[Effect of phosphorylation of cortactin at different sites on secretion by airway mucus 5AC].

OBJECTIVE: To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.
 Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin.
 Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05).
 Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.

[Effects of secretary leukocyte protease inhibitor-transfected bone marrow mesenchymal stem cells on airway inflammation and mucus secretion in chronic obstructive pulmonary disease].

OBJECTIVE: To explore the effects of secretary leukocyte protease inhibitor (SLPI)-transfected bone marrow mesenchymal stem cells (BMSCs) transplantation on airway inflammation and mucus secretion in chronic obstructive pulmonary disease (COPD) rats. METHODS: Sixty rats were equally and randomly divided into negative control, COPD model, BMSCs and SLPI-transfected BMSCs groups. The COPD rat model was established in all rats with the exception of the negative control rats by smoking and intratracheal instillation of lipopolysaccharide (LPS). BMSCs with or without transfection of plasmid containing SLPI gene were delivered through caudal vein of rats at 30 days post-induction. The expression of SLPI was examined with Western blot. The levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA). Goblet cell hyperplasia of lung pathological section was observed on. RESULTS: Compared with the negative control group, the expression of SLPI increased significantly after the administration of SLPI-transfected BMSCs (0.79 ± 0.06 vs 0.24 ± 0.02, P < 0.05). The levels of IL-8 and TNF-α in BMSCs and SLPI-transfected BMSCs group were lower than those in the COPD model group. Compared with the negative control group, the administration of SLPI-transfected BMSCs resulted in a further decrease in IL-8 and TNF-α in bronchoalveolar lavage fluid [(17.6 ± 1.7) vs (36.6 ± 4.0) ng/L, P < 0.05]. SLPI-transfected BMSCs transplantation also significantly attenuated goblet cell hyperplasia in rats (P < 0.05). CONCLUSION: There is a potential role for cell-based SLPI gene therapy in the treatment of COPD.

Role of JNK signal transduction pathway in IL-8 and TNF-α secretion from alveolar macrophages induced by mechanical ventilation with large-tidal volume in rabbits / 中华麻醉学杂志

Objective To investigate the role of c-Jun N-terminal kinase (JNK) signal transduction pathway in IL-8 and TNF-α secretion from alveolar macrophages induced by mechanical ventilation with large-tidal volume in rabbits. Methods Thirty male New Zealand white rabbits weighing 210-260 g were randomly divided into 330-40 bpm, PEEP 0), and SB203580 group (group S). The animals were anesthetized with iv pentobarbital sodium 40 mg/kg, traeheostomized and mechanically ventilated. Group C received no mechanical ventilation. The animals were mechanically ventilated for 3 days in group V. The animals were mechanically ventilated for 3 days and SB203580 (a specific JNK inhibitor) 6 mg/kg was injected via the ear vein every day during ventilation (the ventilation parameters were the same as those in group V). The animals were then sacrificed by exsanguination. The concentrations of IL-8 and TNF-α in bronchoalveolar lavage fluid (BALF) were determined by ELISA and the alveolar macrophages were collected. After the macrophages were cultured for 2 h in vitro, the expression of IL-8 mRNA and TNF-α mRNA was determined by RT-PCR. Results Compared with group C, the levels of IL-8 , TNF-α,IL-8 mRNA and TNF-α mRNA were significantly increased in group V (P＜0.05). Compared with group V, the levels of TNF-α and TNF-α mRNA were significantly decreased ( P ＜ 0.01 ), but no significant change was found in the levels of IL-8 and IL-8 mRNA in group S ( P ＞ 0.05). Conclusion JNK signal transduction pathway plays an important role in TNF-α secretion from alveolar macrophages induced by mechanical ventilation with large-tidal volume in rabbits, but is not involved the secretion of TNF-α.