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Zhang X.,Chinese University of Hong Kong | Han J.,Chinese University of Hong Kong | Han J.,Institute of Liver Disease | Man K.,University of Hong Kong | And 8 more authors.
Journal of Hepatology | Year: 2016

Background & Aims CXC chemokine receptor 3 (CXCR3) is involved in virus-related chronic liver inflammation. However, the role of CXCR3 in non-alcoholic steatohepatitis (NASH) remains unclear. We aimed to investigate the role of CXCR3 in NASH. Methods Human liver tissues were obtained from 24 non-alcoholic fatty liver disease (NAFLD) patients and 20 control subjects. CXCR3 knockout (CXCR3-/-), obese db/db mice and their wild-type (WT) littermates were used in both methionine-and-choline-deficient (MCD) diet and high-fat high-carbohydrate high-cholesterol (HFHC) diet-induced NASH models. In addition, MCD-fed WT mice were administrated with CXCR3 specific antagonists. Results CXCR3 was significantly upregulated in liver tissues of patients with NAFLD and in dietary-induced NASH animal models. Compared with WT littermates, CXCR3-/- mice were more resistant to both MCD and HFHC diet-induced steatohepatitis. Induction of CXCR3 in dietary-induced steatohepatitis was associated with the increased expression of hepatic pro-inflammatory cytokines, activation of NF-κB, macrophage infiltration and T lymphocytes accumulation (Th1 and Th17 immune response). CXCR3 was also linked to steatosis through inducing hepatic lipogenic genes. Moreover, CXCR3 is associated with autophagosome-lysosome impairment and endoplasmic reticulum (ER) stress in steatohepatitis as evidenced by LC3-II and p62/SQSTM1 accumulation and the induction of GRP78, phospho-PERK and phospho-eIF2α. Inhibition of CXCR3 using CXCR3 antagonist significantly suppressed MCD-induced steatosis and hepatocytes injury in AML-12 hepatocytes. Blockade of CXCR3 using CXCR3 antagonists in mice reversed the established steatohepatitis. Conclusions CXCR3 plays a pivotal role in NASH development by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress. © 2015 European Association for the Study of the Liver.


Fu B.-F.,Hebei North University | Li S.-X.,Institute of Liver Disease | Ning S.-B.,Air Force General Hospital of PLA
World Chinese Journal of Digestology | Year: 2013

AIM: To observe the effect of recombinant adenovirus- associated virus (rAAV)-mediated RNA interference on HBV replication and expression in HepG2.2.15 cells. METHODS: The expression box of hu6-shRNA was placed between two ITRs of AAV and then ligated to the basic core promoter (BCP) of HBV and BCP-driven Rep gene of AAV, which resulted in rAAV. The rAAV was transfected into HepG2.2.15 cells (HCC cells in which the HBV gene was inserted). The expression of HBsAg and HBeAg and replication of HBV-DNA in cultured supernatant were determined on days 1, 2, 3 and 10 after transfection, and the AAVS1 region was sequenced on day 3 after transfection. RESULTS: The target sequence-containing vectors PLRBR322-324, PLRBR522-324, PLRBR322- 2424 and PLRBR522-2424 were successfully constructed. All the four vectors had inhibitory effects on the expression of HBsAg and HBeAg and on HBV-DNA replication, with the former two (PLRBR322-324 and PLRBR522-324) having more significant inhibitory effect on HBsAg expression, the latter two on HBeAg expression and the third on HBV-DNA replication. The inhibitory effects on HBsAg and HBeAg expression and HBV-DNA replication were most obvious on day 3 after transfection, and the inhibition rate remained high on day 10. Site-directed integration of the target sequence was located in the AAVS1 region. CONCLUSION: The rAAV constructed by several elements of AAV and HBV, together with the help of site-directed integration mediated by Rep protein, is a good exploration to solve the problem of short-term effect of RNAi against HBV. © 2013 Baishideng. All rights reserved.


Wang J.,Institute of Liver Disease
Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology | Year: 2013

To perform a systematic comparative analysis of two different commercial automated systems using chemiluminescence immunoassay to quantitatively detect hepatitis B virus surface antigen (HBsAg) in patient sera. The Elecsys2010 electrical chemiluminescence immunoassay (ECLIA; manufactured by Roche) and the ARCHITECT il000 chemiluminescence magnetic microparticle immunoassay (CMIA; manufactured by Abbott) were used to detect HBsAg in 100 serum samples of individuals who presented at our department with suspected hepatitis infection between January and May 2012. The manufacturer's protocols were strictly followed. The categorical data was analyzed by Chi-squared test, and linear regression analysis was used to compare the results of the two assay systems. The HBsAg detection results from the two different assay systems showed good correlation (r >or= 0.95), and had good correlation at a low (r = 0.966), medium (r = 0.974) and high (r = 0.984) cutoff values. However, the positive detection rate of CMIA was significantly higher than that of ECLIA(94% vs. 88%, P < 0.05). When the HBsAg content was below 0.10 IU/ml, the ECLIA detection rate and sensitivity were slightly higher than those of CMIA. The ARCHITECT i1000 and Elecsys 2010 immunoassay systems have good correlation in quantitative detection of HBsAg, but the former may be more sensitive.


Ding S.,Ningbo University | Hu A.,Ningbo University | Hu A.,Institute of Liver Disease | Hu Y.,Ningbo University | And 4 more authors.
Tumor Biology | Year: 2014

The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose-time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis. © 2013 International Society of Oncology and BioMarkers (ISOBM).


Cai B.,Anhui Medical University | Wang M.,Anhui Medical University | Wang M.,Institute of Liver Disease | Zhu X.,Huadong Medical Institute of Biotechnology | And 7 more authors.
International Journal of Molecular Sciences | Year: 2015

Lipopolysaccharides (LPS) can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4) signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2) complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linkedimmunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages. © 2015 by the authors; licensee MDPI, Basel, Switzerland.

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