Neutrophils involvements in COPD: Effects of essential oil extracted from Nigella sativa (L.) seeds on human neutrophil functions and elastase activity

Abstract

Chronic Obstructive Pulmonary Disease ( COPD ) is a leading cause of death and disability, but has only recently been extensively explored from a cellular and molecular perspective. Emphysema is a chronic inflammation that leads to fixed narrowing of small airways and alveolar wall destruction, characterised by increased number of alveolar inflammatory cells mainly neutrophils (Noguera and Miralles 2001; Baraldo and Badin 2003; Vachier et al., 2004), this leads to increased elastolysis that appears as a result of proteases secretion mainly Neutrophil Elastase (NE) , (Sommerhof et al., 2000; Lindén et al., 2005) . This study is a contribution aimed mainly to understand COPD at cellular and molecular levels and searching for novel natural drugs for treatment of patients . COPD symptoms, patients lung function status, FEV1, FVC and FEV1 /FVC are used to realize the first part of this research work. α1-AT deficiency in plasma was evaluated only in selected patients with severe COPD symptoms and showed lung injuries. Essential oil was extracted from Nigella sativa (L.) seeds by hydrodistillation. Gas chromatography / Mass spectrometry analysis revealed the detection of 22 compounds most are monoterpens. Human Neutrophils (HNs) are isolated on percoll gradients, counted and tested for viability using trypan blue exclusion method. Chemotactic response is based on multiple blind well assay system. Microassays carried out to evaluate the inhibitory effects of essential oil and its major components on HNE activity , determination of HNE activity is realized using; N-Methoxy-succinyl-(Ala)3-Pro-Val-p-Nitro-Anilide as HNE substrate. Strong correlation between smoking and air pollution with respiratory symptoms is observed. Patients suffering from severe COPD (stage III) with some lung injuries demonstrate α1-AT deficiency compared to mild COPD patients. The development of COPD to emphysema and the deterioration of lung function are found in smokers patients. The control movement and chemotactic response of HNs to 0.1 µM fMLP is reduced at a dose-dependent manner. Essential oil significantly inhibit neutrophil chemotaxis from 0.05 to 0.5 mg/mL. IC50 values showing inhibition to induced neutrophil chemotaxis and control movement are 0.08 and 0.07 mg/mL respectively. Human Neutrophil Elastase (HNE) secretion is inhibited by essential oil at a concentration dependent manner from 0.5 to 2.5 mg/mL. Components of essential oil are potent inhibitors for PMNs functions. The observed inhibition of neutrophils functions occur via intracellular pathway. Active serine protease could be essential for neutrophils responding process and/or signal transduction pathways. Inhibition of HNE activity by essential oil is found to be dose dependant. Highest Inhibitory Concentration (HIC) of essential oil caused total inhibition to HNE activity is 5.8 mg/ml. Results of this study reveal that Carvacrol (5-Isopropyl-2-methyl phenol) is the principal inhibitor against elastolytic activity with a very low IC50 (12 µM).