OBJECTIVETo investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell (ASMCs) migration and cytoskeletal reorganization induced by endothelin-1 (ET-1).

METHODSPrimary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-1 treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation of myosin-phosphatase target 1 (p-MYPT1) was examined using Western blot analysis.

RESULTSAt the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect (P<0.01). Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 micromol/L significantly blocked ASMC migration (P<0.01). ET-1 (10 nmol/L) exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPT1 protein expression after ET-1 exposure for 15 and 30 min (P<0.01), but prolonged exposure failed to result in the expression enhancement (P>0.05).

CONCLUSIONRho-kinase signaling pathway may play an important role in ET-1-induced ASMC migration and reorganization of actin cytoskeleton.

Amides ; pharmacology ; Bronchi ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeleton ; drug effects ; metabolism ; Endothelin-1 ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Humans ; Microscopy, Confocal ; Muscle, Smooth ; cytology ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; rho-Associated Kinases ; antagonists & inhibitors ; metabolism

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

OBJECTIVETo explore the effects of serum of asthmatic patients, dexamethasone, interleukin-4 (IL-4), interferon-gamma (IFN-γ) and transforming growth factor-β (TGF-β) on the expression of interleukin-22 receptor 1 (IL-22R1) mRNA and protein in HASMCs in vitro.

METHODSIL-22R1 mRNA and protein expressions in HASMCs treated with different stimulating agents were measured by real-time PCR and Western blotting, respectively.

RESULTSIL-22R1 mRNA and protein expressions in HASMCs were significantly increased after stimulation by serum from asthmatic patients, but decreased after co-stimulation with dexamethasone. IL-22R1 mRNA and protein expressions in the cells both increased after stimulation by IL-4, IFN-γ and TGF-β.

CONCLUSIONIL-22R1 in HASMCs might be involved in the pathogenesis of asthma, and the therapeutic effect of dexamethasone on asthma is mediated, at least partially, by IL-22R1. The effects of IFN-γ, IL-4, and TGF-β on asthma may also be attributed to their actions on HASMCs.

Asthma ; blood ; Cell Line ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-4 ; pharmacology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Receptors, Interleukin ; metabolism ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo investigate the effect of liver X receptor agonist T0901317 on transforming growth factor-β1 (TGF-β1)-induced expression of α-smooth muscle actin (α-SMA) in normal human lung fibroblasts.

METHODSPrimary normal human lung fibroblast isolated from the lung specimens of lung cancer patients by explant culture technique were identified with immunostaining for vimentin and keratin. The cells in passages 4 to 10 were treated with T0901317 and/or TGF-β1, and RT-PCR, Western blotting and immunofluorescence assay were used to detect α-SMA expression in the fibroblasts.

RESULTSLung fibroblast expressed vimentin but not keratin. The results of RT-PCR, Western blotting and immunofluorescence assay all showed that normal human lung fibroblasts constitutively expressed α-SMA under baseline condition, and TGF-β1 at 5 ng/ml induced a significant upregulation of α-SMA both at the mRNA and protein levels. Liver X receptor agonist T0901317 (5 µg/ml) significantly inhibited TGF-β1-induced upregulation of α-SMA expression.

CONCLUSIONLiver X receptor agonist T0901317 can inhibit the upregulation of α-SMA in normal human lung fibroblasts induced by TGF-β1, suggesting the potential value of liver X receptor agonist in the treatment of lung fibrosis.

Actins ; metabolism ; Cells, Cultured ; Female ; Fibroblasts ; drug effects ; metabolism ; Humans ; Hydrocarbons, Fluorinated ; pharmacology ; Liver X Receptors ; Lung ; cytology ; Middle Aged ; Orphan Nuclear Receptors ; agonists ; RNA, Messenger ; genetics ; Sulfonamides ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology