IDH1/MDH1 deacetylation promotes acute liver failure by regulating NETosis.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disease, but its pathogenesis is not fully understood. NETosis is a novel mode of cell death. Although the formation of neutrophil extracellular traps (NETs) has been found in various liver diseases, the specific mechanism by which NETosis regulates the development of ALF is unclear. In this article, we explore the role and mechanism of NETosis in the pathogenesis of ALF. METHODS: Clinically, we evaluated NETs-related markers in the liver and peripheral neutrophils of patients with ALF. In in vitro experiments, HL-60 cells were first induced to differentiate into neutrophil-like cells (dHL-60 cells) with dimethyl sulfoxide (DMSO). NETs were formed by inducing dHL-60 cells with PMA. In in vivo experiments, the ALF model in mice was established with LPS/D-gal, and the release of NETs was detected by immunofluorescence staining and western blotting. Finally, the acetylation levels of IDH1 and MDH1 were detected in dHL-60 cells and liver samples by immunoprecipitation. RESULTS: Clinically, increased release of NETs in liver tissue was observed in patients with ALF, and NETs formation was detected in neutrophils from patients with liver failure. In dHL-60 cells, mutations at IDH1-K93 and MDH1-K118 deacetylate IDH1 and MDH1, which promotes the formation of NETs. In a mouse model of ALF, deacetylation of IDH1 and MDH1 resulted in NETosis and promoted the progression of acute liver failure. CONCLUSIONS: Deacetylation of IDH1 and MDH1 reduces their activity and promotes the formation of NETs. This change aggravates the progression of acute liver failure.

AGK2 pre-treatment protects against thioacetamide-induced acute liver failure via regulating the MFN2-PERK axis and ferroptosis signaling pathway.

BACKGROUND: Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure. METHODS: Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6ß (ATF6ß), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence. RESULTS: The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6ß, p-PERK, CHOP, ROS and Fe2+ and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells. CONCLUSIONS: The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.

Efficacy and safety of a nanoparticle therapeutic vaccine in patients with chronic hepatitis B: A randomized clinical trial.

BACKGROUND AND AIM: HBV DNA can be reduced using antiviral drugs in patients with chronic hepatitis B (CHB); however, the rate of HBeAg seroconversion remains low. A clinical trial was conducted to assess the efficacy and safety of a de novo designed liposome-based nanoparticle lipopeptide vaccine, εPA-44, for CHB. APPROACH AND RESULTS: A two-stage phase 2 trial, which included a 76-week, randomized, double-blind, placebo-controlled trial (stage 1) and a 68-week open-label extension (stage 2), was conducted in 15 centers across China (Clinicaltrials.gov No. NCT00869778). In stage 1, 360 human leukocyte antigen A2 (HLA-A2)-positive and HBeAg-positive patients were randomly and equally distributed to receive six subcutaneous injections of 600 µg or 900 µg εPA-44 or placebo at week 0, 4, 8, 12, 20, and 28. In stage 2, 183 patients received extended 900 µg εPA-44, and 26 patients were observed for relapse without further treatment. The primary endpoint was the percentage of patients with HBeAg seroconversion at week 76. At week 76, patients receiving 900 µg εPA-44 achieved significantly higher HBeAg seroconversion rate (38.8%) versus placebo (20.2%) (95% CI, 6.9-29.6%; p = 0.002). With a combined endpoint of HBeAg seroconversion, alanine aminotransferase normalization and HBV DNA < 2,000 IU/mL, both 900 µg (18.1%) and 600 µg (14.3%), resulted in significantly higher rate versus placebo (5.0%) (p = 0.002 and p = 0.02, respectively) at week 76. In stage 2, none (0 of 20) of 900 µg εPA-44-treated patients experienced serologic relapse. The safety profile of εPA-44 was comparable to that of placebo. CONCLUSIONS: Among HLA-A2-positive patients with progressive CHB, a finite duration of 900 µg εPA-44 monotherapy resulted in significantly higher HBeAg seroconversion rate than placebo and sustained off-treatment effect. A phase 3 trial is ongoing (ChiCTR2100043708).

NOD2-mediated HDAC6/NF-κb signalling pathway regulates ferroptosis induced by extracellular histone H3 in acute liver failure.

Acute liver failure (ALF) is life-threatening and often associated with high mortality rates. The aim of the present study was to investigate whether extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF and explore its potential mechanism. RAW264.7 macrophages and C57BL/6 mice were used in this study. LPS, D-galactosamine (D-Gal), histone H3, histone H3 antibody, NOD2 agonist Muramyl Dipeptide (MDP) and HDAC6-siRNA were administered in this study. The key molecules of ferroptosis, NOD2, HDAC6 and the NF-κb pathway, were detected. In vitro, histone H3 was released into the extracellular environment from cell nucleus after LPS exposure. In addition, histone H3 could induce ferroptosis in RAW264.7 macrophages with increased level of Fe2+ and ROS and decreased levels of GPX4 and GSH. MDP further aggravated ferroptosis in RAW264.7 macrophages stimulated by histone H3, which was accompanied by elevated NOD2, HDAC6, p-P65 and IκBα. HDAC6-siRNA ameliorated ferroptosis in RAW264.7 macrophages induced by histone H3, which was accompanied by decreased levels of HDAC6, p-P65 and IκBα. However, HDAC6-siRNA did not alter NOD2 levels in RAW264.7 macrophages administered histone H3. In vivo, the levels of NOD2, HDAC6 the NF-κb pathway and ferroptosis were increased in ALF mice, which were downregulated by histone H3 antibody and upregulated by histone H3. Extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF by regulating theNOD2-mediated HDAC6/NF-κb signalling pathway.

Changes of flavin-containing monooxygenases and trimethylamine-N-oxide may be involved in the promotion of non-alcoholic fatty liver disease by intestinal microbiota metabolite trimethylamine.

Evidence shows that trimethylamine (TMA)/trimethylamine-N-oxide (TMAO) is closely related to non-alcoholic fatty liver disease (NAFLD). The conversion of TMA to TMAO is mainly catalyzed by flavin-containing monooxygenases 3 (FMO3) and FMO1. In this study, we explored the role of TMA in the process of NAFLD. The human NAFLD liver puncture data set GSE89632 and rat TMAO gene chip GSE135856 was downloaded for gene differential expression analysis. Besides, oleic acid (OA) combined with palmitate were used to establish high-fat cell model. TMA, TMAO and FMO1-siRNA were used to stimulate L02 cells. Contents of free fatty acid (FFA), triglyceride (TG), TMAO, FMO1 and unfolded protein response (UPR) related proteins GRP78, XBP1, Derlin-1 were detected. Our results showed that FMO1 and PEG10 were important in the progression of NAFLD. Immunohistochemistry showed that FMO1 in NAFLD liver was increased. In addition, the contents of FFA, TG, FMO1 expression, and TMAO were significantly increased after OA + palmitate and TMA stimulation. However, after silencing FMO1 with siRNA, the expressions of these molecules were decreased. Besides, the protein levels of GRP78, XBP1, Derlin-1 were increased after TMAO treatment (all P < 0.05). In Conclusion, high fat and TMA could induce the expression of FMO1 and its metabolite TMAO. When FMO1 is silenced, the effects of high fat and TMA on TMAO are blocked. And the role of TMAO in NAFLD may be through the activation of UPR.

HDAC6 inhibitor ACY1215 inhibits the activation of NLRP3 inflammasome in acute liver failure by regulating the ATM/F-actin signalling pathway.

Acute liver failure (ALF) is a rare and critical medical condition. This study was designed to investigate the protective effects and underlying mechanism of ACY1215 in ALF mice. Our findings suggested that ACY1215 treatment ameliorates the pathological hepatic damage of ALF and decreases the serum levels of ALT and AST. Furthermore, ACY1215 pretreatment increased the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF. Moreover, ACY1215 inhibited the level of NLRP3, ASC, caspase-1, IL-1ß and IL-18 in ALF. The ATM inhibitor KU55933 could decrease the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF with ACY1215 pretreatment. The F-actin inhibitor cytochalasin B decreased the level of F-actin and vinculin in ALF with ACY1215 pretreatment. However, cytochalasin B had no effect on protein levels of ATM, Chk2, p53 and p21 in ALF with ACY1215 pretreatment. Cytochalasin B could dramatically increase the level of NLRP3, ASC, caspase-1, IL-1ß and IL-18 in ALF with ACY1215 pretreatment. These results indicated that ACY1215 exhibited hepatoprotective properties, which was associated with the inhibition of NLRP3 inflammasome, and this effect of ACY1215 was connected with upregulation of the ATM/F-actin mediated signalling pathways.

The relationship between liver pathological inflammation degree and pyroptosis in chronic hepatitis B patients.

The aim of this study is to explore the relationship between liver pathological inflammation degree and pyroptosis in patients with chronic hepatitis B (CHB). One hundred and twenty CHB patients' liver tissue samples, including A0-A3 inflammatory grades, were selected. Six tissue sections were selected for each indicator in each inflammation grade. The results of immunohistochemical analysis on the pyroptosis-related molecules (NLRP3, GSDMD, caspase1, interleukin [IL]-1ß, and IL-18) were determined. The correlation between the pyroptosis-related molecules and liver inflammatory activities was analyzed. The expression of NLRP3, GSDMD, caspase1, IL-18, and IL-1ß was respectively significantly positively correlated with the grade of inflammatory activity (r s = 0.690, p < 0.01; r s = 0.681, p < 0.01; r s = 0.540, p < 0.01; r s = 0.725, p < 0.01; r s = 0.663, p < 0.01) and linear relationship (χ 2 = 56.763, p < 0.01; χ 2 = 55.350, p < 0.01; χ 2 = 34.776, p < 0.01; χ 2 = 62.523, p < 0.01; χ 2 = 52.521, p < 0.01) in liver tissue. The high expression of NLRP3, GSDMD, caspase1, IL-1ß, and IL-18 may be involved in the process of liver tissue inflammation and damage, which is positively correlated with liver tissue inflammation in patients with CHB.

Coronavirus Disease 19 Infection Does Not Result in Acute Kidney Injury: An Analysis of 116 Hospitalized Patients from Wuhan, China.

BACKGROUND: Whether the patients with coronavirus disease 19 (COVID-19) infected by severe acute respiratory syndrome (SARS)-CoV-2 would commonly develop acute kidney injury (AKI) is an important issue worthy of clinical attention. This study aimed to explore the effects of SARS-CoV-2 infection on renal function through analyzing the clinical data of 116 hospitalized COVID-19-confirmed patients. METHODS: One hundred sixteen COVID-19-confirmed patients enrolled in this study were hospitalized in the Department of Infectious Diseases, Renmin Hospital of Wuhan University from January 14 to February 13, 2020. The recorded information includes demographic data, medical history, contact history, potential comorbidities, symptoms, signs, laboratory test results, chest computer tomography scans, and treatment measures. SARS-CoV-2 RNA in 53 urine sediments of enrolled patients was detected by real-time reverse transcription-polymerase chain reaction. RESULTS: Twelve (10.8%) patients showed mild increase of blood urea nitrogen or creatinine (<26 µmol/L within 48 h), and 8 (7.2%) patients showed trace or 1+ albuminuria in 111 COVID-19-confirmed patients without chronic kidney disease (CKD). All these patients did not meet the diagnostic criteria of AKI. In addition, 5 patients with CKD who were undergone regular continuous renal replacement therapy (CRRT) before admission were confirmed infection of SARS-CoV-2 and diagnosed as COVID-19. In addition to therapy for COVID-19, CRRT was also applied 3 times weekly during hospitalization for these 5 patients with CKD. In the course of treatment, the renal function indicators showed stable state in all 5 patients with CKD, without exacerbation of CKD, and pulmonary inflammation was gradually absorbed. All 5 patients with CKD were survived. Moreover, SARS-CoV-2 RNA in urine sediments was positive only in 3 patients from 48 cases without CKD, and 1 patient had a positive for SARS-CoV-2 open reading frame 1ab from 5 cases with CKD. CONCLUSION: AKI was uncommon in COVID-19. SARS-CoV-2 infection does not result in AKI, or aggravate CKD in the COVID-19 patients.

Artificial neural network-based models used for predicting 28- and 90-day mortality of patients with hepatitis B-associated acute-on-chronic liver failure.

BACKGROUND: This study aimed to develop prognostic models for predicting 28- and 90-day mortality rates of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF) through artificial neural network (ANN) systems. METHODS: Six hundred and eight-four cases of consecutive HBV-ACLF patients were retrospectively reviewed. Four hundred and twenty-three cases were used for training and constructing ANN models, and the remaining 261 cases were for validating the established models. Predictors associated with mortality were determined by univariate analysis and were then included in ANN models for predicting prognosis of mortality. The receiver operating characteristic curve analysis was used to evaluate the predictive performance of the ANN models in comparison with various current prognostic models. RESULTS: Variables with statistically significant difference or important clinical characteristics were input in the ANN training process, and eight independent risk factors, including age, hepatic encephalopathy, serum sodium, prothrombin activity, γ-glutamyltransferase, hepatitis B e antigen, alkaline phosphatase and total bilirubin, were eventually used to establish ANN models. For 28-day mortality in the training cohort, the model's predictive accuracy (AUR 0.948, 95% CI 0.925-0.970) was significantly higher than that of the Model for End-stage Liver Disease (MELD), MELD-sodium (MELD-Na), Chronic Liver Failure-ACLF (CLIF-ACLF), and Child-Turcotte-Pugh (CTP) (all p < 0.001). In the validation cohorts the predictive accuracy of ANN model (AUR 0.748, 95% CI: 0.673-0.822) was significantly higher than that of MELD (p = 0.0099) and insignificantly higher than that of MELD-Na, CTP and CLIF-ACLF (p > 0.05). For 90-day mortality in the training cohort, the model's predictive accuracy (AUR 0.913, 95% CI 0.887-0.938) was significantly higher than that of MELD, MELD-Na, CTP and CLIF-ACLF (all p < 0.001). In the validation cohorts, the prediction accuracy of the ANN model (AUR 0.754, 95% CI: 0.697-0.812 was significantly higher than that of MELD (p = 0.019) and insignificantly higher than MELD-Na, CTP and CLIF-ACLF (p > 0.05). CONCLUSIONS: The established ANN models can more accurately predict short-term mortality risk in patients with HBV- ACLF. The main content has been postered as an abstract at the AASLD Hepatology Conference (https://doi.org/10.1002/hep.30257).

Long noncoding RNA LNC473 inhibits the ubiquitination of survivin via association with USP9X and enhances cell proliferation and invasion in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Recent studies reported that lncRNA LINC00473 (LNC473) was involved in cancer progression. However, the clinical significance and functional role of LNC473 in HCC progression is still unknown. In the present study, we found that the LNC473 expression was markedly elevated in HCC tissues and correlated with bigger tumor size, higher BCLC stage, vascular invasion and poor prognosis. Gain- and loss-of-function assay showed that LNC473 enhanced HCC cell proliferation and invasion and induced epithelial-mesenchymal transition (EMT) process. Mechanistically, LNC473 associated with oncoprotein survivin and regulates its stability. Moreover, LNC473 could recruit deubiquitinase USP9X to inhibit the ubiquitination level of survivin and then increase survivin expression. Therefore, our results suggest that LNC473 exerts its functions as an oncogene in HCC progression and may be a therapeutic target for HCC treatment.

Histone Deacetylase 2 Inhibitor CAY10683 Alleviates Lipopolysaccharide Induced Neuroinflammation Through Attenuating TLR4/NF-κB Signaling Pathway.

Neuroinflammation involves in the progression of many central nervous system diseases. Several studies have shown that histone deacetylase (HDAC) inhibitors modulated inflammatory responses in lipopolysaccharide (LPS) stimulated microglia. While, the mechanism is still unclear. The aim of present study was to investigate the effect of HDAC2 inhibitor CAY10683 on inflammatory responses and TLR4/NF-κB signaling pathways in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. The effect of CAY10683 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expressions of inflammatory cytokines were analyzed by western blotting and RT-PCR respectively. The TLR4 protein expression was measured by western blotting, immunofluorescence, immunohistochemistry respectively. The protein expressions of MYD88, phospho-NF-κB p65, NF-κB-p65, acetyl-H3 (AH3), H3, and HDAC2 were analyzed by western blotting. We found that CAY10683 could inhibit expression levels of inflammatory cytokine TNF-α and IL-1ß in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. It could induce TLR4, MYD88, phospho-NF-κB p65, and HDAC2 expressions. Moreover, CAY10683 increased the acetylation of histones H3 in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. Taken together, our findings suggested that HDAC2 inhibitor CAY10683 could suppress neuroinflammatory responses and TLR4/NF-κB signaling pathways by acetylation after LPS stimulation.

The Nephroprotective Effect of MS-275 on Lipopolysaccharide (LPS)-Induced Acute Kidney Injury by Inhibiting Reactive Oxygen Species (ROS)-Oxidative Stress and Endoplasmic Reticulum Stress.

BACKGROUND Histone deacetylase (HDAC) inhibitors can attenuate acute kidney injury (AKI)-mediated damage and reduce fibrosis in kidney disease models. The aim of the present study was to investigate the effects of the HDAC inhibitor MS-275 on lipopolysaccharide (LPS)-induced AKI and the associated mechanisms. MATERIAL AND METHODS A LPS-induced model in 6-8 weeks-old mice was established by intraperitoneal injection of LPS (10 mg/kg), with pre-treatment of MS-275 (2 mg/kg/day) administered intraperitoneally for five days. In addition, HK-2 cells were exposed to LPS (1 µg/mL) at 0.1 nM, 1 nM, 10 nM, and 100 nM. For our in vitro MS-275 study, detection programs included histology, biochemical, immunohistochemistry, mRNA and protein expression as well as apoptosis. RESULTS MS-275 ameliorated renal damage, enhanced the survival rate of the LPS-induced sepsis model, decreased the expressions of TNF-α, IL-1ß, IL-6, COX-2, and NF-κBp65 nucleus translocation, suppressed the HDAC activity which was enhanced in septic AKI mice, and enhanced the acetylation of histone H3 and H4. Reactive oxygen species (ROS) production was enhanced in the kidney of LPS mice compared to control mice, while MS-275 suppressed the production of ROS in kidney tissue. In the in vitro studies, MS-275 reduced the LPS-induced apoptosis of HK-2 cells, inhibited ROS and MDA production, increased the production GSH and SOD activity, decreased the expressions of CHOP, GRP78, caspase3, and capase12, which was related to endoplasmic reticulum stress in LPS stimulated HK-2 cells. CONCLUSIONS MS-275 pre-treatment improved renal function and ameliorated histological alterations, inflammation, and ROS production in LPS-induced AKI mice and may act through inhibiting ROS-oxidative stress and endoplasmic reticulum stress.

The Protective Mechanism of CAY10683 on Intestinal Mucosal Barrier in Acute Liver Failure through LPS/TLR4/MyD88 Pathway.

The purpose of this study was to investigate the protective mechanism of HDAC2 inhibitor CAY10683 on intestinal mucosal barrier in acute liver failure (ALF). In order to establish ALF-induced intestinal epithelial barrier disruption models, D-galactosamine/LPS and LPS were, respectively, used with rats and NCM460 cell and then administrated with CAY10683. Transepithelial electrical resistance (TEER) was measured to detect the permeability of cells. Real-time PCR and Western blotting were employed to detect the key mRNA and protein levels. The intestinal epithelial tissue pathology was detected. After interfering with CAY10683, the mRNA and protein levels of TLR4, MyD88, TRIF, and TRAF6 were decreased compared with model group (P < 0.05), whereas the levels of ZO-1 and occluding were elevated (P < 0.05). The permeability was elevated in CAY10683-interfered groups, when compared with model group (P < 0.05). And the degree of intestinal epithelial tissue pathological damage in CAY10683 group was significantly reduced. Moreover, CAY10683 significantly decreased the TLR4 staining in animal tissue. The HDAC2 inhibitor CAY10683 could promote the damage of intestinal mucosal barrier in ALF through inhibiting LPS/TLR4/MyD88 pathway.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid alleviates liver fibrosis by suppressing the transforming growth factor-ß1 signal pathway.

BACKGROUND: Histone deacetylases (HDACs) inhibitors are new anti-fibrotic drugs that inhibit the activity of hepatic stellate cells. The present study focused on the anti-fibrotic function of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) by suppressing transforming growth factor-ß1 (TGF-ß1) signaling. METHODS: Male Sprague-Dawley rats were used to induce liver fibrosis with carbon tetrachloride (CCl4) and LX2 cell (human hepatic stellate cell line) was stimulated by TGF-ß1. Both animals and cells were treated with SAHA. The Smad7 and connective tissue growth factor (CTGF) mRNA levels were detected by real-time polymerase chain reaction (PCR). Western blotting was used to examine the protein levels of CTGF, Histone H3 (H3), Smad7, Smad2/3, Acetyl-Histone H3 (AH3), HDAC2, α-smooth muscle actin (α-SMA), HDAC6, p-Smad2/3 and HDAC8. In addition, the TGF-ß1 and liver enzyme levels from rat serum were detected. Histopathological changes were examined by hematoxylin and eosin (HE), Sirius red and Masson trichrome staining. The α-SMA expression was detected by immumohistochemical staining. RESULTS: Compared with control group, the TGF-ß1 and liver enzyme levels from rat serum, together with the mRNA levels of CTGF and protein levels of CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were elevated in fibrotic rats (P < 0.01). But the Smad7 mRNA and AH3 protein levels were notably suppressed in the fibrotic rats (P < 0.01). Pathological examination showed the typical changes of liver fibrosis in the fibrotic rats. After the treatment with SAHA, the levels of liver enzymes, TGF-ß1, CTGF, HDAC2, α-SMA, HDAC6, p-Smad2/3 and HDAC8 were reduced (P < 0.01) and Smad7 and AH3 protein contents were elevated in liver fibrotic rats (P < 0.01). Moreover, immumohistochemistry showed that SAHA significantly suppressed the α-SMA protein content in fibrotic liver (P < 0.01). CONCLUSION: The HDAC inhibitor SAHA alleviated liver fibrosis by suppressing the TGF-ß1 signaling.

High serum resistin associates with intrahepatic inflammation and necrosis: an index of disease severity for patients with chronic HBV infection.

BACKGROUND: Studies have revealed that resistin plays a role as an intrahepatic cytokine with proinflammatory activities. This study investigated the association between serum resistin and fibrosis severity and the possible marker role of resistin in the inflammatory process of chronic hepatitis B. METHODS: In this study, 234 subjects with HBV infection were retrospectively selected, including 85 patients with chronic hepatitis B (CHB), 70 patients with HBV-related liver cirrhosis (LC-B), and 79 patients with HBV-related liver failure (LF-B). Serum levels of resistin, IL-1, IL-6, IL-17, IL-23, TNF-α, and TGF-ß1 were assayed by ELISA. Demographic and clinical characteristics of patients were extracted from clinical databases of Taihe Hospital, Hubei University of Medicine, including serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and liver stiffness (LS). RESULTS: All the selected patients with HBV infection showed significantly increased levels of serum resistin, which was rarely detectable in the healthy controls. Serum resistin levels in patients with CHB, LC-B, and LF-B were 4.119 ± 5.848 ng/mL, 6.370 ± 6.834 ng/mL, and 6.512 ± 6.076 ng/mL, respectively. Compared with the CHB group, patients with LC-B or LF-B presented with significantly higher serum levels of resistin (p < 0.01). On the other hand, all of the enrolled patients had high serum levels of IL-1, IL-6, IL-17, TNF-α, and TGF-ß1, but not IL-23. Interestingly, serum levels of resistin was significantly positively correlated with serum levels of TGF-ß1 in LC-B patients (R = 0.3090, p = 0.0290), with IL-17 in LC-B (R = 0.4022, p = 0.0038) and LF-B patients (R = 0.5466, p < 0.0001), and with AST (R = 0.4501, p = 0.0036) and LS (R = 0.3415, p = 0.0310) in CHB patients. CONCLUSIONS: High serum resistin associates with intrahepatic inflammation and necrosis and may be used as an index of disease severity for patients with chronic HBV infection.

Real-world treatment patterns and clinical outcomes of HCV treatment-naive patients in China: an interim analysis from the CCgenos study.

BACKGROUND AND AIM: In China, chronic hepatitis C virus (HCV) infection represents a considerable healthcare burden. Although interferon-based therapy has been the standard-of-care for many years, few long-term, real-life studies have assessed interferon-based treatment in China. The objective of CCgenos follow-up study was to analyze long-term treatment patterns and outcomes in a cohort of treatment-naïve, Han ethnic, patients with chronic HCV infection. METHODS: Patients who had participated in the CCgenos cross-sectional study were invited to enter this 5-year follow up. Clinical information and centralized HCV-RNA measures were collected at scheduled study visits every 6 months for untreated patients and every 3 months for treated patients. RESULTS: Among 512 patients enrolled, 334 (65.2%) received interferon-based treatment and 178 (34.8%) remained untreated over a median of 4.1 (1.2-4.3) years. A total of 82.8% (424/512) of patients had an IL28B CC genotype (GT); 60.7% (311/512) had HCV GT1b infection, including 121 (38.9%) untreated. Most patients with baseline cirrhosis were untreated (26/46, 56.5%). Among patients who completed treatment and 24 weeks of post-treatment follow up, the duration of interferon-based therapy was frequently longer than recommended (52.9% [92/174] of GT1b-infected were treated for > 1 year). Rates of sustained virologic response (SVR24) were 71.1% (226/318) overall; 62.4% (111/178) among patients with HCV GT1b infection; and 42.9% (15/35) among patients with cirrhosis. CONCLUSIONS: There remains a high unmet need for effective HCV treatment in China, evidenced by a high proportion of patients remaining untreated by the current standard-of-care and relatively low SVR24 rates for patients with both GT1b infection and cirrhosis.

Trichostatin A protects against intestinal injury in rats with acute liver failure.

BACKGROUND: Histone deacetylase (HDAC) inhibitors have been widely applied in the clinic as anticancer drugs against multiple neoplasms and proved their anti-inflammation under different pathology recently. Trichostatin A (TSA) is an HDAC inhibitor specific in class I and II HDAC enzymes. The aim of the present study was to elucidate the protective effects of TSA on acute liver failure (ALF) in rats and its potential mechanism. METHODS: A total of 18 female Sprague-Dawley rats were separated into control, model, and TSA groups. We used Western blotting to determine the expression of HDACs, inflammatory cytokines, and acetylation of histone in liver and small intestine. The gene expression of inflammatory factors and Cox-2 was detected by a polymerase chain reaction. Colonic motility was assessed by spatiotemporal mapping. Histologic analysis and immunohistochemistry were performed. Intestinal permeability examination and levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were also observed. RESULTS: ALF procedure caused harm to histology of liver and small intestine, increased the intestinal permeability and serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin. It also interrupted the normal organization of colonic motor patterns by hurting enteric nervous system and pacemaker cells. Along with the decrease of inflammatory factors in ALF rats by TSA administration, all the damage to the liver, the small intestine, and the colon was repaired. CONCLUSIONS: TSA alleviates the lesion in liver, as well as in small intestine and colon in ALF rats by directly inhibiting inflammatory response.

Fabrication of Alginate/Calcium Carbonate Hybrid Microparticles for Synergistic Drug Delivery.

A hybrid drug delivery system coloaded with different drugs for synergistic drug delivery was developed. Alginate/calcium carbonate (CaCO3) hybrid microparticles (MPs) were fabricated via a facile coprecipitation method under mild conditions without using any organic solvent and surfactant. Due to the incorporation of negatively charged alginate chains onto the surface, the obtained hybrid MPs with spherical morphology showed good colloidal stability in an aqueous solution. An antitumor drug (doxorubicin, DOX) and a drug resistance reversal agent (verapamil, VP) were coloaded in the hybrid MPs simultaneously to obtain dual-drug-loaded MPs (DOX/VP/MP). Due to the presence of inorganic CaCO3 (∼54 wt%), the drugs could be loaded in the hybrid MPs with high encapsulation efficiency and the drug release could be effectively sustained. The cell growth inhibition of the drug-loaded MPs was evaluated in HeLa cells. An in vitro study showed DOX/VP/MP exhibited higher cell growth inhibition as compared with DOX monodrug-loaded MPs (DOX/MP). These results suggest the hybrid MPs can potentially be used as a synergistic drug delivery platform for cancer chemotherapy.

Amphiregulin promotes the immunosuppressive activity of intrahepatic CD4+ regulatory T cells to impair CD8+ T-cell immunity against hepatitis B virus infection.

Hepatitis B virus (HBV) infection causes liver diseases and hepatocellular carcinoma. Immunotolerance in HBV-infected patients is one of the factors that incur failure of HBV clearance and persistent HBV amplification. However, the mechanisms underlying immunotolerance after HBV infection are yet to be thoroughly understood. Using a novel HBV mouse model, we found for the first time that epidermal growth factor receptor (EGFR) is up-regulated on intrahepatic regulatory T (Treg) cells in HBV-infected mouse livers. The EGFR-positive Treg cells are more immunosuppressive than EGFR-negative Treg cells, demonstrated by higher expression of immunosuppressive cytokines and robust inhibition of CD8+ T-cell proliferation in vitro. Furthermore, EGFR-positive Treg cells potently restrain CD8+ T-cell-mediated anti-viral activity, leading to higher HBV burden in hepatocytes. Amphiregulin, a cytokine of the EGF family, is significantly up-regulated in HBV-infected livers, but the cellular sources of amphiregulin are still elusive. Amphiregulin promotes the immunosuppressive activity of EGFR-positive Treg cells in vitro, so as to profoundly inhibit production of anti-viral components in CD8+ T cells. Taken together, our discovery elucidated a novel mechanism contributing to immunotolerance and viral amplification after HBV infection. Our study may provide new clues for developing therapeutic strategies against HBV infection.

Coexistence of hepatitis B virus quasispecies enhances viral replication and the ability to induce host antibody and cellular immune responses.

UNLABELLED: Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and used for construction of HBV replication-competent plasmids. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion, and subcellular localization of viral proteins in vitro. Viral replication intermediates were detected by Southern blotting. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a nonexportable hepatitis B virus surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when coapplied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection. IMPORTANCE: Hepatitis B virus (HBV) is an important human pathogen. HBV quasispecies with genetically heterogenous variants are thought to play a role in the progression of HBV-associated liver diseases. So far, direct evidence is available in only a few cases to confirm the proposed role of HBV variants in the pathogenesis. We report here that the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased HBV replication and induced significantly stronger intrahepatic cytotoxic T lymphocyte responses and antibody responses specific to HBV surface antigen (HBsAg) in mice. Our discovery uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses and may enhance immune-mediated liver damage under some circumstances, representing a novel mechanism for the immunopathogenesis of HBV infection.