U 3010

The assay of the generation of superoxide anion was based on the SOD-inhibitable reduction of ferricytochrome c [65 (link),66 (link)]. In brief, after supplementation with 0.5 mg/mL ferricytochrome c and 1 mM Ca²⁺, neutrophils (6 × 10⁵ cells/mL) were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated with 100 nM fMLP during the preincubation of 1 µg/mL cytochalasin B (fMLP/CB) for 3 min. Changes in the absorbance with a reduction in ferricytochrome c at 550 nm were continuously monitored in a double-beam, six-cell positioner spectrophotometer with constant stirring (Hitachi U-3010). Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm at the concentration of 1 mM in cuvette with 1-cm optical path length).

The assay for detection of O₂^•– generation was based on the SOD-inhibitable reduction of ferricytochrome c [32 (link)]. In short, neutrophils (1 × 10⁶ cells/mL) pretreated with the various test agents (50 and 5 μM) at 37 °C for 5 min were stimulated with fMLP (1 μmol/L) in the presence of ferricytochrome c (0.5 mg/mL). Extracellular O₂^•– production was evaluated with a UV spectrophotometer at 550 nm (Hitachi U-3010, Tokyo, Japan). The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control − resting) − (compound − resting)}/(control − resting) × 100. The software SigmaPlot was used for determining the IC₅₀ values [31 (link)].

The superoxide anion generation in activated human neutrophils was measured using the reduction of ferricytochrome c. In addition, Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide was used as the elastase substrate to detect elastase release. Neutrophils were incubated with ferricytochrome c or elastase substrates at 37°C, and then treated with N11 for 5 min. FMLP (30 nM) with cytochalasin B (0.5 or 1 µg/ml) or phorbol myristate acetate (PMA, 5 nM) was used to stimulate neutrophils. The change in absorbance was continually monitored at 550 nm or 405 nm using a spectrophotometer (U-3010, Hitachi, Tokyo, Japan) [23] (link), [24] (link).
Neutrophils were incubated with FMLP (100 nM) for 15 min and supernatants were collected for extracellular elastase activity assay. The supernatants were incubated with DMSO (as the control group) or testing drugs for 2 min and reacted with elastase substrate for 10 min. The change in absorbance was continually monitored at 405 nm by spectrophotometry.
Hydroethidine (HE) was employed to detect intracellular ROS production. Neutrophils were labeled with HE (10 µM) for 15 min at 37°C. HE-labeled neutrophils were treated with N11 for 5 min before adding FMLP (30 nM). The fluorescence intensity was assayed using flow cytometry.

Morphology of the products was investigated by scanning electron microscopy (SEM, Hitachi, S4800, Tokyo, Japan), and transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, Hillsboro, OR, USA). The crystallographic structure of the products was measured by an X-ray diffraction (XRD, SHIMADZU, Kyoto, Japan). The size distribution of the products was characterized by dynamic laser light scattering (DLS, Malvern, Nano ZS90, Worcestershire, UK). The composition of the products was confirmed by Fourier transform infrared spectroscopy (FTIR, SHIMADZU, Kyoto, Japan) with the KBr disk method. UV data was recorded by UV/Vis spectrophotometer (U3010, Hitachi). The confocal laser scanning microscopy (CLSM, Leica TSC SP5 confocal unit, Buffalo Grove, IL, USA) was used to the cell uptake behavior of the products.