Objective To investigate the effects of heme oxygenase-1 (HO-1) on the signal transduction pathway of cigarette smoke-induced mucin (MUC) expression in airway mucous hypersecretion. Methods The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by cigarette smoke extract (CSE),and treated with hemin or ZnPPIX,an inductor or inhibitor of HO-1. The expression of HO-1,dual oxidase 1(Duox1),MUC5AC,epidermal growth factor receptor (EGFR) and p-EGFR were detected. The cell viability treated with hemin or ZnPPIX was assessed by methyl thiazolyl tetrazolium method (MTT). The cells were divided into 4 groups:a negative control group,a CSE treatment group,an hemin pre-treatment group and a ZnPPIX pre-treatment group. The changes of EGFR mRNA,Duox1 mRNA,HO-1 mRNA and MUC5AC mRNA were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression changes of Duox1,HO-1,p-EGFR and EGFR were measured by Western blot,while the protein of MUC5AC in supernate and endochylema were detected by enzyme-linked immunosorbent assay (ELISA). Results The expression levels of MUC5AC mRNA and its protein in supernate liquid and endochylema in the CSE group were (0.660?0.044),(62.80?6.85) ?g/mg and (157.00?3.26) ?g/mg,and those of EGFR were 0.596?0.061 and 0.620?0.051,both of them increased significantly (P0.05),while the protein of p-EGFR,the mRNA and protein of EGFR,Duox1 and MUC5AC increased significantly (P

Objective To investigate the effects of hyaluronan (HA) and CD44 on airway mucous hypersecretion,and explore the molecular mechanism of activation of epidermal growth factor(EGF)/epidermal growth factor receptor(EGFR) signal transduction pathway by signal factors. Methods BEAS-2 B airway epithelial cells were cultured in vitro,and were stimulated by neutrophil elastase(NE).Reactive oxygen species(ROS)scavenger(DMTU),hyaluronidase(Hase),CD44 antibody and tissue kallikrein(TK)inhibitor(PI)were served as interventional factors,and control group(serum free culture),NE stimulation group(50 nmol/L NE),DMTU+NE group(20μmol/L DMTU+50 nmol/L NE),DMTU+Hase+NE group(20 μmol/L DMTU+10μg/mL Hase+50 nmol/L NE),CD44Ab+NE group(5 μg/mL CD44Ab+50 nmol/LNE)and PI+NE group(1 00μg/mL PI+50 nmol/L NE)were established.The expression of mucin(MUC)5AC mRNA was detected by RT-PCR,the expression of MUC5AC and EGF protein was determined by ELISA,and the expression of phosphorylated epidermal growth factor receptor(p-EGFR)protein was analysed by Western blotting. Results The expression of MUC5AC,EGF and p-EGFR protein and MUC5AC mRNA in NE stimulation group was significantly higher than that in control group(P<0.01),the expression in DMTU+NE group was significantly lower than that in NE stimulation group(P<0.01),the expression in MTU+Hase+NE group was significantly higher than that in DMTU+NE group(P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA,and P<0.01 for EGF protein),the expression in CD44Ab+NE group and PI+NE group was significantly higher than that in NE stimulation group(P<0.05 for MUC5AC and p-EGFR protein and MUC5AC mRNA,and P<0.01 for EGF protein). Conclusion NE up-regulates the expression of MUC5AC gene via ROS/HA/CD44/TK/EGF/EGFR signal transduction pathway in airway epithelial cells.

Objective To clone the human mucin (MUC)5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. Methods The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software.After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced.By promoter deletion analysis, 3 promoter segments with diferent lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors.Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-кB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Results Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end.The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully.Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P＜0.05 vs. contro1) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. Conclusion This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression.There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.

Objective To explore the molecule mechanism of protective effects of transfecting human elafin gene recombinant plasmid to NCI-H292.Methods Constructe eukaryotic expression vector pEGFP-C1-elafin and transfectied it into NCI-H292 cells,then co-incubated with polymorphonuclear granulocyte(PMN) with stimulation by lipopolysaccharide(LPS).Afater 24 hour,Western bolt and MTT technique were respectively performed to detect expression of ZO-1 in cells and adhesion capability of cells with collagen.Results After stimulation with LPS,Western blot showed that the 220 ku onula occludens-1(ZO-1) protein in transfecting pEGFP-C1 NCI-H292 cells extracts were obviously decreased,in transfecting pEGFP-C1-elafin NCI-H292 cells extracts.And MTT technique showed that compared with transfecting pEGFP-C1-elafin cells,adhesion capability of transfecting pEGFP-C1 cells with collagen were obviously decreased.Conclusion Transfection of recombinant human elafin gene into airway epithelial cells could maintain airway epithelium and enhance the capability of airway to inflammatory injury.

Objective To explore the upstream signal transduction pathway of neutrophil elastase(NE)-induced mucin(MUC)5AC gene expression.Methods A549 cells were either incubated with NE alone or with DMTU,aprotinin or AG1478.The level of MUC5AC mRNA was measured with RT-PCR.The activation of epidermal growth factor receptor(EGFR) and signaling were assessed by measuring the release of epidermal growth factor(EGF) and the phosphorylation of EGFR.Results NE increased MUC5AC gene expression accompanied by an increase of EGF and phosphorylated EGFR.DMTU,aprotinin and AG1478 significantly inhibited MUC5AC gene expression.DMTU and aprotinin significantly decreased the level of EGF and phosphorylated EGFR.ConclusionNE can induce MUC5AC gene expression via EGFR signalling in A549 cells.The upper stream involves oxidants,activation of tissue kallikrein and EGF.

The method for examining the microcirculation bloodflow of mice mesentery was used to evaluate the action of Shujinhuoxuedingtong wine against microcirculation disturbance caused by adrenaline. The result indicates that the wine can obviously improve the microcirculation function and is responsible for the antistatic action observed. The wine's effect is found to be superior to that of Shujinhuoxuedingtong Powder. The above fingings suggest that the wine have the action of promoting blood circulation to remove blood stasis and improving microcirculation and the effect of promoting the bloodflow perfusion quantity in tissue organs. It's effect is prior to that of powder.

Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P

Objective To synthesize a marine natural product, 24-methylenecholesterol. Methods The target molecule was synthesized in 7 steps using hyodeoxycholic acid as the starting material, 3?-(tetrahydropyran-2-yloxy)-cholest-5-ene-24-ketone as the key intermediate and isopropylation as the key step. Results The target molecule was synthesized with a total yield of 66% and confirmed by MS, IR, 1HNMR and 13CNMR. Conclusion Our synthesis of 24-methylenecholesterol is characterized by easily acquired starting material, short synthetic route, high yield and atom-economic.

Objective To explore the mutual effect of proinflammatory factor tumor necrosis factor(TNF)-? and cigarette smoke extract(CSE) in the induction of mucin 5AC in BEAS-2B human airway epithelial cells.MethodsThe BEAS-2B cells were transfected with nuclear factor(NF)-?B decoy oligonucleotides(ODNs) or transfected with NF-?B scrambled ODNs as negative control,then treated with TNF-?,CSE,and TNF-? plus CSE,respectively. The expression of phosphorylated epidermal growth factor receptor(p-EGFR),mucin 5AC(MUC5AC) protein production and gene expression were detected by Western blot,ELISA and RT-PCR respectively.Results Significant increasing of p-EGFR protein production in scrambled ODNs transfected cells with elevation of MUC5AC protein production and mRNA expression was recorded and TNF-? and CSE co-incubated groups were much higher than single TNF-? or CSE stimulated group(P

0.05).Conclusion Hypertonic stress can induce the airway epithelial cells to develop a high mucus secretion with the concentration-dependent manner in the level of transcription,P38 signal pathway plays a major role.