Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the ßγ subunit of Gi-protein of fMLP receptor in human neutrophils.

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein ßγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 µM)-induced superoxide anion production (IC50:1.98 ± 0.14 µM), the release of cathepsin G (IC50:2.76 ± 0.15 µM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the ßγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCß by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the ßγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

In vitro evaluation of aerosol delivery by different nebulization modes in pediatric and adult mechanical ventilators.

BACKGROUND: Aerosol delivery through mechanical ventilation is influenced by the type of aerosol generator, pattern of nebulization, and a patient's breathing pattern. This study compares the efficiency of pneumatic nebulization modes provided by a ventilator with adult and pediatric in vitro lung models. METHODS: Three pneumatic nebulization modes (inspiratory intermittent [IIM], continuous [CM], and expiratory intermittent [EIM]) provided by the Galileo Gold ventilator delivered medical aerosol to collection filters distal to an endotracheal tube with adult and pediatric test lungs. A unit dose of 5 mg/2.5 mL albuterol was diluted into 4 mL with distilled water and added to a jet nebulizer. The nebulizer was placed proximal to the ventilator, 15 cm from the inlet of the heated humidifier chamber with a T-piece and corrugated aerosol tubing and powered by gas from the ventilator in each of the 3 modes. Time for nebulization was recorded in minutes. Albuterol samples collected in the inhalation filter, nebulizer, T-piece, and corrugated tubing were eluted with distilled water and analyzed with a spectrophotometer. RESULTS: The inhaled drug, as a percentage of total dose in both lung models, was 5.1-7.5%, without statistical significance among the 3 modes. Median nebulization times for IIM, CM, and EIM were 38.9, 14.3, and 17.7 min, respectively, and nebulization time for the 3 modes significantly differed (P < .001). The inhaled drug mass for the 3 modes with the adult lung model was similar to that with the pediatric lung model (7.39 ± 0.76 vs. 6.27 ± 0.69%, P = .77). CONCLUSIONS: Aerosol drug delivery with a jet nebulizer placed proximal to the ventilator was not dependent on nebulization mode during simulated pediatric and adult conventional mechanical ventilation. Use of expiratory intermittent mode and continuous nebulization should be considered to reduce treatment time.