Objective To investigate Bcl-2 expression in the peripheral blood mononuclear cells and its clinical significance of liver fibrosis (LF). Methods Tested Bcl-2 protein levels in T, B cells of 47 patients (male 17, female 30) with LF, 35 patients (male 24, female 11) with chronic hepatitis B and no LF and 41 cases (male 29, female 12) normal controls by two color cytofluorography. Results Among them, LF patients, chronic hepatitis B without LF and normal controls, the proposition of T cells (including CD3~+, CD4~+ and CD8~+ subgroups) and CD19~+ B cells expressed Bcl-2 protein increased significantly in LF patients (P

Objective To observe the effects of adiponectin on mRNA and protein expressions of connective tissue growth factor (CTGF) in hepatic stellate cells (HSCs) and the levels of procollagen type Ⅲ (PC Ⅲ) and hyaluronic acid (HA).Methods Cultured rat HSCs were treated with different concentrations of adiponectin.CTGF mRNA and protein expressions were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot.The levels of PCⅢ and HA were detected by enzymelinked immunosorbent assay (ELISA).Results Compared with the control group,the result of RT-PCR showed that the four groups had different degrees of inhibitory effect,of which group D exhibited the strongest inhibitory effect.The absorbance ratio was 1.54 ±0.18,1.21 ±0.14,0.96 ±0.10,and 0.79 ± 0.08,respectively (t =2.42,P ＜0.05;t =2.73,P ＜0.05;t =3.28,P ＜0.01;t =4.67,P ＜0.01).Western Blot also indicated that four groups had different degrees of inhibitory effect,of which D group exhibited the strongest inhibitory effect.The ratio of integral absorption was 1.54 ± 0.18,1.21 ±0.14,0.96±0.10,and 0.79 ±0.08,respectively (t =2.84,P ＜0.01;t =4.05,P ＜0.01;t =6.25,P ＜0.01;t =9.72,P ＜0.01).The levels of PCⅢ and HA secreted in culture media were also decreased.It was significantly decreased with the concentration of adiponectin increased.Conclusion Adiponectin can inhibit the levels of PC Ⅲ and HA,which may be achieved through reducing CTGF mRNA and protein expressions.

Objective To investigate the effects of ginsenoside Rg3 on growth and apoptosis of gastric cancer cell line MKN-45 and SGC-7901 in vitro. Methods MKN-45 and SGC-7901 cells at logarithmic growth phase were obtained, and were cultured with ginsenoside Rg3 of different concentrations (20, 30, 40, 50 μg/mL) for 24, 48 h or 24, 48 and 72 h. Cells cultured without ginsenoside Rg3 were served as controls. The inhibition rates of ginsenoside Rg3 on MKN-45 and SGC-7901 cells were detected by MTT assay, apoptosis rate of SGC-7901 cells was determined by Annexin V/PI double staining flow cytometry, cell cycles of SGC-7901 cells were analysed by flow cytometry, and morphological changes of SGC-7901 cells in 50 μg/mL ginsenoside Rg3 treatment group were observed by transmission electron microscopy. Results The inhibition rates on MKN-45 and SGC-7901 cells in each ginsenoside Rg3 treatment group were significantly higher than those in control group (P < 0.05), and the inhibition rates increased with the concentrations of ginsenoside Rg3 and time of culture ( P < 0.05). Compared with control group, the apoptosis rates of SGC-7901 cells and percentages of cells in G_1/G_1 cell cycle in each ginsenoside Rg3 treatment group were significantly increased in a concentration and time dependent manner. Typical morphology of SGC-7901 cell apoptosis was observed by transmission electron microscopy in 50 μg/mL ginsenoside Rg3 treatment group. Conclusion Ginsenoside Rg3 has significant inhibition effect on gastric cancer cell lines in vitro with a concentration and time dependent manner, the mechanism of which may involve the induction of gastric cell line apoptosis.

Objective To investigate the effect of rapamycin(RPM) on the gene expression of Toll-like receptor (TLR)-4 by inhibiting Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in rats with acute liver failure (ALF).Methods The ALF model of rats was induced by D-galactosamine (D-GalN) 800 mg/kg and lipopolysaccharide (LPS) 8 μg.The blood and liver tissue samples were collected at 2,6,12,24 and 48 h after D-GalN/LPS injection.SD rats were randomly divided into three groups:normal control group (n=6),ALF group (n=30) and RPM treatment group (n=30).The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at each time points were tested.The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were tested by enzyme-linked immunosorbent assay (ELISA),and the mRNA expressions of TLR-4 in liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR).The data analysis was performed using t test.Results The levels of TNF-α and IL-6 were increased markedly 2 h after GalN/LPS iniection and peaked at 6 h.Treatment with RPM could significantly inhibit the levels of TNF-α and IL-6.The TLR-4 mRNA expression levels in liver tissues at 6,12,24,48 h in ALF group were 0.745±0.135,1.092±0.175,1.115±0.152 and 0.812±0.13,respectively;those in RPM group were 0.545±0.118,0.798±0.124,0.857±0.109 and 0.595±0.152,respectively.The differences between two groups at each time points were all significant (t=2.726,3.349,3.382 and 2.567,respectively,all P＜0.05).In addition,TLR-4 mRNA expression was positively correlated with ALT and AST levels(r=0.722 and 0.712,respectively,both P＜0.01).Conclusions Inhibition of JAK/STAT pathway can markedly down regulate TLR-4 gene expression in liver tissue of rats with ALF,which indicates that JAK/STAT pathway may be involved in the regulation of TLR-4 mRNA expression during ALF.

Objective To observe the expression and significance of hypoxia inducible factor (HIF)-1α,matrix metalloproteinase(MMP)-9 and transforming growth factor(TGF)-β1 in liver tissues of rats with hepatic fibrosis and the effects of TGF-β1 vaccine on the expressions of HIF-1α,MMP-9 and TGF-β1.Methods The hepatic fibrosis rat model was set up by dimethylnitrosamine (DMN).The rat model was injected with TGF-β1 vaccine.The expressions of HIF-1α,MMP-9 and TGF-β1 were detected by immunohistochemistry in rat fibrosis tissues after 6 weeks.Results The expressions of HIF-1α,MMP-9 and TGF-β1 in fibrosis model group and TGF-β1 vaccine group were all significantly higher than control group(P＜0.01),which was positively correlated with the fibrosis degree(r=0.911,0.814 and 0.836,respectively,P＜0.01).However,the expressions of HIF-1α,MMP-9 and TGF-β1 in TGF-β1 vaccine group were all significantly decreased than fibrosis model group (t=2.62,4.03,3.43;P＜0.01).Conclusion During the development of liver fibrosis,HIF-1α promotes the transcriptions and expressions of MMP-9 and TGF-β1.

Objective To investigate the effect of endotoxin tolerance(ETT) on the rats with acute liver failure (ALF) and Janus kinase/signal transducer and activator of transcription (JAK/ STAT) signal transduction pathway. Methods S-D male rats were divided randomly into three groups: control group, ALF model group anti ETT group, lipopolysacharide (LPS) 0.1 mg/kg(ETT groups) or saline(ALF groups)was administered by five consecutive intraperitoneal injections at 24 h intervals. On the sixth day all animals were treated with intraperitoneal injection of D-galactosamine (D-GaIN) 800 mg/kg and LPS 8 μg/rat. The blood was gathered from portal vein and livers were take out before and 2, 6,12, 24 and 48 h after the injection of D-GalN/LPS. Liver function and liver histopathology of each group were observed. The gene expressions of STAT3 and SOCS3 in the livers were measured by semi-quantitative reverse transcriptionpolymerase chain reaction(RT-PCR). The tumor necrosing factor(TNF)-α and interleukin(IL)-6 level were determined by enzyme-linked immunosorbent assay(ELISA). The data analysis was performed by using t test. Results The histological damage in the liver tissue was significantly milder in ETT group compared to ALF group, but still severer than that of control group (TNF-α: 6 h: t=2. 670,P<0.05,12 h: t=3. 604,24 h: t=6. 426, 48 h: t=3. 274,all P<0.01;IL-6:6 h: t=2. 333,P<0. 05,12 h: t=4. 266, 24 h: t=8. 063,48 h: t=4. 177, all P<0. 01). The gene expressions of STAT3 and SOCS3 in the liver were increased significantly in ALF group, however, in ETT group the expression of STAT3 was inhibited while the expression of SOCS3 was increased and much higher than those in ALF groups. Conclusions LPS pretreatment can induce ETT in rats, which will reduce the expression of TNF-α and IL-6. In ETT groups, the gene expression of STAT3 is lower while the gene expression of SOCS3 is higher compared to those in ALF groups. It suggests that JAK/STAT pathway may involve in mechanisms of ETT.

Objective To explore the influences of plumbagin on phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signaling pathway in rats with carbon tetrachloride induced liver fibrosis.Methods Forty male SD rats were assigned to control group,model group,2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group,with 10 rats in each group.Rats of the control group were injected with 0.9％ NaCl solution (2 mL/kg) intraperitoneally,while rats of the other three groups were injected with 60％ carbon tetrachloride/peanut oil (2 mL/kg,3 times a week for 6 weeks) intraperitoneally.Since the third week after modeling,rats of plumbagin treated groups were treated with intraperitoneal injection of plumbagin at a dose of 2 mg/kg and 3 mg/kg (twice a week for 4 weeks),respectively.Serum levels of alanine aminotransferase (ALT),aspartate aminotransferase (AST),albumin (Alb) were monitored routinely.Hyaluronic acid (HA) and laminin (LN) were measured by radioimmunoassay after 6 weeks; protein expression of liver PI3K,Akt and phosphorylated Akt (p-Akt) were evaluated by Western blotting and immunohistochemistry,respectively.Comparison of means among groups was performed by univariate analysis of variance.Results The hepatic fibrosis model was successfully established after 6 weeks.Serum levels of ALT,AST,HA and LN of model group were significantly higher than those of control group (all P＜0.05).Serum levels of ALT,AST,HA and LN of 2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group were significantly lower than those of model group,and the differences were statistically significant (all P＜0.05).The protein expressions of PI3K and Akt in each group were comparable (all P＞0.05).The p-Akt protein was mainly expressed in nucleus of hepatocytes.The levels of p-Akt protein in control group,model group,2 mg/kg plumbagin treated group and 3 mg/kg plumbagin treated group were 0.0821±0.0003,0.7374±0.0037,0.3679 ±0.0332 and 0.1327±0.0561,respectively,and that in model group was significantly higher than control group (t =851.302,P＜0.05),but those in plumbagin treated groups were both lower than model group (t=71.858 and 28.363,all P＜0.05).Conclusions Plumbagin presents anti-fibrotic effects in the liver,by down-regulating the expression of p-Akt during the development of fibrosis,which might be one of the antifibrotic mechanisms.

Aim To construct a eukaryotic expression vector of human MCHR2 and to analyze the stable expression and signal transduction pathways of MCHR2 in CHO cells. Methods The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and inserted into eukaryotic expres-sion vector pcDNA3.1(+), resulted in the recombinant expression vector pcDNA3.1-MCHR2. The recombinant plasmid was confirmed by the restriction enzymatic digestion and DNA sequencing analysis, and then transfected into CHO cells by lipofectamine. A stably-transfected cell line was obtained by the dominant G418 selection, and the expression of the MCHR2 gene on transcription and translation levels were identified by RT-PCR and Western blot. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was stably transfected into CHO cells. A stably-transfected cell line was established and the MCHR2 gene was efficiently transcribed and translated. MCH stimulation had no effect on the production of cAMP, however, it could induce a clear and transient increase of intracellular calcium, suggesting that MCHR2 was only coupled to Gq protein. Conclusion The stable expression of MCHR2 and analysis of its signal transduction pathways in CHO cell line provided a solid experimental foundation for further studies on the function of the MCHR2 gene in vitro.

Objective: To investigate the effects of andrographolide on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-activated macrophages. Methods: LPS-activated mouse peritoneal macrophages were cultured in media with different concentrations of andrographolide. Cytotoxicity of andrographolide was detected by cell counting kit-8. The macrophages were lysed, and then expressions of phosphorylated ERK1/2, JNK and p38 and nuclear factor-κB inhibitor (IκBα) protein were detected by Western blotting and TNF-α mRNA expression was detected by reverse transcription-polymerase chain reaction. Supernatants of the macrophages were used to detect content of TNF-α protein by enzyme-linked immunosorbent assay. Results: Andrographolide at 1-100 μg/mL showed no cytotoxicity on LPS-activated mouse peritoneal macrophages. Andrographolide inhibited ERK1/2 phosphorylation in LPS-activated murine peritoneal macrophages, which was concentration-dependent (P<0.01). Andrographolide at 1-25 μg/mL had no effects on phosphorylation levels of JNK and p38 and IκBα degradation in LPS-stimulated mouse peritoneal macrophages. In activated macrophages, TNF-α expression was inhibited by 12 μg/mL andrographolide and 20 μmol/L PD98059 (inhibitor of ERK1/2 signaling pathway) at both mRNA expression and protein secretion levels. Conclusion: In LPS-activated macrophages, andrographolide may inhibit the expression of TNF-α by inhibiting ERK1/2 signaling pathway.

Objective To evaluate the feasibility to screen donor with hepatitis C virus (HCV) infection by HCV core antigen ELISA (HCV-cAgELISA).Methods 183 anti-HCV positive,6370 anti-HCV negative (including 120 specimens with HbsAg +) serum specimens by using HCV-cAgELISA and HCV RT-PCR methods.Results Comparing with HCV RT-PCR,the detection results of HCV-cAgELISA consistency were 92.34％ in anti-HCV positive serum specimens and 99.97％ in anti-HCV negative serum specimens,respectively.In 6370 anti-HCV negative specimens,1 serum sample was positive HCV RNA identification in 3 serum specimens which were positive for HCV-cAgELISA.HCV RNA copies and HCV-cAg positive rate was a positive correlation.Conclusion Sensitivity of HCVcAg ELISA is silllilar to HCV RT-PCR.HCVcAgELISA is a simple,fast,and reliable memod to screen donor with HCV infection in blood transfusion medicine.