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Objective:To investigate the effect of Rab7 on cytokine induced by TLR7 (Toll like receptor-7) R848 activated in Raw264.7,and discusses the influence of Rab7 on MAPK signal transduction.Methods: TLR7 downstream cytokines such as TNF-α,IL-6,IFN-α,IFN-β and IP-10 activated by R848 were detected through Q-PCR in Rab7 silenced mouse macrophages,and then analysis of phosphorylation of MAPK determined with Western blot showed the effect of Rab7 on signal transduction of MAPK.Results: Rab7 inhibit production of cytokine activated by TLR7,and also,Rab7 had an inhibitory effect on MAPK signal pathway.Conclusion: The experimental results further illustrate that the Rab7 is the TLR7 signal transduction pathway negative regulatory factor,and to participate in MAPK signaling pathway.
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Objective To establish a kind of simple,rapid,accurate and reliable method to analyze the concentration of alcohol in blood by headspace gas chromatography (HS-GC) with dual-column and dual-detector.Methods The samples were pre-treated by headspace sampler,which was the basis on the extraction principle of the gas extracting volatile substances.Next,these samples were analyzed by HS-GC that the tertiary butyl alcohol was acted as the internal standard substance.The HS-GC was equipped with two chromatographic column (the DB-ALC2 chromatographic column of 001 channel;the DB-ALC1 chromatographic column of 002 channel).At the same time,the HS-GC was also equipped with two hydrogen flame ionization detector (FID1 detector;FID2 detector).The retention time of the peak was finally performed as qualitative parameter and the standard curves method of internal standard were acted as quantitative basis.Results The liner range of the method was 0.2-2.0 mg/mL.The linear regression equation of 001 channel was Y=1.057 7X+0.048 2 and the correlation coefficient was R2=0.999 05.Besides,the linear regression equation of 002 channel was Y=1.039 5X+0.046 5 and the correlation coefficient was R2=0.999 25.In short,the average recovery rate of the method was 99.70%.Relative standard deviation(RSD) was less than 4% between the analysis results of 001 channel and 002 channel for the determination of the plan sample.Conclusion The method shown satisfactorily that it could not only be applied to determine the alcohol of blood of forensic toxicological analysis,but also be applied to determine the plan sample of ability test and verify of laboratory ability accreditation.
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Objective:To study the relationship between CYP4F2 gene polymorphism and coronary heart disease (CHD) in Mongolia patients ,and investigate clinical characteristics of these patients . Methods:All subjects received questionnaire . Gene amplification and genotyping were performed in 234 Mongolian CHD patients (CHD group) and 221 non-CHD pa‐tients (normal control group) using high temperature ligase detection reaction technique .The relationship between Mongo‐lian CHD and CYP4F2 gene polymorphisms of two single nucleotide polymorphism (SNP) sites (rs1558139 ,rs2108622) was analyzed .Results:Compared with normal control group ,there were significant rise in percentages of male (41.18% vs . 67.95% ) ,smoking history (32.13% vs .41.88% ) ,body mass index [BMI ,(21.66 ± 4.53 ) kg/m2 vs .(25.34 ± 5.37 ) kg/m2 ] and triglyceride level [ (1.66 ± 0.90) mmol/L vs .(1.92 ± 1.38) mmol/L] ,and significant reduction in level of high density lipoprotein cholesterol [ (1.18 ± 0.28) mmol/L vs .(1.07 ± 0.29) mmol/L] in CHD group , P<0.05 or <0.01.There were no significant difference in genotype and allele frequencies of rs 1558139 and rs2108622 between two groups . Conclusion:Clinical characteristics of Mongolian CHD patients include high male percentage ,smoking history ,high body mass index and high triglyceride level .CYP4F2 gene polymorphisms of rs1558139 and rs2108622 are not related to coronary heart disease in Mongolian patients .
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Objective:Using the macrophage cell lines RAW264.7 stably expressing Rab5a and its dominant negative mutant Rab5aN133I as models to analyze the effect and the mechanism of Rab 5a,Rab5aN133I on CpG-induced production of pro-inflammatory cytokines and type Ⅰ IFN.Methods: The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7 cells by liposome,and screened with G418.The G418-resistant colonies were obtained and amplified.The transformed cell lines were i-dentified by RT-PCR,Real time-PCR and Western blot.The production of cytokines were measured after transformed cell lines of Rab5a and Rab5aN133I was stimulation with CpG for 8 h.Results: Rab5a expression in transfected cells was significantly higher than the control cell group (P<0.05).Overexpression of Rab5a significantly promoted the production of TNF -α,IL1-β(P<0.01) and IFN-β( P<0.05) in CpG stimulated RAW264.7.The production of cytokines was restored in Rab 5aN133I transfected cell line.Conclusion:Rab 5a promotes CpG-induced pro-inflammatory cytokines and typeⅠIFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.
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Objective:To explore the inhibitory effects of seabuckthorn polysaccharide on hepatic oxidative stress in a mice model of acute liver injury induced by intraperitoneal injection of LPS and D -GalN and detect the expression on hepatic BCL-2/Bax and PPAR-γ.Methods: C57BL/6 male mice were randomly divided into six groups:control group ( CTRL), model group ( L/G), dexamethasone positive control group ( DXM ) , low ( SPL ) , medium ( SPM ) and high dose group ( SPH ) of seabuckthorn polysaccharide.Mice in the SPL,SPM and SPH group were gavaged with 50,100 and 200 mg/kg seabuckthorn polysaccharide for 14 days respectively.Acute liver injury model were established by intraperitoneal injection of LPS (10 μg/kg) and D-GalN (700 mg/kg) .Serum and liver samples were collected 4 h after model establishment .Serum levels of ALT and AST and the content of MDA were de-tected.Hepatic expression of SOD 2 BCL-2 and Bax was determined by Western blot and the expression of PPAR-γwas detected by im-munohistochemistry .Results:ALT and AST levels significantly increased in the model group and decreased dose-dependently after pre-treatment with seabuckthorn polysaccharide .The level of MDA in the model group increased significantly as compared with the control group and decreased in seabuckthorn polysaccharide groups ,while the level of SOD 2 decreased in the model group and recovered in sea-buckthorn polysaccharide groups .The expression of Bax decreased after pretreatment with seabuckthorn polysaccharide .There was no obvious effect on BCL-2 expression after sea buckthorn polysaccharide supplementation .The expression of PPAR-γreduced in the sea-buckthorn polysaccharide group as compared with the model group .Conclusion:Seabuckthorn polysaccharide protects against LPS /D-GalN-induced liver injury.The effect is associated with an upregulation of SOD 2 and downregulation of Bax .
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Objective:To study the protective effects of sea buckthorn polysaccharide extracts on lipopolysaccharide( LPS)/D-galactosa mine ( D-GalN )-induced liver injury in mice and investigate the regulation on hepatic TLR4 and SOCS3 expression.Methods:C57BL/6 male mice were randomly divided into six groups:control group,model group,dexamethasone positive control group, low, medium and high dose group of sea buckthorn polysaccharide.Mice in the sea buckthorn polysaccharides low, medium and high dose group were gavaged with 50, 100 and 200 mg/kg sea buckthorn polysaccharide extracts for 14 days respectively.Acute liver injury model were established by intraperitoneal injection of LPS(10 μg/kg) and D-GalN (700 mg/kg).The mice in the dexamethasone positive control group were intraperitoneally injected with dexamethasone (10 mg/kg) before model estab-lishment.Serum and liver samples were collected after model establishment for 4 h .Serum levels of ALT and AST were detected.Histological changes of liver tissue were observed by HE staining.Hepatic expression of TLR4 and SOCS3 was detected by Western blot.Results:Sea buckthorn polysaccharide significantly inhibited LPS/D-GalN-induced elevation in serum levels of ALT and AST.It also alleviated liver cell injury and inflammatory infiltration.Western blot results showed that sea buckthorn polysaccharide inhibited LPS/D-GalN-induced TLR4 expression.SOCS3 expression was not dramatically influenced by sea buckthorn polysaccharide supplementation.Conclusion:Sea buckthorn polysaccharide protects against LPS/D-GalN-induced liver injury.This protective effects may be achieved by inhibiting the expression of TLR4 but not associated with modulation on SOCS3 expression.
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Objective:To establish cell lines stably expressing Rab5a and its the inactive mutant Rab5aN133I,analyze the effect of Rab5a on the expression of cytokines in LPS-stimulated RAW264.7 cells .Methods:RAW264.7 cells were transfected with Rab5a and its inactive mutant vector Rab5a N133I separately,and then screened by G418.Rab5a stable expressing cell lines were identified by Real time-PCR.The growth of the stable cell lines was analyzed by MTT assay.After the stable cell lines were stimulated by LPS for different time periods,the expression of iNOS,TNF-αand IL-6 was detected.Results:Rab5a and Rab5aN133I transfection resulted in elevated Rab5a mRNA expression compared with the control cells ( P<0.05 ).Rab5a overexpression enhanced the proliferation of RAW264.7 cells.However,the proliferation of Rab5aN133I cells was significantly slower than the control cells ( P<0.05).Overexpression of Rab5a promoted LPS-induced production of iNOS,TNF-αand IL-6 in RAW264.7 cells (P<0.01). Conversely,overexpression of Rab5aN133I abolished the stimulating effects of Rab5a.Conclusion: Rab5a promoted LPS-induced expression of iNOS,TNF-αand IL-6 in RAW264.7 macrophages in a GTP-binding ability-dependent manner.