Objective To analyze the effect of N-acetyl-L-cysteine(NAC)on endoplasmic reticulum stress mediated HepG2 cells apoptosis and evaluate the role of NAC in the treatment of liver injury.Methods HepG2 cells were treated with thapsigargin(TG)to establish the model of oxidative endoplasmic reticulum stress mediated apoptosis,and NAC was used to intervene in apoptosis.To evaluate the apoptosis,various methods such as MTT assay,flow cytometry,DNA ladder and Western blot were performed.Results After treated with 2 μmol/L TG for 0,24,36 and 48 hours,the vitality of HepG2 cells decreased.The ratio of apoptotic cells increased along with the prolonged treatment duration of TG(0.7%±0.5%,27.6%±6.3%,29.7%±3.3%,47.9%±3.5% respectively,P<0.05),and the production of reactive oxygen species(ROS)also increased in time-dependent manner(14.0%±0.5%,36.1%±3.0%,38.2%±6.0%,48.3%±12.4%,P<0.05).The HepG2 cells showed typical morphologic change of endoplasmic retieulum stress induced by 2 μmol/L TG after 36 h and 48 h.DNA ladder was observed at the same concentration and time point correspondingly.Endoplasmic reticulum stress mediated-apoptosis was confirmed by Western blot.Both 10 mmol/L and 20 mmol/L NAC could protect ceils from apoptosis.The ratio of apoptotic cells decreased to 14.0%±1.3% and 11.0%±0.3%,respectively.The production of ROS decreased to 34.7%±0.8% and 31.5%±2.9%,respectively.The effect was related to the concentration of NAC.Conclusions As a Ca2+-adenosine triphoshatase inhibitor,TG may disrupt intracellular calcium homeostasis,which can induce endoplasmie reticulum stress and apoptosis.NAC,the precursor of the synthesis of-SH,can directly inhibit the ROS reaction and alleviate liver damage,which may play a role in the treatment of liver failure.

Objective To observe the regulatory role of microRNA-1187(miR-1187)in hepatocyte apoptosis through miR-1187 targeting regulation of caspase-8 mRNA expression.Methods The acute liver failure model was established by injection of D-galactosamine plus lipopolysaccharides(LPS)in BALB/c mice.The liver tissues were collected for LNA-miRNA array analysis and functional analysis of genes targeted by miRNA.Embryonic murine hepatocyte cell line 2(BNL-CL2)was cultivated in vitro and treated with tumor necrosis factor(TNF)-α and D-galactosamine to induce the transfection of miR-1187 in transfected group or untransfected group.The expressions of miR-1187 and caspase-8 mRNA were detected by real-time polymeramse chain reaction(PCR)and caspase-8 protein was determined by Western blot.The apoptosis rate was detected by flow cytometry.The comparison of means between groups was done by t test.Results The miR-1187 signal was deceased with the development of acute liver failure.The 3'UTR of caspase-8 mRNA had direct binding sites with miR1187.In BNL-CL2 cell experiments,miR-1187 was down-regulated in untransfected group and decreased more slowly in transfected group(t=6.371,P<0.01).The expression of caspase-8 mRNA was up-regulated in untransfected group and increased less in transfected group(t=4.539,P<0.01).The apoptosis rate in transfected group was significantly lower than untransfected group(t=3.365,P<0.05).Conclusios miR-1187 is one of inhibitors of hepatocyte apoptosis.High expression of miR-1187 could regulate the expression of caspase-8 mRNA,thus inhibit the apoptosis of hepatocytes.

Objective To investigate the expression and function of retinoic acid-inducible gene Ⅰ(RIG-Ⅰ) in monocyte-derived dendritic cells (MoDC) at different stages of hepatitis B virus(HBV)infection and to explore the role of RIG-Ⅰ in the disease progression after HBV infection. Methods Peripheral blood samples were collected from 28 hepatitis B virus-infected persons, including 21 cases of chronic hepatitis B (CHB) and 7 of acute hepatitis B (AHB). Eighteen healthy subjects were recruited as controls. Purified CD14~+ monocytes were isolated by CD14 microbeads. MoDCs were induced from CD14~+ monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 7 days, and then were infected with vesicular stomatitis virus (VSV) to stimulate RIG-Ⅰ expression. The mRNA expression levels of RIG-Ⅰ, interferon (IFN )-promoter stimulating factor-1 (IPS-1) and IFN-β at 16 hours and 24 hours after infection with VSV were measured by real-time quantitative polymerase chain reaction (PCR). Data with normal distribution were tested by analysis of variance. Continuous variables between groups were compared using Mann-Whitney U test. Comparison among multiple groups was done by Kruskal-Wallis test. Results The expression levels of RIG-Ⅰ in MoDCs from CHB patients were significantly lower than those in AHB patients and healthy controls at 16 hours (2.44±2.03, 19. 54±3. 15, 21. 48±8. 39, respectively; F=7.451,P=0.002) and 24 hours (2. 68±2. 93, 10. 31 ±3. 88, 14. 01 ±5. 04, respectively, F = 7. 908, P = 0. 001)following VSV stimulation. The IPS-1 levels in both CHB patients and AHB patients were higher than those in healthy controls at 16 hours (2. 05±l. 08, 1. 99±1. 56, 0. 60±0. 31, respectively) F=7.246,P =0.003) and 24 hours (2. 27±2. 16, 3.24 ± 1.21, 1. 08±0. 73, respectively; F= 13. 598, P = 0. 001).Furthermore, the IFN-β expression levels were significantly lower in CHB patients compared to AHB patients and healthy controls at 16 hours and 24 hours after VSV stimulation. Conclusions The expressions of RIG-Ⅰ and IPS-1 in MoDC are abnormal in HBV infected persons, which indicates that RIG-Ⅰ signaling pathway might be blocked by HBV. The impaired function of MoDC may play a role in HBV infection and chronicity.

Objective To elucidate the roles of TANK-binding kinase-1(TBKl)in hepatitis B virus (HBV)infection induced interferon antiviral immunity.Methods Peripheral blood monocytes were separated by CD14 magnetic microbeads from healthy volunteers(HV)and chronic hepatitis B(CHB)patients.Purified mDCs were induced and proliferated in the culture medium with human granulocyte-macrophage concentration of 25 mg/L were stimulated.The mRNA expressions of TBK1,interferon regulatory factor (IRF)3 and interferon(IFN)-βwere quantified by real time polymerase chain reaction(PCR).The levels of IFN-β in supernatants were determined by enzyme-linked immunosorbent assay(ELISA).Reslllts The mRNA levels of TBK1,IRF3 and IFN-β did not change significantly at 0,12,24 and 48 h after the significantly at 0, 12, 24 and 48 h in CHB group, whereas, it was significantly up-regulated at 12 h in HV group. Conclusions Our results suggest that there may be some disorders in host antiviral signal transduction pathways downstream the binding between ligands and receptors on mDC surface. The insufficient IFN-β expression after HBV infection may result in persistent chronic infection.

Objective To explore the relationship among cytokine levels and Toll-like receptor (TLR) 2,4 in hepatitis B virus (HBV) related cirrhosis. Methods Heparin anticoagulated venous blood of 35 randomly selected HBV related cirrhosis and 35 healthy volunteers were collected aseptically. Plasma tumor necrosis factor (TNF)-α concentration was determined using enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMC) were separated and stained with anti-TLR2,4 monoclonal antibodies, then analysed by flow cytometry. Total RNA was extracted from PBMC and TLR2,4 mRNA expression levels were evaluated by real-time quantitative polymerase chain reaction (PCR) using SYBR Green detection. Means of normal distribution were compared by t test and one factor analysis of variance. The data of abnormal distribution were analyzed using Mann-Whithey U test, Kruskal-Wallis H test and Spearman correlation analysis. Results The plasma concentration of TNF-α in the cirrhotic patients was significantly higher than that in the healthy controls (33.52 ng/L vs 6.07 ng/L, Z=-6.584, P<0. 01), which was parellel with Child-Pugh score. TLR2 positive rate in PBMC from the cirrhotic patients was significantly higher than that from the healthy controls (20.65% vs 12.04%, Z=-4.458, P<0.01), which was positively correlated with plasma TNF-α level (r= 0.448 3, P<0.05), and parellel with Child-Pugh score. Between the cirrhotic and healthy groups, there was no significant difference of TLR4 positive rate in PBMC. The mRNA expression level of TLR2/GAPDH in PBMC from the cirrhotic patients was significantly higher than the controls (0.234 2 vs 0.043 1, Z=-6.83, P<0.01), which was positively correlated with plasma TNF-α level (r=0.411 1, P<0.05), and parellel with Child-Pugh score. Between the two groups, there was no significant difference of TLR4 mRNA expression level in PBMC. Conclusions The expression of TLR2 in PBMC from cirrhotic patients is significantly elevated, which is positively correlated with plasma TNF-α level and the severity of liver disease. The expression of TLR4 in PBMC from cirrhotic patients is unchanged. It suggests that TLR2 but not TLR4, plays an important role in the progression of liver cirrhosis.

OBJECTIVETo investigate the changes in circulating plasmacytoid dendritic cells (pDCs) in patients with chronic hepatitis B (CHB) during the course of treatment with pegylated-interferon alfa-2s (peg-IFNa-2a) and to determine the correlations with therapeutic response.

METHODSForty-one patients with CHB who were receiving peg-IFNa-2a antiviral treatment for 48 weeks were enrolled in the study.Expression of the Toll-like receptor 9 (TLR9) on and frequency and functionality of the pDCs were analyzed at treatment weeks 0, 2, 12, 24, 36 and 48.

RESULTSAll patients exhibited an initially rapid decrease in the numbers of circulating pDCs and showed CpG-induced endogenous IFNa production within the first 2 weeks of treatment.Subsequently, all responders displayed a continuous increase in pDC numbers as well as functionality, both of which peaked around week 12 of treatment; in addition, these treatment responses were accompanied by significantly increased levels of type 1 T helper cytokines (P less than 0.05), which did not occur in the non-responders.

CONCLUSIONpDCs are involved in the initial therapeutic immune response stimulated by peg-IFNa-2a treatment.Recovery of blood pDC number and functionality may represent a predictor of favorable response to peg-IFNa-2a antiviral treatment in patients with CHB.

Antiviral Agents ; Dendritic Cells ; Hepatitis B, Chronic ; Humans ; Interferon-alpha ; Polyethylene Glycols ; Recombinant Proteins ; Toll-Like Receptor 9 ; Treatment Outcome