ABSTRACT
Objecitve:To investigate effects of urotensin Ⅱ( UⅡ)/UT system on innate immune inflammatory signal pathway TLR4-IRF3 in the lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs).Methods: Rat KCs were isolated and cultured.Pro-in-flammatory cytokines including IL-6,IFN-βand IFN-γwere assayed by ELISA in culture supernatant of KCs.Cell surface TLR4 were tested with flow cytometry technique.Expression of IRF3 were tested with real-time PCR and Western blot.Results: Significant increases were showed in IL-6, IFN-βand IFN-γsecretion, TLR4-expressed positive rates and IRF3 mRNA levels in KCs after stimulated by LPS,but were inhibited via urantide pretreatment.In addition,LPS induced upregulation of nuclear IRF3 protein and downregulation of cytoplasm IRF3 protein in KCs,which were blocked by urantide pretreatment.Conclusion:UⅡ/UT system mediates immune inflammatory response in part through activating TLR 4-IRF3 pathway in LPS-stimulated KCs.
ABSTRACT
Objective To evaluate the expression and clinical significance of miRNA in plasma of patients with primary biliary cirrhosis.Methods Plasma from 19 PBC patients,10 healthy volunteers and 10 viral hepatitis patients were selected from Shanghai Songjiang Hospital from december 2010 to January 2013.Among them 3 PBC patients' plasma and 3 healthy volunteers' plasma were detected by miRNA microarray for miRNA expression profile examination.Real-time PCR was used to verify the results of microarray,miRNA target gene predictior software was used to predict the target genes of differentially expressed miRNA.ROC was used to determine the clinical value of plasma miRNA.Results According to microarray,a total of 16 miRNAs were found to be differentially expressed.As revealed by qRT-PCR,the expression of miRNA-92a-3p and miRNA-4516 decreased while the expression of miRNA-572 and miRNA-575 were up-regulated in PBC group compared with healthy controls (P ＜ 0.05).In comparison with nonPBC cirrhosis group,only miRNA-92a-3p and miRNA-4516 were down-regulated (P ＜ 0.05).The area under the curve (AUC)of miRNA-92a-3p for the diagnosis and differential diagnosis of PBC were 0.92 and 0.84,respectively.while for The area under the ROC curve of miRNA-4516,the AUC for diagnosis and differential diagnosis PBC were 0.89 and 0.76,respectively.The optimal cut-off values for identifying PBC from healthy controls were defined as 1.26 ng/μl.for miRNA-92a-3p (sensitivity,92％ ;specificity,80％)and 1.16ng/ul for miRNA-4516 (sensitivity,85％ ;specificity,70％)respectively.The optimal cut-off values for identifying PBC from viral hepatitis were defined as 1.08 ng/μl.for miRNA-92a-3p (sensitivity,89％ ; specificity,81％)and 1.06 ng/μl for miRNA-4516 (sensitivity,77％ ;specificity,68％).Conclusion The results indicate that plasma from patients with PBC has a unique miRNA exprssion profile and these differentially expressed miRNA can be used as clinical biomarkers of PBC.
ABSTRACT
Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor ( UⅡ/UT) system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase ( p38 MAPK) and nuclear factor-κB ( NF-κB ) in lipopolysaccharide ( LPS )-stimulated Kupffer cells ( KCs ) . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1:UⅡ(-) urantide (-)LPS(-), group 2:UⅡ(+)urantide(-)LPS(-), group 3: UⅡ(-)urantide(+)LPS(-), group 4:UⅡ(-)urantide(-)LPS(+), group 5:UⅡ(+) urantide(-) LPS(+) and group 6:UⅡ(-)urantide(+) LPS(+) .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay (EMSA).Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups (P>0.05).The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 (P0.05), but that were decreased in group 6 than those in group 4 (all P<0.01).Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells .
ABSTRACT
Objective To investigate the liver repair effects of Pim-3 gene in rat with fulminant hepatic failure (FHF). Methods Thirty-two rats were divided into four groups (eight for each group). Three groups of rats were pretreated with Ringer's solution, vector plasmid or Pim-3 gene recombinant plasmid respectively and, one day later, received intraperitoneal injections with lipopolysacchride (LPS) and D-galactosamine (D-GalN). The fourth group served as normal control.Eight hours after the LPS/D-GalN injection, the liver tissues and blood samples were collected. The contents of serum transaminase was tested by automatic blood biochemistry meter. The morphological changes were observed by light microscopy using hematoxylin and eosin (HE) staining. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β gene expression was detected by reverse transcriptionpolymerase chain reaction(RT-PCR). The serum levels of TNF-α and IL-1β were measured by enzyme linked immunosorbent assay (ELISA), and cell apoptosis by TdT-mediated dUTP nick end labeling (TUNEL) assay. Comparisons between groups were done by analysis of variance. ResultsThe over expressions of Pim-3 gene and reporter gene, green fluorescent protein (GFP) were induced by injection with recombinant plasmid solution.In comparison with the rats retreated with Ringer' s solution or vector plasmid, those pretreated with recombinant plasmid had a lower mortality and lower serum transaminase levels. The injection of recombinant plasmid significantly reduced hemorrhage, necrosis and inflammatory infiltration in the liver. Liver apoptotic index (AI) was dramatically lower in rats treated with recombinant vectors compared to the rats treated with Ringer's solution or vector plasmids [(10. 2±6.9)％ vs (83. 1±12.6) ％ and (79.9±13.4) ％ respectively, P＜0. 01]. In addition, the expression of exogenous Pim-3 gene remarkable inhibited the transcriptions and expressions of TNF-α and IL-1β. ConclusionsPim-3 gene can protect rats from LPS/D-GalN-induced FHF possibly by inhibiting expressions and secretions of inflammatory cytokines, such as TNF-α and IL-13, in liver tissues.