AIM: To investigate the level of ET-1 produced by cultured human bronchial epithelial cells (HBECs) under injury and the effects of injured HBECs on ET-1 production in sub-epithelial fibroblasts. The interaction between ET-1 and matrix metalloproteinase-9(MMP-9) was detected in HBECs under damage. The purpose of the study is to evaluate the effect of injured HBECs related to ET-1 release on airway remodeling in asthma. METHODS: ET-1 level was detected in supernatants from cultured HBECs 12 h after being treated with either mechanical scraping or LPS stimulation or mechanical scraping plus LPS, as well as from subepithelial fibroblasts cocultured with mechanical damaged HBECs. It was also measured in the supernatant from HBECs transfected with MMP-9 expression plasmid. MMP-9 activity was assessed in supernatants from HBECs stably transfected with pEGFPc1 -antisense-ET-1 converting enzyme(ECE) RNA. RESULTS: It was found that there was an increase in ET-1 level in supernatants from HBECs either treated with mechanical scraping plus LPS or transiently transfected with MMP-9 plasmid, as well as from sub-epithelial fibroblasts cocultured with mechanical scraping HBECs compared with that in controls. Gelatin zymography showed a obviously attenuated gelatinolytic activity of MMP-9 in conditioned media of HBECs expressing antisense ECE RNA after mechanical damage. CONCLUSIONS: Airway epithelial cells under injury are able to overproduce ET-1 as well as initiate ET-1 release from sub-epithelial fibroblasts, MMP-9 produced by injured bronchial epithelial cells may also increase ET-1 processing leading to ET-1 production further. The interaction between ET-1 and MMP-9, both of which enhanced in damaged HBECs, may play an important role in airway inflammation related to airway remodeling in asthma.

AIM: To explore the effects of p38 mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt on interleukin (IL)-6, the endothelin (ET)-1-mediated process of airway fibroblast activation induced by injured human bronchial epithelial cells (HBE). METHODS: Human primary cultured airway fibroblasts were co-cultured with HBE pre-treated with or without poly-L-arginine (PLA). The procedure was also performed in the presence or absence of p38 MAPK selective inhibitor SB203580, PI3K selective inhibitor LY294002 or ETA receptor blocker BQ123, respectively. Immunostaining, Western blotting or ELISA were used for detecting α-smooth muscle actin (α-SMA) expression, the activities of p38 MAPK and Akt in fibroblasts or IL-6 levels in supernatants of fibroblasts. In addition, fibroblasts were mixed with soluble collagen and cultured with HBE treated as the same mentioned above, the gel contraction was measured by serial area measurements. RESULTS: ET-1 and IL-6 levels [(13.69±1.36) ng/L, (56.7±10.7) ng/L] in the supernatants of fibroblasts cultured with injured HBE were significantly higher than those in the supernatants of fibroblasts cultured with HBE [(3.79±0.64) ng/L, (15.5±3.2) ng/L]. BQ123, SB203580 or LY294002 decreased IL-6 levels [(27.2±3.1) ng/L, (31.5±3.6) ng/L, (41.3±3.2) ng/L] differently in the supernatants of fibroblasts induced by injured HBE. Activation of p38 MAPK preceded Akt in fibroblasts cultured with injured HBE. BQ123 reduced the phosphorylation levels of p38 MAPK and Akt. SB203580 concentration-dependently attenuated Akt phosphorylation, while LY294002 had little effect on p38 MAPK phosphorylation. Fibroblasts expressed more α-SMA after cultured with injured HBE and showed significant increase in the gel contraction compared to fibroblasts cultured with HBE [percentage of gel contraction: (61.2±2.7)% vs (15.4±7.3)%], all these effects were diminished or inhibited by BQ123, SB203580 or LY294002. Furthermore, the effects of BQ123 and SB203580 on decreased gel contraction were stronger than the effect of LY294002. CONCLUSION: ET-1 exerts a key role in the airway fibroblasts activation induced by injured HBE through activating p38 MAPK, PI3K/Akt signaling and promoting IL-6 expression.

Objective To observe the effects of Huoxue Chubi Decoction on contents of PDGF-A and TGF-βin skin lesion of scleroderma mimicking mice models;To discuss its possible mechanism. Methods Thirty mice were randomly assigned into blank group, model group, TCM high-, medium-, low-dose group, with six mice in each group. Other than blank group, the rest of the 4 groups were given bleomycin-hydrocholoride 0.1 mL intradermal injection daily for 3 weeks to establish scleroderma mice models. From week 4 to 7, blank group and model group received gavage with equal amount of distilled water daily;TCM high-, medium-, low dosage group received gavage with Huoxue Chubi Decoction with different concentrations of 44.8, 22.4, 11.2 g/kg daily. The pathological changes in the skin tissue samples taken from each group were observed under microscope;the thickness of skin from each group was measured and the expression of PDGF-A, TGF-β, type-Ⅰ collagen (COL-Ⅰ) and type-Ⅲ collagen (COL-Ⅲ) were tested through immunohistochemical staining. Results Compare with blank group, the dermis tissue of model group was thicker, with the presence of thicker and greater number of collagen fibers, as well as infiltration of inflammatory cells. Compare with model group, thickness of skin and dermis collagen growth of TCM high-, medium-, low-dose group were milder varied by the concentration of the decoction. The result of immunohistochemical staining showed that the expressions of PDGF-A, TGF-β, COL-Ⅰ and COL-Ⅲ of modelgroup significantly increased than blank group (P<0.01); the expression of the mentioned indicators were statistically significant lower in the TCM high-, medium-, low-dose group than model group; The expressions of PDGF-A and TGF-β showed a positive correlation with the amount of COL-Ⅰ and COL-Ⅲpresented in each group. Conclusion Huoxue Chubi Decoction can suppress collagen expression and fibroblast formation in skin lesion of scleroderma mice models by down regulating the expressions of TGF-β and PDGF-A, thus demonstrate its potential in scleroderma treatment.

Objective To investigate changes in the intraocular pressure(IOP) and anterior chamber angle in patients with chronic renal failure before and after hemodialysis. Methods Fifty?eight patients(116 eyes) with chronic renal failure were measured with Goldmann applanation tonometer and anterior segment optical coherence tomograph. The patients were divided into three groups based on gonioscopy results:the narrow angle group(22 eyes),the Iris neovascular group(3 eyes)and the open angle group(91 eyes). IOP was measured by Goldmann applanation tonometer in patients in the three groups before and after hemodialysis. Anterior chamber angle opening distance (AOD) was detected by anterior segment optical coherence tomograph before and after hemodialysis. The blood urea nitrogen,creatinine,albumin were also determined before and after hemodialysis. All changes in the parameters were analyzed with a paired t test. Results The IOP in narrow angle eyes and in neovascular eyes increased significantly after hemodialysis ,while the IOP in open angle eyes showed no significant changes. The AOD decreased significantly after hemodialysis. The blood urea nitrogen and creatinine decreased significantly in 58 patients,while albumin increased after hemodialysis. Conclusion The IOP in narrow angle eyes and in neovascular eyes increased after hemodialysis ,correlating with the resistance increase of aqueous outflow pathway and the change of plasma osmotic pressure. As a result ,it is recommended that the condition of eye of patients with chronic renal failure should be checked ,and patients should receive appropriate controlling measure or treatment before hemodialysis.

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride ( CoCl2 ) on cave-olin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcino-ma A549 cells.METHODS:The concentrations of Cav-1 and hypoxia-inducible factor ( HIF)-1αin pleural effusion of the patients with lung cancer ( MPE) or tuberculous pleurisy ( TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1αinhibitor YC-1.The concentrations of Cav-1 and HIF-1αin the cell supernatants were measured by ELISA.The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respec-tively.RESULTS:The levels of Cav-1 and HIF-1αin MPE were significantly higher than those in TBPE.There was a highly positive correlation between Cav-1 and HIF-1αlevels in the pleural effusion.CoCl2 induced the generation of Cav-1 and HIF-1αin A549 cells in a concentration-and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration-or time-dependent inhibition was ob-served.HIF-1αinhibitor YC-1 concentration-dependently inhibited the generation of HIF-1αand Cav-1 induced by CoCl2 in A549 cells.CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1.CONCLUSION:The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells.A possible role for HIF-1αis indicated in Cav-1 generation.

Objective To investigate the influence of simvastatin on inflammatory indices in nasal lavage,sputum and blood and clinical index in patients with chronic obstructive pulmonary diseases (COPD). Methods Thirty-seven stable COPD patients were randomly divided into simvastatin-treatment group (n=17),orally given simvastatin tablets for 4 weeks in addition to basic therapy,40 mg,qd) and control group (n=20),given usual med-ication). Total cell counts,percentage of leukocytes (N%) and levels of interleukin IL-8,IL-6 in nasal lavage and sputum at pre-post-treatment were compared;Serum C-reactive protein (CRP),total cholesterol (TC),low-density lipoprotein-cholesterol (LDL-C) as well as IL-8,IL-6 concentrations were measured,the variation of lung function,Sino-Nasal Outcome Test 20(SNOT-20) and St George's Respiratory Questionnaire(SGRQ) score were analyzed. Results After the treatment,the nasal lavage and sputum total cell counts,N%,IL-8 and IL-6 levels[nasal lavage: (0.7±0.3)×107/L,(41.1±10.9)%,(105.8±74.5) ng/L,(3.8±1.6) ng/L;sputum: (0.8±0.3)×109/L,(56.6±9.6) %,(2565.5±831.9) ng/L,(109.8±42.3) ng/L] dropped slightly in the simvastatin group com-pared with that at pretreatment [nasal lavage: (0.8±0.3)×107/L,(43.2±10.8) %,(107.6±86.3) ng/L,(4.1±1.9)ng/L;sputum: (0.8±0.3)×109/L,(58.1±9.3)% ,(2659.4±885.2) ng/L,(111.8±46.6) ng/L] (P>0.05) ;There were significant decreases in serum CRP [(4.3±3.7) mg/L vs (2.6±1.8) mg/L],IL-6 [(4.8±2.0)ng/L vs(4.7±1.9)ng/L] ,TC[(4.2±1.0) mmol/L vs(3.7±0.8)mmol/L] ,LDL-C[(2.4±0.5) mmol/L vs (2.2±0.5)mmol/L] (P>0.05) ;IL-8 concentrations in serum were lower gently[(6.2±1.8) ng/L vs (6.4±1.9) ng/L] (P>0.05). Significant change of simvastatin treatment on SGRQ was only reflected in the symp-tom score [pre-post-treatment:39.6±10. 8 vs 32.3±11.6,P<0.05,respectively],while other observation items (SNOT-20,FEV1%,FEV1/FVC) changed not notably (P>0.05). No marked changes in inflammatory markers and quality of life scores,lung function were observed in control group (P>0.05). Conclusion Simvastatin may be as-sociated with the potential to alleviate systemic inflammation and relieve symptoms in COPD patients.

Objective Studies show that the ERCC1 gene may be involved in secondary cisplatin resistance.This article aims to investigate the effects of shRNA targeting silencing excision repair cross-complementation group 1 (ERCC1-shRNA) on the proliferation and apoptosis of lung cancer A549/DDP cells treated with different concentrations of cisplatin.Methods Lung cancer A549/DDP cells were divided into a negative control, a blank control, an ERCC1-shRNA1, and an ERCC1-shRNA2 group.Human interfering RNA (RNAi) targeting the human ERCC1 gene was constructed and transfected into the A549/DDP cells using Lipofectamine 2000.The mRNA and protein expressions of ERCC1 in the A549/DDP cells were detected by real-time PCR and Western blot respectively, the proliferation-inhibition rate was assessed by MTT, and their cell cycle and apoptosis were determined by flow cytometry.Results ERCC1-shRNA was successfully constructed and transfected into the A549/DDP cells.Both the mRNA and protein expressions of ERCC1 were significantly lower in the ERCC1-shRNA1 (0.20±0.04 and 0.24±0.10) and ERCC1-shRNA2 (0.47±0.28 and 0.37±0.11) than in the negative control (0.96±0.12 and 1.32±0.13) and blank control groups (0.84±0.07 and 1.45±0.23) (P<0.01).Compared with the negative and blank control groups, the ERCC1-shRNA1 group showed a significantly decreased IC50 value (16.71±2.33 and 16.69±1.69 vs 7.78±0.54, P<0.01) and an increased proportion of G0/G1 phase cells ([72.87±3.23] and [71.75±4.56] vs [82.99±4.23]%, P<0.05), with the cell cycle arrested in the G0/G1 phase.The apoptosis rate of the cells in the ERCC1-shRNA1 group was remarkably lower after treated with cisplatin at the concentrations of 6.25 and 12.5 μg/mL than at 0 μg/mL ([8.17±0.65] and [11.91±1.41] vs [29.97±3.14]%, P<0.05).Conclusion ERCC1-shRNA can inhibit the proliferation and enhance the apoptosis of A549/DDP cells by silencing the expression of the ERCC1 gene.

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Objective To explore the roles of PKCθ(protein kinase Cθ)signaling pathway on the activation,proliferation and TH1/TH2 cytokines production of the γδT lymphocytes stimulated by Mycobacterium tuberculosis antigen(Mtb-Ag) in vitro.Methods Peripheral blood mononuclear cells(PBMC)were pretreated with or without Rottlerin at 5.0 μmol/L,and stimulated with Mtb-Ag and cultured in rhIL-2 containing medium.After different time of culture,activation molecules and cytokines production of γδT cells were measured by flow cytometry.The proliferation proportion and the percentage of each generation of γδT cells were determined by carboxylfluoreseein diacetate succinmidyl ester(CFSE)labeling technique and flow cytometry.Results After 3d of stimulation with Mtb-Ag,the expression rates of CD69 and CD25 of γδT cells were 46.2%and 45.6%,respectively.Pretreatment of 5.0 μmol/L Rottlerin markedly inhibited the both expressions of CD69 and CD25 in γδT cells(P＜0.01).After stimulation and expansion in vitro for 5,10,and 15 d,the percentages of the γδT cell were 9.6%,54.6%and 82.4%,respectively.There was a few γδT cells in propagation on the 5th day of culture,and almost all γδT cell divisions were above 6 generations on the 10th and 15th day of culture.Pretreatment of the Rotflerin significantly suppressed the γδT cell proliferation,but after 10 d culture,there were still a few parts of γδT cells in propagation.Meanwhile,after 7,14,and 21d of culture,upon stimulation with PMA+Ionomycin,the IFN-γ producing-γδT cells were about 80%at all times.But only after 21d culture,IL-4-producing γδT cells was 2.6%.,The percentage of IFN-γ producing γδT cells markedly reduced in Rottlerin group(P＜0.01).IL-4 secretion of the γδT cells was almost completely blocked.Conclusion PKCO signal pathway involves in activation,proliferation and differentiation of the γδT lymphocytes stimulated by Mtb-Ag.

To understand the genetic origin and variation of porcine epidemic diarrhea virus (PEDV) prevailing in Henan province,a total of 22 PEDV positive samples from suckling piglets with severe diarrhea were collected from 16 pig farms and amplified by RT-PCR.ORF3 genes were then cloned into pMD18-T vector and sequenced.The result of sequencing showed that the length of ORF3 genes all were 675 bp which encoded 224 amino acids.The strains in this study had eight amino acid substitutions when compared with CV777 strain.The nucleotide homologies of the 22 strains were 95.9％-100％ and amino acid homologies were 96.4％-100％.By comparing with those of European strain CV777 and vaccine strain truncated CV777,the nucleotide homologies were 97.1％-97.9％ and 94.8％-95.4％,respectively.Based on the phylogenetic relationship of ORF3 genes,the PEDV field strains and PEDV reference strains could be divided into two groups,and all the field strains identified in this study belong to group 2.It suggested that prevalent PEDV strains in this study seem to be closely related to Henan isolates in previous years,domestic strains,Janpanese strains,American strains as well as Korean strains but differ genetically from European strains or vaccine strains used in China.This experiment analyzed the ORF3 gene sequence features in prevailing of Henan region in recent years,which provides a new support of epidemiology study and protein function research of PEDV ORF3 gene.