Elastase, a serine proteinase released by activated human neutrophils, can degrade a wide variety of biomacromolecules including elastin, and is considered a marker of inflammatory diseases. As the logical strategy to protect tissue is to inhibit excessive elastase activity, experimental and clinical researches have concentrated on trying to find efficient elastase inhibitors. As thymol, one of the major components of thyme oil with a phenolic structure, has been credited with a series of pharmacological properties, that include antimicrobial and antioxidant effects, the aim of this study was to explore whether it can also interfere with the release of elastase by human neutrophils stimulated with the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). After the neutrophils were incubated with increasing amounts of thymol (2.5, 5, 10, 20 µg/ml), elastase release was initiated by fMLP and measured using MeO-Suc-Ala-Ala-Pro-Val-MCA. The results showed that thymol inhibited fMLP-induced elastase release in a concentration-dependent manner, with the effects of 10 and 20 µg/ml being statistically significant. The behavior of cytosolic calcium mobilization revealed by fura-2 closely resembled that of elastase, thus suggesting that they may be related. The hydrophobic nature of thymol means that it can approach ion channel proteins through the lipid phase of the membrane, alter the local environment of calcium channels and thus inhibit capacitative calcium entry. In brief, thymol inactivates calcium channels machinery, thus triggering a corresponding reduction in elastase. The antibacterial and antimycotic activity of thymol is already well known, but our findings that it inhibits elastase extend our knowledge of the anti-inflammatory activity of this interesting molecule that is already credited with antioxidant activity. These two latter characteristics make thymol a molecule that can have helpful effects in controlling the inflammatory processes present in many infections.

Activated neutrophils can release superoxide anion and nitric oxide (NO), which subsequently combine with each other to yield peroxynitrite anions, powerful and harmful oxidants that preferentially mediate the oxidation of the thiol groups in proteins and non-protein molecules. These oxidants play a direct role in the inflammatory process in chronic obstructive pulmonary disease and asthma by increasing the number of neutrophils and macrophages that induce a self-sustaining phlogogenic loop. Budesonide (BUD) and erdosteine (a muco-active drug which, after metabolization, produces an active metabolite (Met I) with a sulfhydryl group) are both active in reducing the release of superoxide anion, NO and peroxynitrite, and can be administered to patients with respiratory diseases. The aim of this study was to investigate the possible synergistic in vitro effect of BUD and Met I on chemiluminescence generation during fMLP-stimulated respiratory bursts of human neutrophils with the NO donor L-arginine, added to the incubating medium. The investigated BUD concentrations ranged from 6 × 10^–8 to 1 × 10^–6 mol/l in logarithmic scale and a significant and progressive reduction in luminol-amplified chemiluminescence (LACL) was observed at concentrations ranging from 2.5 × 10^–7 to 1 × 10^–6 mol/l. The investigated concentrations of Met I varied from 0.62 to 10 µg/ml. No significant changes were observed at 0.62, 1.25, and 2.5 µg/ml, but a significant decrease in LACL was observed at 5 and 10 µg/ml. When the two drugs were combined, there was a greater significant decrease in LACL versus the single drugs with the combinations of BUD 1 × 10^–6 mol/l plus Met I 10 µg/ml, BUD 5 × 10^–7 mol/l plus Met I 5 µg/ml, BUD 2.5 × 10^–7 mol/l plus Met I 2.5 µg/ml, and BUD 1.25 × 10^–7 mol/l plus Met I 1.25 µg/ml. A further interesting finding was that the combination of BUD 2.5 × 10^–7 mol/l plus Met I 2.5 µg/ml and BUD 1.25 × 10^–7 mol/l plus Met I 1.25 µg/ml significantly decreased LACL, whereas the single concentrations had no significant effect, thus indicating the possibility of extending the duration of the effect. Our findings indicate a synergistic antioxidant effect when BUD and Met I are given together, which is of interest for counteracting the airway phlogosis involved in many respiratory diseases.

Previous claims concerning the activity of sulphurous water have been based on the patients' subjective sense of wellbeing and on symptomatic (or general) clinical improvements that are not easy to define or quantify exactly. Our findings indicate that, in addition to its known mucolytic and antioxidant activity, sulphurous water also has an anti-elastase activity that may help to control the inflammatory processes of upper and lower airway diseases.

Polymorphonuclear neutrophils (PMNs) can generate superoxide anions and nitric oxide (NO), which is not only an important mediator of various cellular activities, but can also react with superoxide anions to produce peroxynitrite anions (ONOO^–). Peroxynitrite is a potent and potentially toxic oxidant that damages various types of biomolecules. It preferentially mediates the oxidation of thiolic groups in protein and non-protein molecules, thus altering their functions. The aim of this study was to examine whether, in addition to its ability to reduce the respiratory bursts of human PMNs, the SH metabolite I (Met I) of erdosteine, can interfere with NO and NO-derived peroxynitrite production, thus extending its antioxidant activity. This was done by means of the luminol amplified chemiluminescence (LACL), which has been widely used to detect the production of reactive oxidant species (ROS) by PMNs under various conditions. At 5 and 10 µg/ml, Met I significantly reduced LACL after fMLP and PMA stimulation. When L-Arg was added to the reaction medium, as a NO donor, the chemiluminescence of fMLP increased by up to 67% and that of PMA by up to 132%, but was once again significantly reduced by 5 and 10 µg/ml of Met I. In a cell-free system, the use of linsidomine (SIN-1) makes it possible to investigate the behavior of LACL induced by peroxynitrite release, which was significantly reduced by Met I concentrations ranging from 1.25 to 10 µg/ml. Our findings indicate that Met I, a molecule with a SH group, reacts with ROS, NO and NO-derived peroxynitrite, and has both antioxidant and scavenging activity. This is of interest for the strategy of protecting against damage induced by radical species in the pulmonary cell environment, in which they can induce a phlogogenic loop, and suggests that adding exogenous thiols may be useful in antagonizing the toxic effects of reactive molecules on endogenous thiols.

Many lung disorders are characterized by airway inflammation involving the recruitment of inflammatory cells, and leading to the release of oxidant and inflammatory mediators. The overproduction of superoxide anion (O₂^–DELETE) and nitric oxide (NO) during the respiratory bursts of neutrophils leads to production of peroxynitrite, a highly damaging oxidant with an important role in the inflammatory loop causing airway hyper-reactivity in respiratory diseases like asthma. The aim of this study was to investigate in vitro the effects of a 1-hour incubation with budesonide at 2.5 × 10^–7, 5 × 10^–7, 1 × 10^–6, 2 × 10^–6 and 4 × 10^–6 mol/l on O₂^–DELETE, NO, and peroxynitrite production during the respiratory burst of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10^–7 mol/l) or phorbol 12-myristate 13-acetate (PMA, 2 × 10^–6 mol/l), as documented by luminol-amplified chemiluminescence (LACL). In absence of L-arginine, budesonide (5 × 10^–7 to 4 × 10^–6 mol/l) dose-dependently reduced both fMLP- and PMA-induced LACL (18.3–50.6%). In the presence of L-arginine (100 µg/ml), a NO donor increasing peroxynitrite production, LACL increased 3–5 times compared with baseline, but budesonide dose-dependently reduced LACL (25.5–59.6%). Mifepristone (4 × 10^–6 mol/l), a glucocorticoid receptor antagonist, inhibited the effect of budesonide on LACL, thus confirming that budesonide reacts with glucocorticoid receptors to exert an antioxidant activity. These results suggest that budesonide target rapidly human neutrophils leading to a fast reduction in both NO and peroxynitrite production, and are consistent with decrease in exhaled NO levels after treatment with budesonide in patients with asthma.