Objective To explore different pathogenesis of chronic hepatitis B(CHB)and asymptomatic H BV carriers(ASCs)by identifying differentially expressed genes.Methods Subtracted library was constructed by suppression subtraetive hybridization(SSH),and α-defensin was identified by dot blot hybridization.Peripheral blood was collected from 46 CHB patients and 11 ASCs.and the expressions of α-defensin mRNA in peripheral blood mononuclear cell and protein in plasma were determined by the real time RT-PCR and enzyme-linked immunosorbent assay(ELISA).Results Real time RT-PCR showed that the expression of α-defensin mRNA in blood samples of CHB was 1.4-fold higher than that of ASCs.As shown by ELISA,the plasma level of α-defensin in CHB was higher than that of ASCs [(216.40±81.25)μg/L vs.(156.00±57.26)μg/L,t=2.23,P<0.05].Conclusion α-defensin may involve in the pathogenesis of CHB,for it iS over-expressed in CHB patients.

Objective To study morphological characteristics of placenta in severe pregnancy induced hypertension (SPIH) and its relationship to pregnant outcome. Methods Morphological changes were observed by light microscopy; Blood biochemical analyses were used to predispose pregnant outcome. Results Difference in pathological changes of placental bed between normal term pregnancy (NTP) and SPIH groups was significant: the placental weight, proliferation of cytotrophoblasts, numbers of the placental villi with syncytial knots, thickness of basal lamina, fibrinoid necrosis and deposition of matrix, stromal edema and fibrosis of villi, the vascular numbers of villi, stasis; lack of physiologic changes in decidual spiral arteries. Clinical examination showed that the rate of anemia, thrombocytopenia, blood concentration, hypoproteinemia, ascites, eye ground artery spasm in SPIH were higher in NIT. Conclusions Pathological changes of placenta play important roles in development of SPIH, and the pathological changes are paralleled with the severity of the disease.

Objective:To investigate the anti-inflammation effects by activation of the cholinergic anti-inflammatory pathway and its mechanisms in non-alcoholic steatohepatitis (NASH)model mice.Me-thods:6-week-old male C57BL/6J (B6)mice were randomly divided into four groups:the first group was normal mice,injected with saline;the second group was normal mice,injected with nicotine;the third group was NASH model mice,injected with saline;the fourth group was NASH model mice,injec-ted with nicotine.The experimental mice were fed with either standard chow (SC)or high-fat and high-fructose (HFHF)for 17 weeks to generate an NASH model mice.The mice received injection once daily for 3 weeks [nicotine dose,400 μg/kg].Then,their pathological characteristics and function of the liver were assessed.The expressions of interleukin-6 (IL-6)and tumor necrosis factor-α(TNF-α)in se-rum were analyzed by enzyme linked immunosorbent assay (ELISA).The expressions of alpha 7 nicotinic acetylcholine receptors (α7nAChR),Toll-like receptors-4 (TLR-4)and nuclear factor κB of phosphory-lation (p-NF-κB)in Kupffer cells were determined by Western blot and immunofluorescence assays.Re-sults:We successfully generated NASH model mice by imitating the high-fat and high-fructose dietary style of NASH patients.The results of our investigation demonstrated that nicotine could reduce signifi-cantly the levels of IL-6,and TNF-αin serum (P <0.05).The expression of p-NF-κB protein in the group which was NASH model mice injected with nicotine declined significantly as compared with the group which was NASH model mice injected with saline (P <0.05).And the expression of α7nAChR protein elevated significantly conversely (P <0.05 ).Conclusion:Activation of the cholinergic anti-inflammatory pathway could inhibit the release of inflammatory factors as TNF-αand IL-6 in NASH model mice,and the mechanism for the inhibition of inflammatory was mediated by NF-κB pathway.

Objective To investigate the effect of rat macrophage α7-acetylcholine receptor (α7-AChR )-mediated cholinergic anti-inflammatory pathway on endotoxin-induced inflammation reaction . Methods Density gradient centrifugation method was used to isolate rat primary macrophages and flow cytometry was used to identify the cell purity .α7-AChR in macrophages was detected by fluorescence confocal microscopy and Western blot .After 1ipopolysaccharides ( LPS) was added to the culture media of primary culture of macrophages , the concentration of tumor necrosis factor ( TNF)-αin the supernatant was determined by enzyme linked immunosorbent assay (ELISA).The expression of p-NF-κB protein in primary cultured macrophages was detected by Western blot .ANOVA was used to analyze TNF-αlevels after adding different concentrations of nicotine .Results α7-AChR was observed by fluorescence confocal microscope in primary macrophages .Nicotine could significantly reduce the concentration of TNF-αin culture supernatants of macrophages after LPS stimulation .When the concentrations of nicotine were 0, 1, 10, 100μmol/L, the concentrations of TNF-αwere (2 123 ±86), (1 486 ±80), (1 316 ±83) and (1 090 ±77)pg/mL, respectively (t=16.33, 20.18 and 26.83, P<0.05).The level of p-NF-κB in macrophages was also reduced when nicotine added .Conclusion Activation of macrophage α7-AChR can inhibit the endotoxin-induced release of inflammatory factor TNF-α, which may be through NF-κB signal pathway .

OBJECTIVETo investigate the effects and mechanisms of the inflammatory reaction related to nonalcoholic steatohepatitis (NASH) and induced by activation of the cholinergic anti-inflammatory pathway.

METHODSA mouse model of NASH was established by feeding a high-fat and high-sugar diet.Activation of the cholinergic anti-inflammatory pathway was achieved by nicotine administration to the NASH modeled mice and normal controls. Liver biopsies were taken and the concentrations of cytokines were measured. Isolated liver primary Kupffer cells and RAw264.7 cells were cultured, pre-treated or not with lipopolysaccharide (LPS) and exposed to nicotine, after which the supernatant concentrations of IL-6 and TNFa were determined by ELISA. The protein expression levels of phosphorylated (p)-NF-kB and I k B were detected in primary cultured Kupffer cells by western blotting.

RESULTSThe mouse model of NASH was successfully established, as evidenced by findings from liver biopsy and serum liver function tests. The degree of liver inflammation in the NASH mice decreased after nicotine administration, and the level of serum TNFa also significantly decreased. The levels of serum TNFa were 21.95+/-0.8 pg/mL in nicotine-treated mice and 38.07+/-1.7 pg/mL in the non-nicotine-treated NASH mice (P less than 0.05). The nicotine treatment also significantly reduced the concentration of TNFa in the culture supernatants of Kupffer cells after LPS stimulation; moreover, the supernatant level of TNFa decreased significantly after the nicotine treatment (Pless than 0.05). LPS stimulation of the RAw264.7 cells led to an increased level ofp-NF-kB and a reduced level ofI-kB, suggesting that the NF-kB pathway had been activated; different doses of nicotine pre-treatment led to down-regulation of the p-NF-kB level and up-regulation of the I-kB level, both in dose-dependent manners.

CONCLUSIONActivating the cholinergic anti-inflammatory pathway inhibits the NASH-related inflammatory reaction, and the mechanism for this inhibition involves the NF-kB signaling pathway.

Animals ; Cholinergic Agents ; Down-Regulation ; Inflammation ; Interleukin-6 ; Kupffer Cells ; Lipopolysaccharides ; Mice ; NF-kappa B ; Non-alcoholic Fatty Liver Disease ; Phosphorylation ; Up-Regulation

OBJECTIVETo investigate the effects of activation of the GLP-1 receptor on the p38MAPK signaling pathway in hepatic stellate cells (HSCs).

METHODSHSCs were isolated and identified according to morphological features; the levels of GLP-1R protein were determined by western blotting.The HSCs were randomly divided into a control grouP (normal saline treatment) and experimental grouP(liraglutide treatment); after 120 hours, the expression of p38MAPK mRNA was examined by RT-PCR and of phosphorylated (p)-p38MAPK protein was detected by western blotting.

RESULTSGLP-1R proteins were detected in the HSCs. Compared with the control group, the experimental group showed significantly decreased p38MAPK mRNA and p-p38MAPK protein (both P < 0.01).

CONCLUSIONThe p38MAPK signaling pathway could be down-regulated when GLP-1R is activated in HSCs.

Cells, Cultured ; Glucagon-Like Peptide 1 ; analogs & derivatives ; pharmacology ; Glucagon-Like Peptide-1 Receptor ; Hepatic Stellate Cells ; metabolism ; Humans ; Liraglutide ; MAP Kinase Signaling System ; RNA, Messenger ; Receptors, Glucagon ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Objective To investigate the effect of nicotinic acetylcholine receptor αt7 (a7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms.Methods C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model,and the mice were sacrificed to isolate and culture the primary liver macrophages.After the treatment with nicotine and endotoxin,ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supematant;indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway,and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages.An analysis of variance was used for comparison of means between multiple groups.Results The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice,the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6:1 599±65 pg/ml vs 1 465±45 pg/ml,P ＜ 0.05;TNF-α:1 567±66 pg/ml vs 1 433±50 pg/ml,P ＜ 0.05).The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice,the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB:69 425±600 vs 51 1334200,P ＜ 0.05;TLR-4:93 387±684 vs 64 198±630,P ＜ 0.05).The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice.The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14,P ＜ 0.05).Conclusion The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH,and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited,which aggravates inflammatory reaction.

Objective:To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors ofnasopharyngeal carcinoma cells.Methods:Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed,and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously.The integration of target genes were detected by PCR,the expressions of E-cad and Bmi-1 were detected by Western blot.The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay.T-he cell cycle and apoptosis were detected by flow cytometry.The cell migration and invasion were detected by Transwell assay.Results:The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells,the protein expression level of E-cad was up-regulated,and the protein expression level of Bmi-1 was down-regulated.The intervention of E-cad and Bmi-1 didn't affect the proliferation,cell cycle and apoptosis of CNE-2 cells,but it significantly inhibited the migration and invasion ability of CNE-2 cells.Furthermore,the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P＜0.01).Conclusion:The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro,which may lay the preliminary experimental basis for gene therapy of human cancer.