Guangfeng Shi

Promoter Hypermethylation Mediated Downregulation of FBP1 in Human Hepatocellular Carcinoma and Colon Cancer

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Impaired TLR3/IFN-β signaling in monocyte-derived dendritic cells from patients with acute-on-chronic hepatitis B liver failure: Relevance to the severity of liver damage

Toll-like receptors (TLRs) are a class of proteins that play key roles in innate immunity through recognition of microbial components. TLR3 is expressed abundantly in dendritic cells, and is responsible for recognizing viral pathogens and inducing interferon beta (IFN-beta) production. Although TLR3 has been reported to be involved in several diseases caused by viral infections, its role in hepatitis B virus (HBV)-induced hepatitis is still largely unknown. We found that expression of TLR3 and IFN-beta was decreased significantly in monocyte-derived dendritic cells (MoDCs) from patients with chronic hepatitis B (CHB, n=40) or acute-on-chronic hepatitis B liver failure (ACHBLF, n=60), compared with normal controls (n=20). We observed a further decrease in TLR3 and IFN-beta in ACHBLF compared to CHB patients. Compared with surviving patients, TLR3 and IFN-beta expression was significantly lower in non-surviving ACHBLF patients, which strongly indicated a correlation between TLR3 signaling impairment in MoDCs and disease severity in ACHBLF patients. Further linear correlation analysis demonstrated significant correlations between expression of TLR3 signaling components (TLR3 and IFN-beta) and disease severity markers (prothrombin activity and total bilirubin) for individual ACHBLF patients. To the best of our knowledge, this is the first study to show that MoDC impairment is correlated with severe liver damage in ACHBLF patients, which suggests the potential of TLR3/IFN-beta expression in MoDCs as a diagnostic marker.

Fuzheng Huayu inhibits carbon tetrachloride-induced liver fibrosis in mice through activating hepatic NK cells

AIM OF THE STUDY Fuzheng Huayu (FZHY) is a Chinese compound herbal preparation which consists of six Chinese herbs. This study examines the preventative effects of FZHY on liver fibrosis induced by carbon tetrachloride (CCl(4)) and explores its possible mechanisms of action. MATERIALS AND METHODS Liver fibrosis was induced in male C57BL/6N mice by injecting a 10% CCl(4) solution intraperitoneal twice a week for six weeks. After 6 weeks of treatment, serum ALT and AST assay, liver tissue histological examination and immunostaining were carried out to examine the liver function and fibrosis degree. The expression levels of alpha-smooth muscle actin (SMA) were measured by quantitative real-time PCR and western blot. Hepatic natural killer (NK) cells were isolated from liver and evaluated by FACS. RESULTS Upon pathological examination, the FZHY-treated mice showed significantly reduced liver damage. The expression of α-SMA increased markedly upon treatment with CCl(4) and the increase was reversed by FZHY treatment. FZHY treatment also enhanced the activation of hepatic NK cells and the production of interferon-gamma (IFN-γ). The protective effects of FZHY were reversed in the mice that were depleted of NK cells by anti-ASGM-1 Ab treatment. CONCLUSIONS FZHY can efficiently inhibit CCl(4)-induced liver fibrosis. Furthermore, the depletion of NK cells attenuates the protective effects of FZHY. We conclude that FZHY could be an effective drug for liver fibrosis, and its mechanism of action involves the activation of hepatic NK cells.

Validation of prognostic scores to predict short-term mortality in patients with HBV-related acute-on-chronic liver failure: The CLIF-C OF is superior to MELD, CLIF SOFA, and CLIF-C ACLF

Abstract Acute-on-chronic liver failure (ACLF) in chronic hepatitis B (CHB) patients has a high short-term mortality. Identification of effective models to predict the short-term mortality may enable early intervention and improve patients’ prognosis. We aim to assess the performance of the CLIF Consortium Organ Failure score (CLIF-C OFs), CLIF sequential organ failure assessment score (CLIF-SOFAs), CLIF Consortium ACLF score (CLIF-C ACLFs), ACLF grade, and model for end-stage liver disease score (MELDs) in predicting the short-term mortality in CHB patients with ACLF. Among the 155 consecutive adult patients with liver failure as a discharge diagnosis were screened, and all the patients were treated at the Department of Infectious Diseases, Huashan Hospital, Fudan University (Shanghai, China) from January 2010 to February 2016. The diagnosis of ACLF was based on the criteria formalized by the ACLF consensus recommendations of the Asian Pacific Association for the Study of the Liver (APASL). Diagnostic accuracy for predicting short-term (28-day) mortality was calculated for CLIF-C OFs, CLIF-SOFAs, CLIF-C ACLFs, ACLF grade, and MELDs in all patients. One hundred fifty-five consecutive adult liver failure patients were screened and 85 patients including 73 males and 12 females were enrolled. Overall, the 28-day transplant-free mortality was 32% in all patients, and 100% in those with severe early course (ACLF-3). The area under the receiver operating characteristic curve (AUROC) of CLIF-C OFs (AUROC: 0.906, P = .0306, compared with MELDs) was higher than those of CLIF-SOFAs (AUROC: 0.876), CLIF-C ACLFs (AUROC: 0.858), ACLF grade (AUROC: 0.857), and MELDs (AUROC: 0.838) for predicting short-term mortality. The cut-point for baseline CLIF-C OFs in predicting death was 8.5, with 67% sensitivity, 90% specificity, and AUROC of 0.906 (95% CI: 0.8450–0.9679). The results indicate that short-term mortality is high in patients with ACLF and CLIF Consortium Organ Failure score is superior to MELD, CLIF SOFA, and CLIF-C ACLF in predicting its short-term mortality.

Serum MicroRNA Levels as a Noninvasive Diagnostic Biomarker for the Early Diagnosis of Hepatitis B Virus-Related Liver Fibrosis.

Background/Aims To investigate the role of selected serum microRNA (miRNA) levels as potential noninvasive biomarkers for differentiating S0–S2 (early fibrosis) from S3–S4 (late fibrosis) in patients with a chronic hepatitis B virus (HBV) infection. Methods One hundred twenty-three treatment-naive patients with a chronic HBV infection who underwent a liver biopsy were enrolled in this study. The levels of selected miRNAs were measured using a real-time quantitative polymerase chain reaction assay. A logistic regression analysis was performed to assess factors associated with fibrosis progression. Receiver operating characteristic (ROC) curve and discriminant analyses validated these the ability of these predicted variables to discriminate S0–S2 from S3–S4. Results Serum miR-29, miR-143, miR-223, miR-21, and miR-374 levels were significantly downregulated as fibrosis progressed from S0–S2 to S3–S4 (p<0.05), but not miR-16. The multivariate logistic regression analysis identified a panel of three miRNAs and platelets that were associated with a high diagnostic accuracy in discriminating S0–S2 from S3–S4, with an area under the curve of 0.936. Conclusions The levels of the studied miRNAs, with the exception of miR-16, varied with fibrosis progression. A panel was identified that was capable of discriminating S0–S2 from S3–S4, indicating that serum miRNA levels could serve as a potential noninvasive biomarker of fibrosis progression.

Role of interleukin-23 in monocyte-derived dendritic cells of HBV-related acute-on-chronic liver failure and its correlation with the severity of liver damage.

BACKGROUND The role of interleukin-23 (IL-23) in monocyte-derived dendritic cells (MoDCs) from hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients remains unclear. The aim of this study was to observe the correlation between the activation of the IL-23 signaling pathway and the prognosis of HBV-ACLF patients. MATERIALS AND METHODS The baseline levels of serum IL-6, IL-12, IL-17, IL-23, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) from immune tolerant (IT), chronic hepatitis B (CHB), HBV-ACLF patients and healthy individuals who served as healthy controls (HCs) were analyzed using the Luminex system, whereas serum IL-23 from HBV-ACLF patients was measured by ELISA before and after treatment. Purified monocytes were isolated from peripheral blood and were induced into MoDCs, IL-23, IL-23R, NF-κB and TRAF6 expression in MoDCs from 4 groups was analyzed using real-time PCR, and IL-23R and intracellular IL-23 were evaluated by flow cytometry. RESULTS Serum IL-6, IL-12, IL-17, IL-23 and TNF-α levels were upregulated in HBV-ACLF patients compared with IT patients, CHB patients and HCs (P<0.05 for all). Serum IL-23 was closely correlated with elevated serum IL-17 in HBV-ACLF patients (r=0.5935, P<0.0001). Moreover, IL-23 and IL-23R levels were significantly upregulated in MoDCs from patients with CHB or HBV-ACLF compared with HCs, and further upregulation of IL-23 and IL-23R was observed in HBV-ACLF patients compared to CHB patients (P<0.05 for all). IL-23 expression was markedly enhanced and was correlated with elevated NF-κB and TRAF6 in MoDCs from HBV-ACLF patients (P<0.05 for both). Linear correlation analysis demonstrated significant correlations between the expression of IL-23 and disease severity markers (MELD scoring system, international normalized ratio, prothrombin time and total bilirubin, r=0.6874, r=0.6475, r=0.6249, r=0.3771, respectively, P<0.05 for all) for individual HBV-ACLF patients, and IL-23 levels were significantly upregulated in non-survival HBV-ACLF patients compared with survival HBV-ACLF patients (P<0.05). CONCLUSION IL-23 in serum and MoDCs is significantly elevated in HBV-ACLF patients, TRAF6/NF-κB may play a role in IL-23 production by MoDCs in HBV-ACLF patients and high pre-treatment IL-23 levels in MoDCs are associated with mortality in HBV-ACLF patients.

Interleukin-23 mediates the pathogenesis of LPS/GalN-induced liver injury in mice

Background Interleukin‐23 (IL‐23) is required for T helper 17 (Th17) cell responses and IL‐17 production in hepatitis B virus infection. A previous study showed that the IL‐23/IL‐17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL‐23 in acute liver injury remains unclear. Objective The purpose of this study was to determine the role of the inflammatory cytokine IL‐23 in lipopolysaccharide/d‐galactosamine (LPS/GalN)‐induced acute liver injury in mice. Methods Serum IL‐23 from patients with chronic hepatitis B virus (CHB), acute‐on‐chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL‐23p19 neutralizing antibody or an IL‐23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10 &mgr;g/kg) and GalN (400 mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17‐related cytokines, including IL‐17 and TNF‐&agr;; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue. Results Serum IL‐23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P < 0.05 for both). Serum IL‐23 was significantly upregulated in the non‐survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN‐induced acute hepatic injury in mice (P < 0.05 for both). Moreover, after treatment, serum IL‐23 was downregulated in the survival group of ACLF patients (P < 0.001). Compared with LPS/GalN mice, mice treated with either an IL‐23p19 neutralizing antibody or an IL‐23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17‐related inflammatory cytokine, including IL‐17 and TNF‐&agr;; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P < 0.05 for all). Conclusion High serum IL‐23 was associated with mortality in ACLF patients and LPS/GalN‐induced acute liver injury in mice. IL‐23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17‐related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL‐23 likely functions upstream of Th17‐related cytokine and chemokine expression to recruit inflammatory cells into the liver. HighlightsIL‐23 was associated with mortality in acute on chronic liver failure patients.IL‐23 neutralizing antibodies attenuated LPS/GalN‐induced acute liver injury in mice.The mechanism of attenuating liver injury involves regulation of Th17‐related cytokine and chemokine expression in liver.

MicroRNA-548j inhibits type I interferon production by targeting ZBTB11 in patients with chronic hepatitis B

Type I IFNs play crucial roles in antiviral immune responses. By inducing cellular resistance to viral infection and apoptosis of virus-infected cells, they impair virus replication and eliminate the invading pathogens. However, in CHB patients, generation of type I IFN was significantly impaired and the underlying mechanisms have not been fully understood. MicroRNA-548 family has been implicated in regulating antiviral response upon various infections. Here we explored the potential role of miR-548j in modulating type I IFN production in CHB. We found that miR-548j expression increased in MoDCs from CHB patients than that from healthy volunteers and transient transfection of miR-548j mimic led to a marked decrease of IFN-α/β production in MoDCs from healthy volunteers while blockade of miR-548j by anti-miR-548j significantly restored IFN-α/β secretion in MoDCs from CHB patients. In addition, Zinc finger and BTB domain containing 11 (ZBTB11) was predicted and finally confirmed to be a target of miR-548j. Forced expression of ZBTB11 also restored IFN-α/β secretion in MoDCs from CHB patients. These results indicate the involvement of miR-548j in aberrant production of type I IFN in CHB patients, and provide a potential target for developing anti-HBV therapies.

miR-96-5p prevents hepatic stellate cell activation by inhibiting autophagy via ATG7

Activation of hepatic stellate cell (HSC), which is the main source of extracellular matrix, plays a pivotal role in liver fibrogenesis. Autophagy of hepatic stellate cell has been recently implicated in liver fibrosis, but the regulation of hepatic stellate cell autophagy during this process remains poorly understood. Here, we first identified miR-96-5p as an aberrantly expressed miRNA in fibrotic liver tissues. Next, we transfected miR-96-5p mimic into human hepatic stellate cell line LX-2 and observed decreased protein and mRNA levels of α-SMA and Col1A1. In addition, transfection of miR-96-5p mimic significantly reduced autophagy activity of LX-2 cells, while transfection of miR-96-5p inhibitor promoted LX-2 cell autophagy. Moreover, autophagy-related protein 7 (ATG7) was predicted as a potential target of miR-96-5p and luciferase assay confirmed its direct interaction with miR-96-5p. Finally, reintroduction of ATG7 into LX-2 cells reversed miR-96-5p-mediated inhibition of autophagy as well as α-SMA and Col1A1 expression. In conclusion, we demonstrated that miR-96-5p can inhibit hepatic stellate cell activation by blocking autophagy via ATG7. These findings provide new insight into the development of miRNA-based anti-fibrotic strategies.Key messages• Altered miRNA expression profile is observed in fibrotic liver tissues.• miR-96-5p can inhibit HSC activation.• Autophagy of HSC is repressed by miR-96-5p during activation.• ATG7 is a direct target of miR-96-5p.• ATG7 can rescue miR-96-5p-mediated inhibition of autophagy and HSC activation.

The role of T helper 17 cells in the pathogenesis of hepatitis B virus-related liver cirrhosis (Review)

In chronic hepatitis B virus (HBV)-infected patients, T helper 17 (Th17) cells are significantly elevated. Th17 cells initiate immune-mediated pathogenesis and have a critical role in the process of HBV-related liver cirrhosis (HBV-LC). The mechanisms underlying this process are attributed to Th17-secreted cytokines, which include interleukin (IL)-17, IL-21 and IL-22; however, a systemic analysis regarding these mechanisms has yet to be conducted. Therefore, the present study aimed to investigate the role of Th17 cells in the pathogenesis of HBV-LC. All randomized clinical trials, case series, case reports and meta-analyses that contained the aforementioned keywords were included in the review process. In addition, unpublished information from the Food and Drug Administration was included. The findings indicated that Th17-secreted cytokines, including IL-17, IL-21 and IL-22, function by activating or silencing hepatic stellate cells, modulating proinflammatory and pro- or antifibrogenic effectors, regulating extracellular matrix formation, upregulating chemokine expression, and inducing hepatocellular damage or hepatoprotection during the HBV-LC process. In addition, Th17 cells and Th17-secreted cytokines may be considered a potential tool in the diagnosis or treatment of HBV-LC. The present review summarized the role of Th17 cells in the pathogenesis of HBV-LC in order to deepen the clinical understanding of the role of Th17 cells and also to support the development of effective therapies for patients with HBV-LC.