are being widely used in the treatment of human immunodeficiency virus (HIV)/hepatitis C virus (HCV) co-infected patients; however, the comparative efficacy and safety of different types and combinations of DAAs are not completely clear. There is still a lack of integration of evidence for optimized therapies for HIV/HCV co-infection. METHODS: We conducted a systematic literature search in several databases up to January 1, 2020. All the studies that reported the sustained virologic response (SVR) and adverse events of DAAs in HIV/HCV co-infected patients were included. The Bayesian Markov Chain Monte Carlo method was used for the pooled estimates of network meta-analysis. RESULTS: We identified 33 eligible articles with 7 combinations of all-oral DAAs for the analyses of efficacy and safety. Grazoprevir-elbasvir ± ribavirin (GZR/EBR ± RBV: 95.6%; 95% CrI, 91.7-98.1%), ombitasvir/paritaprevir/ritonavir and dasabuvir ± ribavirin (3D ± RBV: 95.3%; 95% CrI, 93.4-96.9%), sofosbuvir-ledipasvir ± ribavirin (SOF/LDV ± RBV: 95.2%; 95% CrI, 93.7-96.6%), and sofosbuvir-daclatasvir ± ribavirin (SOF/DCV ± RBV: 94.8%; 95% CrI, 92.5-96.6%) were the most effective combinations for HIV/HCV co-infected patients, with SVR rates of approximately 94% and above while severe adverse events were rare. However, the SVR rates of sofosbuvir-ribavirin (SOF/RBV) and sofosbuvir-simeprevir ± ribavirin (SOF/SMV ± RBV) both failed to reach 90%, and the incidences of adverse events were higher than 5%. CONCLUSIONS: Efficacy and safety of all-oral DAAs were in prospect for HIV/HCV co-infection patients. GZR/EBR ± RBV was the optimal combination recommended for HIV/HCV co-infected patients based on the excellent treatment effects and insignificant adverse events.

Potential predictors for prognosis and postpartum recovery time of acute fatty liver of pregnancy.

BACKGROUND: Acute fatty liver of pregnancy (AFLP) is a potentially lethal condition of pregnant women with a high mortality rate. Potential predictors related to postpartum recovery time and prognostic factors of AFLP are still unclear. This study aimed to evaluate potential predictors for prognosis and postpartum recovery time of AFLP. METHODS: We retrospectively analyzed the clinical data of 76 AFLP patients in our hospital from 2002 to 2017 and investigated potential predictors using univariate analysis and multivariate logistic regression analysis. RESULTS: Hepatic encephalopathy (HE) was found to be associated with prognosis in AFLP patients (P = 0.005, OR = 26.844). The postpartum recovery time analysis showed that AFLP patients with a age < 25 had the shortest recovery time, but no significant difference (P = 0.134, OR = 5.952). The postpartum recovery time of patients with liver failure (LF) was significantly prolonged compared to those without LF (P = 0.036, OR = 10.052). Cryoprecipitate, and plasma infusion showed no significant effect on prognosis or recovery time. Artificial liver support therapy (ALST) had no effect on prognosis, but it might affect postpartum recovery time with no statistical significance (P = 0.128, OR = 5.470). CONCLUSION: HE is a potential predictor for prognosis of AFLP. LF is a potential predictor for postpartum recovery time.

High mobility group protein B1 controls liver cancer initiation through yes-associated protein -dependent aerobic glycolysis.

Emerging studies have suggested that the Hippo pathway is involved in the tumorigenesis of hepatocellular carcinoma (HCC). However, the key regulator of the Hippo pathway in liver tumor metabolic reprogramming remains elusive. Here, we provide evidence that high mobility group box 1 (HMGB1), a chromosomal protein, plays a role in the regulation of the Hippo pathway during liver tumorigenesis. Cre/loxP recombination-mediated HMGB1 depletion in hepatocytes blocks diethylnitrosamine-induced liver cancer initiation in mice, whereas short hairpin RNA-mediated gene silencing of HMGB1 inhibits HCC cell proliferation. Mechanistically, the binding of HMGB1 to GA-binding protein alpha promotes the expression of yes-associated protein (YAP), a major downstream effector of the Hippo pathway that contributes to liver tumorigenesis by inducing hypoxia-inducible factor 1α (HIF1α)-dependent aerobic glycolysis. Like wild-type YAP-complementary DNA, YAP-5SA-S94A can restore HIF1α DNA binding activity, glycolysis-associated gene expression, and HIF1α-YAP complex formation in YAP-knockdown HCC cell lines. In contrast, verteporfin, a reagent targeting the interface between YAP and TEA domain transcription factor, has the ability to block YAP-HIF1α complex formation. Notably, genetic or pharmacologic inhibition of the HMGB1-YAP-HIF1α pathway confers protection against excessive glycolysis and tumor growth in mice. CONCLUSION: These findings demonstrate that HMGB1 plays a novel role in modulating the YAP-dependent HIF1α pathway and shed light on the development of metabolism-targeting therapeutics for HCC chemoprevention. (Hepatology 2018;67:1823-1841).

Protective effects of specific cannabinoid receptor 2 agonist GW405833 on concanavalin A-induced acute liver injury in mice.

Cannabinoid receptor 2 (CB2R) is highly expressed in immune cells and plays an important role in regulating immune responses. In the current study, we investigated the effects of GW405833 (GW), a specific CB2R agonist, on acute liver injury induced by concanavalin A (Con A). In animal experiments, acute liver injury was induced in mice by injection of Con A (20 mg/kg, i.v.). The mice were treated with GW (20 mg/kg, i.p., 30 min after Con A injection) or GW plus the selective CB2R antagonist AM630 (2 mg/kg, i.p., 15 min after Con A injection). We found that Con A caused severe acute liver injury evidenced by significantly increased serum aminotransferase levels, massive hepatocyte apoptosis, and necrosis, as well as lymphocyte infiltration in liver tissues. Treatment with GW significantly ameliorated Con A-induced pathological injury in liver tissue, decreased serum aminotransferase levels, and decreased hepatocyte apoptosis. The therapeutic effects of GW were prevented by AM630. In cell experiments, we showed that CB2Rs were highly expressed in Jurkat T cells, but little expression in L02 liver cells. Treatment with GW (10-40 µg/mL) dose-dependently decreased the viability of Jurkat T cells and induced cell apoptosis, which was reversed by AM630. In the coculture of Jurkat T cells with L02 liver cells, GW dose-dependently protected L02 cells from apoptosis induced by Con A (5 µg/mL). The protective effect of GW was reversed by AM630 (1 µg/mL). Our results suggest that GW protects against Con A-induced acute liver injury in mice by inhibiting Jurkat T-cell proliferation through the CB2Rs.

Prevalence of hepatitis B virus, hepatitis C virus, human immunodeficiency virus and Treponema pallidum infections in hospitalized patients before transfusion in Xiangya hospital Central South University, China from 2011 to 2016.

BACKGROUND: Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Treponema pallidum (TP) infections are considered classic transfusion-transmissible infections (TTIs). Few data are available about the prevalence of TTIs in patients before blood transfusion in China. This study aimed to investigate the seroprevalence of four TTIs among patients before blood transfusion in Xiangya Hospital Central South University, China. METHODS: From 2011 to 2016, 442,121 hospitalized patients before possible blood transfusion were tested for hepatitis B surface antigen (HBsAg), anti-HCV, syphilis antibody (anti-TP) and anti-HIV. RESULTS: Of the 442,121 patients, the overall positivity of the four TTI serum markers was 15.35%. The positive rates of HBsAg, anti-HCV, anti-HIV and anti-TP were 10.98, 1.43, 0.16 and 2.78%, respectively. TTI serum markers showed a significant difference by gender, with positive rates of 17.98% for males and 12.79% for females. The prevalence of TTI serum markers varied significantly by age. The overall co-infection rate was 0.63%, and the top three multiple infections were HBV-TP, HBV-HCV, and HCV-TP. The co-infection rates of HBV-TP and HBV-HCV showed a significant decrease from 2011 to 2016, while the rates of other co-infections remained stable. CONCLUSIONS: The prevalence of TTIs in patients before blood transfusion is much higher compared to that in blood donors in the region. The infection rates of HIV and TP increased, and the infection rate of HBsAg decreased in recent years.

High mobility group box 1 promotes sorafenib resistance in HepG2 cells and in vivo.

BACKGROUND: Primary liver cancer is a lethal malignancy with a high mortality worldwide. Currently, sorafenib is the most effective molecular-targeted drug against hepatocellular carcinoma (HCC). However, the sorafenib resistance rate is high. The molecular mechanism of this resistance has not been fully elucidated. High mobility group box 1 (HMGB1) is a multifaceted protein that plays a key role in the proliferation, apoptosis, metastasis and angiogenesis of HCC cells. In addition, HMGB1 has been suggested to contribute to chemotherapy resistance in tumours, including lung cancer, osteosarcoma, neuroblastoma, leukaemia, and colorectal cancer. This study investigated the association between HMGB1 and sorafenib resistance in HCC. METHODS: HepG2 cells with HMGB1 knockdown or overexpression were generated. The efficacy of sorafenib in these cells was tested using flow cytometry and a cell counting assay. The subcellular localization of HMGB1 in HepG2 cells following sorafenib treatment was measured by western blotting and confocal microscopy. A murine subcutaneous HCC model was generated to examine the association between HMGB1 and the sensitivity of sorafenib treatment. RESULTS: The HMGB1 knockdown cells exhibited a significantly higher apoptotic level and lower cell viability than the normal HMGB1 expressing cells following the sorafenib treatment. In addition, the cell viability observed in the HMGB1 overexpressing cells was higher than that observed in the control cells following the sorafenib intervention. Sorafenib had a better tumour inhibition effect in the HMGB1 knockdown group in vivo. The amount of mitochondrial HMGB1 decreased, while the amount of cytosolic HMGB1 increased following the exposure to sorafenib. Altogether, HMGB1 translocated from the mitochondria to the cytoplasm outside the mitochondria following the exposure of HepG2 cells to sorafenib. CONCLUSIONS: A novel potential role of HMGB1 in the regulation of sorafenib therapy resistance in HCC was observed. The knockdown of HMGB1 restores sensitivity to sorafenib and enhances HepG2 cell death, while HMGB1 overexpression blunts these effects. The translocation of HMGB1 from the mitochondria to the cytosol following sorafenib treatment provides new insight into sorafenib resistance in HCC.

Performance of scoring systems to predict mortality of patients with acute-on-chronic liver failure: A systematic review and meta-analysis.

BACKGROUND AND AIM: Acute-on-chronic liver failure (ACLF) has characteristic feature of multisystem organ failure, rapid progression, and low early transplant-free survival. We performed a meta-analysis to determine the accuracy of five scoring systems in predicting mortality of ACLF patients. METHODS: A systematic database search was performed, and retrieved articles were graded according to methodological quality. Collated data was meta-analyzed by hierarchical summarized receiver operating characteristic model and bivariate model to evaluate the diagnostic accuracy of scoring systems. RESULTS: Of 4223 studies identified, 26 studies involving 4732 ACLF patients were included. The model of end-stage liver disease (MELD) score was found to have largest the area under summarized receiver operating characteristic (AUROC) (0.82) compared with other estimated scoring systems, especially for 3-month mortality. MELD serum sodium (MELD-Na) score showed homologous high accuracy, with the AUROC was 0.81. However, meta-analyses of 16 studies showed that Child-Pugh-Turcotte score had least AUROC (0.71). Sequential organ failure assessment (SOFA) score presented moderately lower diagnostic accuracy, with AUROC being 0.73. Moreover, chronic liver failure-SOFA score presented excellent accuracy of prognostication with highest diagnostic odds ratios. CONCLUSION: This review demonstrated that MELD had moderate diagnostic accuracy to predict mortality of ACLF patients. Considering the expectative diagnostic value, chronic liver failure-SOFA could be regarded as a promising replacement of MELD. To improve the predictive power of scoring systems, multicenter prospective studies of large sample sizes with long-term follow-up are needed.

Hepatitis B virus X protein in liver tumor microenvironment.

Encoded by the hepatitis B virus, hepatitis B virus X protein (HBx) is a multifunctional, potentially oncogenic protein that acts primarily during the progression from chronic hepatitis B to cirrhosis and hepatocellular carcinoma (HCC). In recent decades, it has been established that chronic inflammation generates a tumor-supporting microenvironment. HCC is a typical chronic inflammation-related cancer, and inflammation is the main risk factor for HCC progression. The viral transactivator HBx plays a pivotal role in the initiation and maintenance of hepatic inflammatory processes through interactions with components of the tumor microenvironment including tumor cells and the surrounding peritumoral stroma. The complex interactions between HBx and this microenvironment are thought to regulate tumor growth, progression, invasion, metastasis, and angiogenesis. In this review, we have summarized the current evidence evaluating the function of HBx and its contribution to the inflammatory liver tumor microenvironment.

TM4SF1 Promotes Proliferation, Invasion, and Metastasis in Human Liver Cancer Cells.

Transmembrane 4 superfamily member 1 (TM4SF1) is a member of tetraspanin family, which mediates signal transduction events regulating cell development, activation, growth and motility. Our previous studies showed that TM4SF1 is highly expressed in liver cancer. HepG2 cells were transfected with TM4SFl siRNA and TM4SF1-expressing plasmids and their biological functions were analyzed in vitro and in vivo. HepG2 cells overexpressing TM4SF1 showed reduced apoptosis and increased cell migration in vitro and enhanced tumor growth and metastasis in vivo, whereas siRNA-mediated silencing of TM4SF1 had the opposite effect. TM4SF1 exerts its effect by regulating a few apoptosis- and migration-related genes including caspase-3, caspase-9, MMP-2, MMP-9 and VEGF. These results indicate that TM4SF1 is associated with liver tumor growth and progression, suggesting that TM4SF1 may be a potential target for treatment of liver cancer in future.

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice.

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor κB, degradation of inhibitor of κB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor κB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage.

Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death.

High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-l-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma.

UNLABELLED: Epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of hepatocellular carcinoma (HCC). BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins implicated in HCC progression. However, the role(s) of BTBD7 in HCC have not been identified. Using highly metastatic HCC HCCLM3 cells, immortalized L02 hepatocytes, metastatic HCC animal models, and three independent cohorts of HCC patient specimens, we aimed to determine the involvement of BTBD7 in HCC metastasis. We show that BTBD7 messenger RNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred through activation of a RhoC-Rock2-FAK-signaling pathway, resulting in matrix metalloproteinase-2/9 production and microvessel formation. Applying a predictive risk score model, Cox regression analysis revealed that high BTBD7 expression integrated with high microvessel density was a powerful independent predictive factor of HCC clinical outcome. CONCLUSION: The present study identifies BTBD7 as a novel candidate prognostic factor and a potential therapeutic target of HCC. (HEPATOLOGY 2013; 57:2326-2337).

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines.

High mobility group protein box1 (HMGB1) and its receptor-receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.

Congo red modulates ACh-induced Ca(2+) oscillations in single pancreatic acinar cells of mice.

AIM: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca(2+) oscillations in mouse pancreatic acinar cells in vitro. METHODS: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca(2+) oscillations were monitored by whole-cell recording of Ca(2+)-activated Cl(-) currents and by using confocal Ca(2+) imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established. RESULTS: Bath application of ACh (10 nmol/L) induced typical Ca(2+) oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 µmol/L) dose-dependently enhanced Ach-induced Ca(2+) oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca(2+) oscillations induced by ACh (10-30 nmol/L), but did not alter the Ca(2+) oscillations induced by ACh (100-10000 nmol/L). Congo red also enhanced the Ca(2+) oscillations induced by bath application of IP3 (30 µmol/L). Intracellular application of congo red failed to alter ACh-induced Ca(2+) oscillations. CONCLUSION: Congo red significantly modulates intracellular Ca(2+) signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug.

Emerging role of high-mobility group box 1 (HMGB1) in liver diseases.

Damage-associated molecular pattern (DAMP) molecules are essential for the initiation of innate inflammatory responses to infection and injury. The prototypic DAMP molecule, high-mobility group box 1 (HMGB1), is an abundant architectural chromosomal protein that has location-specific biological functions: within the nucleus as a DNA chaperone, within the cytosol to sustain autophagy and outside the cell as a DAMP molecule. Recent research indicates that aberrant activation of HMGB1 signaling can promote the onset of inflammatory and autoimmune diseases, raising interest in the development of therapeutic strategies to control their function. The importance of HMGB1 activation in various forms of liver disease in relation to liver damage, steatosis, inflammation, fibrosis, tumorigenesis and regeneration is discussed in this review.

Combined detection of mRNA expression of Alpha-fetoprotein in peripheral blood and telomerase activity of monocytes in hepatocellular carcinoma patients.

BACKGROUND/AIMS: The detection of hepatocellular carcinoma (HCC) cells in the peripheral blood is not only important for the diagnosis of FICC, but is also of critical importance for early detection of hematogenous metastasis of HCC. The purpose of this study was to measure Alpha-fetoprotein (AFP) mRNA in the peripheral blood and telomerase activity in peripheral blood monocytes to determine their utility, in isolation and concert, in the early diagnosis of HCC. METHODOLOGY: Nested RT-PCR was employed to detect the mRNA expression of AFP in the peripheral blood, and PCR-ELISA was used to measure the telomerase activity of peripheral blood mononuclear cells (PBMCs) in patients with HCC, benign hepatic tumors, chronic liver diseases and healthy subjects. RESULTS: Of the 30 HCC patients, 22 (73.3%) were positive for AFP, and 28/30 (93.3%) HCC patients were positive for telomerase activity. The mRNA expression of AFP was positively correlated with telomerase activity in HCC patients (r=0.6742, p<0.05). CONCLUSIONS: Detection of both mRNA expression of AFP in the peripheral blood and telomerase activity in PBMCs may prove to play an important role in achieving an early diagnosis of HCC.

Repurposing Niclosamide as a Therapeutic Drug against Acute Liver Failure by Suppressing Ferroptosis.

Acute liver failure (ALF) is a severe liver disease with a high mortality rate without effective therapeutic drugs. Ferroptosis is a form of programmed cell death that plays an important role in ALF. In this study, we aimed to identify ferroptosis-related genes in ALF, thereby predicting promising compounds to treat ALF. First, mRNA microarray data were utilized to identify the ferroptosis-related differentially expressed genes (DEGs). Hub genes were screened in the protein-protein interaction network and validated. Subsequently, potential drugs to treat ALF were predicted. One of the predicted drugs was tested in an ALF model of mice. Ferroptosis examination and molecular docking were analyzed to explore the mechanism. A total of 37 DEGs were identified, ten hub genes were extracted, and their expression in ALF was validated. The predicted drug niclosamide mitigated lipopolysaccharide/D-galactosamine-induced hepatotoxicity, and decreased mortality of mice in the ALF model. Mechanically, niclosamide may combine with signal transducer and activator of transcription 3 to inhibit ALF progression by suppressing ferroptosis. This study may help advance our understanding of the role of ferroptosis in ALF, and niclosamide may be promising for therapeutic efficacy in patients with ALF.