OBJECTIVETo study the inhibitory effect of shikonin on the proliferation of human airway smooth muscle cells (HASMCs) in vitro.

METHODSHASMCs from the trachea were obtained by primary culture of the tissue explants and adherent culture. The HASMCs were exposed to shikonin at 0 (control group), 0.5, 1, 2, 5, 10, 20, and 40 micromol/L for 12, 24, and 48 h, after which the cell proliferation was assessed by 3-(4,5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay. Flow cytometry was used for cell cycle analysis of the HASMCs exposed to shikonin at 40, 20, 10, 5 micromol/L and 0 micromol/L (control group) for 24 h. Immunocytochemistry with SP method was performed to detect the expression of proliferating cell nuclear antigen (PCNA) in the HASMCs treated with shikonin at 20 micromol/L and 0 micromol/L (control group) for 24 h.

RESULTSShikonin at the concentrations of 20 and 40 micromol/L significantly inhibited the proliferation of the cells (P<0.05), and the effect was especially obvious after 48 h of cell exposure, with inhibition rates of 30.1% and 42.9%, respectively. No significant difference was found between the two concentrations for their cell growth inhibition effects (P>0.05). Shikonin at the concentrations of 20 and 40 micromol/L caused significant cell cycle arrest in G(0)/G(1) phase (P<0.05), the effect of which, however, was not concentration-dependent (P>0.05). Shikohin at 20 micromol/L significantly down-regulated the expression of PCNA in the cells (P<0.05).

CONCLUSIONShikonin can inhibit the proliferation of HASMCs in vitro.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flow Cytometry ; Humans ; Immunohistochemistry ; Muscle, Smooth ; cytology ; drug effects ; metabolism ; Naphthoquinones ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Trachea ; cytology

Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelin-1 ; pharmacology ; Glyburide ; pharmacology ; Pinacidil ; pharmacology ; Potassium Channels ; drug effects ; Rabbits ; Thymidine ; metabolism ; Trachea ; cytology ; drug effects

To investigate the effect of lithium on cell cycle progression of airway epithelial cells, primary pig tracheobronchial epithelial cells were incubated with lithium chloride (LiCl) at different concentrations (0, 5 mmol/L, and 10 mmol/L) and time (12 h, 16 h and 24 h). After the treatment, cells were counted, cell cycle profile was measured by BrdU labeling and flow cytometry, and expression of cyclin D1 and cyclin B1 were detected by Western blotting. The results showed that after 24h of 10mmol/L but not 5mmol/L LiCl treatment, proliferation of cells was slowed down as manifested by delayed confluence and cell number accumulation (P<0.05). Lithium did not change the percentage of cells in S phase (P>0.05), but 24 h incubation with 10 mmol/L LiCl induced a G2/M cell cycle arrest. Furthermore, 10mmol/L LiCl elevated cyclin D1 expression after 12h treatment, while expression of cyclin B1 increased more significantly after 24h incubation. These data demonstrate that lithium inhibits proliferation of pig airway epithelial cells by inhibiting cell cycle progression, and suggest that lithium-sensitive molecule(s) such as glycogen synthase kinase 3 may have a role in the regulation of growth of airway epithelial cells.
Animals ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Epithelial Cells ; cytology ; drug effects ; Flow Cytometry ; Glycogen Synthase Kinase 3 ; metabolism ; Lithium Chloride ; pharmacology ; Swine ; Trachea ; cytology

OBJECTIVETo study the analgesic, expectorant and antitussive effects of the compatible use of Aconiti Radix Cocta and Fritillaria cirrhosa or F. thunbergii with different matching ratio or dose in mice.

METHODThe two-factor, seven-level uniform design method was adopted to observe the analgesic, expectorant and antitussive effects of the oral administration with the two combined decoctions in rats, with frequency of body torsions induced by acetum, secretion of phenol red in tracheas and frequency of coughs as indexes. Significant matching proportions and doses were collected for verification.

RESULTThe effect on the frequency of body torsions: The combined decoctions could effectively reduce the frequency of body torsions. According to a regression analysis, Aconiti Radix Cocta and F. cirrhosa had the synergistic effect, which was maximized with a ratio of 1: 1. The 1: 1 combined decoction played the least role in reducing the frequency of body torsions with a total dose of more than 5 g x kg(-1). The effect on the secretion of phenol red in tracheas. The combined decoctions could effectively increase the secretion of phenol red in tracheas. According to a regression analysis, Aconiti Radix Cocta and F. thunbergii had the antagonism, which was maximized at the ratio of 1: 1, and minimized with a total dose of less than 10 g x kg(-1) and a ratio of 5: 1 between F. thunbergii and Aconiti Radix Cocta. The effect on the frequency of coughs. The combined decoctions could effectively reduce the frequency of coughs. According to a regression analysis, Aconiti Radix Cocta and F. cirrhosa had the antagonism, which was maximized at the ratio of more than 1: 5 and less than 10: 1. There was no interaction between Aconiti Radix Cocta and F. thunbergii. F. thunbergii could reduce the frequency of coughs, whereas Aconiti Radix Cocta showed no effect.

CONCLUSIONThe compatible application of Aconiti Radix Cocta and F. cirrhosa could enhance the analgesic effect of Aconiti Radix Cocta and reduce the expectorant and antitussive effects of F. cirrhosa, which vary according to different matching ratio and dose. The compatible application of Aconiti Radix Cocta and F. thunbergii shows no effect on the antitussive effect of F. thunbergii. This study provides experimental basis for in-depth studies on the combined effect of Aconiti Radix Cocta and Fritillaria--two of eighteen incompatible pairs.

Aconitum ; chemistry ; Analgesics ; pharmacology ; Animals ; Antitussive Agents ; pharmacology ; Behavior, Animal ; drug effects ; Drug Synergism ; Drugs, Chinese Herbal ; pharmacology ; Expectorants ; pharmacology ; Fritillaria ; chemistry ; Male ; Mice ; Phenolsulfonphthalein ; metabolism ; Trachea ; drug effects ; metabolism

OBJECTIVEIn this study, we pretreated the mice ASMCs by dexamethasone (Dex) within 10 min, to test the peak of [Ca2+]i and phospho-PLCbeta (ser1105) in the cells by treated with Ach.

METHODSThe peak of [Ca2+]i was measured by Fura-2/AM methods and the phospho-PLCbeta-ser1105 was by Western blot, and compared with dexamethasone pretreated groups. Glucocorticoid receptor antagonist RU486 and the protein synthesis inhibitor cycloheximide groups were settled in our study.

RESULTSGlucocorticoids (GCs) significantly decreased the resting values and peak of [Ca2+]i elevation and elevated the intracellular levels of phospho-PLCbeta (ser1105) in 10 min. Neither the RU486 nor cycloheximide could alter the inhibitory effects of glucocorticoids stated above.

CONCLUSIONOur results demonstrate that glucocorticoids exert rapid inhibitory effects. The series of signal changes in this process that restrain the peak of [Ca2+]i may be responsible for the rapid nongenomic inhibitory effects of GCs by reducing the activity of PLC.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Guinea Pigs ; Male ; Mifepristone ; pharmacology ; Muscle, Smooth ; drug effects ; metabolism ; Phospholipase C beta ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trachea ; cytology

BACKGROUND: Hypersecretion of mucin due to goblet cell hyperplasia is frequently encountered in many chronic airway diseases, such as chronic bronchitis, bronchiectasis, bronchial asthma and cystic fibrosis. Even in normal individuals, viral infection or bacterial pneumonia frequently provoke huge amounts of bronchial secretions which may cause airway obstruction. The production of mucin was regulated by epidermal growth factor (EGF) in vitro. To know whether this EGF system regulates mucin secretion in vivo and Pseudomonas also stimulates the mucin secretion by the same pathway, we studied these relationships in the cultured rat tracheal epithelial cells. METHODS: Rat tracheal epithelial cells were obtained by pronase dissociation from the male Fisher 344 rats. When cells became confluent, they were divided into 6 groups and stimulated with either EGF for 24 hours or Pseudomonas extracts for 12 hours with or without selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478. RESULTS: We found that both EGF and Pseudomonas extracts phosphorylated the tyrosine residue in the EGF receptor from the rat tracheal epithelial cells and this tyrosine phosphorylation was nearly completely blocked by selective EGF-R tyrosine kinase inhibitor tyrphostin AG1478. The mucin secretion was also stimulated by either EGF or Pseudomonas extracts but more strong secretion of mucin and MUC5AC gene expression in the rat tracheal epithelial cell was done by Pseudomonas extracts. CONCLUSION: These data suggest that Pseudomonas secretes the mucin by way of the EGF receptor and MUC5AC gene expression and the inhibitors of EGF receptor tyrosine phosphorylation would be useful to prevent the huge production of mucin due to Pseudomonas aeruginosa lung infection.
Animal ; Blotting, Western ; Cells, Cultured ; Comparative Study ; Epidermal Growth Factor/*metabolism/pharmacology ; Epithelial Cells/drug effects/*secretion ; Gene Expression ; Male ; Models, Animal ; Mucins/drug effects/*genetics/*secretion ; *Pseudomonas aeruginosa ; RNA, Messenger/analysis ; Rats ; Rats, Inbred F344 ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Trachea/cytology/drug effects/*microbiology/*secretion

BACKGROUNDAn important physiological feature of asthma is the phenotypic change of airway smooth muscle cells (ASMCs), but the precise mechanisms behind the ASMCs' change remains unknown. Our study assessed whether p21Ras can directly modulate the phenotype of ASMCs.

METHODSRat ASMCs were treated with FTP III, a highly specific p21Ras inhibitor. ASMCs were identified via immunocytochemistry. The ultrastructure of cells was observed by electron microscopy, and the expression of α-actin was evaluated by Western blotting analysis. The levels of IL-6 and RANTES were measured by enzyme linked immunosorbent assay (ELISA).

RESULTSIt was observed that ASMCs in asthma exhibited a proliferative/secretory phenotype and were larger, denser and had many pseudopods, as well as increased signs of secretory organelles. Additionally, the level of α-actin, a marker of ASMCs, was reduced in asthmatic ASMCs and the secretion of IL-6 and RANTES was increased. When FTP III was added to asthmatic ASMCs it induced a contractile phenotype, with increased α-actin levels and reduced secretion of IL-6 and RANTES.

CONCLUSIONSIt appears that p21Ras induces asthmatic ASMCs to a proliferative/secretory phenotype, but its inhibitor FTP III, can significantly reverse this phenotype. The role of p21Ras in the ASMCs may be a new target for asthma treatment.

Actins ; metabolism ; Animals ; Asthma ; metabolism ; Blotting, Western ; Cells, Cultured ; Chemokine CCL5 ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Interleukin-6 ; metabolism ; Lung ; cytology ; Male ; Microscopy, Electron, Transmission ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; ultrastructure ; Organophosphonates ; pharmacology ; Proto-Oncogene Proteins p21(ras) ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trachea ; cytology

OBJECTIVETo investigate the mechanism underlying myofibroblast differentiation induced by transforming growth factor (TGF) beta1 in obliterative bronchiolitis following lung transplantation.

METHODSHeterotopic tracheal transplantation was performed in Smad3 wild-type and knock-out mice to simulate the lung transplantation in human. Murine tracheal fibroblasts cultivated in primary culture were used for in vitro study. Immunohistochemistry, immunocytochemistry, Western Blotting, RT-PCR and DNA electrophoresis mobility gel shift assay were conducted to detect the expression of alpha-smooth muscle actin (alphaSMA), the marker of fibroblast-myofibroblast differentiation, and the activation of Smad3, p38 and ERK1/2.

RESULTSIn affected airways of experimental obliterative bronchiolitis, abundant expression of alphaSMA were found. In vitro study for tracheal fibroblasts, the activation of Smad3 by TGF-beta1 presents as three major forms, phosphorylation, nuclear translocation and DNA binding. In Smad3 wild-type fibroblasts, TGF-beta1 induces the increase of the myofibroblasts transformation, characterized by the elevation of alphaSMA, both at transcription and protein level. While in Smad3 knock-out fibroblasts, the transformation of myofibroblasts induced by TGF-beta1 is significantly decreased (t = 2.080, P = 0.027; t = 1.982, P = 0.032), but not completely abolished. Further study in Smad3-deficient fibroblasts demonstrates that p38 and ERK1/2 could be activated by TGF-beta1 and result in fibroblast differentiation.

CONCLUSIONSTGF-beta1 could promote the transformation of fibroblasts into myofibroblasts in Smad3 dependent and independent signal pathways, especially the Smad3 dependent path, and result in the development of obliterative bronchiolitis.

Actins ; genetics ; metabolism ; Animals ; Blotting, Western ; Bronchiolitis Obliterans ; genetics ; pathology ; surgery ; Cell Differentiation ; drug effects ; Cells, Cultured ; Disease Models, Animal ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smad3 Protein ; genetics ; metabolism ; Trachea ; cytology ; transplantation ; Transforming Growth Factor beta1 ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

PURPOSE: This study aims to investigate the additive effect of the Hedera helix (HH) and Rhizoma coptidis (RC) extracts mixture on antitussive and expectorant activities in animals. MATERIALS AND METHODS: The expectorant assay was performed with phenol red secretion in mice trachea. Mice or guinea pigs were randomly divided into groups of 8 each, including negative and positive control groups. After gastric administration of the test extracts in mice, 2.5% phenol red solution (0.2 mL) was intraperitoneally injected. Trachea was dissected and optical density of tracheal secretion was measured. After gastric administration of the test extracts in guinea pigs, the antitussive activities were assessed using a citric acid-induced cough measurement. RESULTS: The extracts of HH and RC significantly increased tracheal secretion and inhibited cough. The mixture of HH and RC extracts in a 1:1 concentration at a dose of 200 mg/kg showed a more potent effect on phenol red secretion (25.25+/-3.14) and cough inhibition (61.25+/-5.36) than the individual use of each extracts [phenol red secretion; HH 13.39+/-4.22 (p=0.000), RC 20.78+/-2.50 (p=0.010), cough inhibition; HH 9.89+/-4.14 (p=0.010), RC 30.25+/-7.69 (p=0.000)]. A 3:1 ratio mixture of HH to RC demonstrated an optimal expectorant effect (p<0.001), and this mixture showed expectorant and antitussive effects in a dose-dependent manner. CONCLUSION: This study provides evidence for antitussive and expectorant effect of a 3:1 mixture of HH and RC, which may be a useful therapeutic option for respiratory diseases.
Animals ; Antitussive Agents/*administration & dosage/pharmacology/therapeutic use ; *Behavior, Addictive ; Cough/*drug therapy ; Drugs, Chinese Herbal/*administration & dosage/pharmacology/therapeutic use ; Ethanol ; Expectorants/*administration & dosage/pharmacology/therapeutic use ; Guinea Pigs ; Hedera/*chemistry/metabolism ; Male ; Mice ; Phytotherapy ; Plant Extracts/*pharmacology ; Plant Roots/chemistry ; Trachea/drug effects/metabolism

Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.
Animals ; Benzoates ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; metabolism ; Exocrine Glands ; secretion ; Flavones ; administration & dosage ; pharmacology ; Humans ; Mice ; Patch-Clamp Techniques ; Rats ; Rats, Inbred F344 ; Thiazolidines ; pharmacology ; Thyroid Gland ; cytology ; Time Factors ; Trachea ; secretion