Preventive Effect of Garlic Oil and Its Organosulfur Component Diallyl-Disulfide on Cigarette Smoke-Induced Airway Inflammation in Mice

Abstract

Garlic (Allium sativum) has traditionally been used as a medicinal food and exhibits various beneficial activities, such as antitumor, antimicrobial, hypolipidemic, antiarthritic, and hypoglycemic activities. The aim of this study was to explore the preventive effect of garlic oil (GO) and its organosulfur component diallyl disulfide (DADS) on cigarette smoke (CS)-induced airway inflammation. Mice were exposed to CS daily for 1 h (equivalent to eight cigarettes per day) for two weeks, and intranasally instilled with lipopolysaccharide (LPS) on day 12 after the initiation of CS exposure. GO and DADS were administered to mice by oral gavage, both at rates of 20 and 40 mg/kg, for 1 h before CS exposure for two weeks. In the bronchoalveolar lavage fluid, GO and DADS inhibited the elevation in the counts of inflammatory cells, particularly neutrophils, which were induced in the CS and LPS (CS + LPS) group. This was accompanied by the lowered production (relative to the CS + LPS group) of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. Histologically, GO and DADS inhibited the CS- and LPS-induced infiltration of inflammatory cells into lung tissues. Additionally, GO and DADS inhibited the phosphorylation of extracellular signal-regulated kinase and the expression of matrix metalloproteinase-9 in the lung tissues. Taken together, these findings indicate that GO and DADS could be a potential preventive agent in CS-induced airway inflammation.

Keywords: airway inflammation; cigarette smoke; diallyl disulfide; extracellular signal-regulated kinase; garlic oil; matrix metalloproteinase-9.

Figure 1

Garlic oil (GO) and diallyl disulfide (DADS) inhibited the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) of the mice that were exposed to cigarette smoke (CS) and lipopolysaccharide (LPS). Inflammatory cell counts were determined by counting the cells in the BALF of all the animals (n = 6 per group) in five squares at 400x magnification under a microscope. NC: normal control; CS + LPS: mice exposed to CS and LPS; ROF: roflumilast (10 mg/kg) administered to mice exposed to CS and LPS; GO-20 and GO-40: GO (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS; DADS-20 and DADS-40: DADS (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS. Values are expressed as means ± standard deviation (SD) (n = 6). Significantly different from NC: ^## (p < 0.01); significantly different from CS + LPS: *, ** (p < 0.05 and < 0.01, respectively).

Figure 2

GO and DADS inhibited levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the BALF of mice that were exposed to CS and LPS. (A) Level of IL-1 β; (B) Level of IL-6; (C) Level of TNF-α. Levels of IL-6, IL-1β, and TNF-α were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. NC: normal control; CS + LPS: mice exposed to CS and LPS; ROF: ROF (10 mg/kg) administered to mice exposed to CS and LPS; GO-20 and GO-40: GO (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS; DADS-20 and DADS-40: DADS (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS. Values are expressed as means ± SD (n = 6). Significantly different from NC: ^## (p < 0.01); significantly different from CS + LPS: *, ** (p < 0.05 and < 0.01, respectively).

Figure 3

GO and DADS inhibited the accumulation of inflammatory cells in the lung tissues. A representative figure of a peribronchial lesion in lung tissue stained with hematoxylin and eosin (H&E) solution (100× magnification). Scale bar = 50 µm. Quantitative analysis of inflammatory response was performed using IMT i-Solution software (IMT i-Solution Inc.). (A) Representative figure of lung tissue; (B) quantitative analysis of inflammation. NC: normal control; CS + LPS: mice exposed to CS and LPS; ROF: ROF (10 mg/kg) administered to mice exposed to CS and LPS; GO-20 and GO-40: GO (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS; DADS-20 and DADS-40: DADS (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS. Values are expressed as means ± SD (n = 6). Significantly different from NC: ^## (p < 0.01); significantly different from CS + LPS: ** (p <0.01).

Figure 4

GO and DADS inhibited extracellular signal-regulated kinase (ERK) phosphorylation and matrix metalloproteinase (MMP)-9 expression, which were induced in the CS + LPS group. Protein expression was analyzed by western blot and the relative ratios of protein expression were evaluated. Densitometric values of protein expression were determined using the Chemi-Doc imaging system (Bio-Rad Laboratories). (A) Gel images showing protein expression; (B) densitometric values of protein expression. NC: normal control; CS + LPS: mice exposed to CS and LPS; ROF: ROF (10 mg/kg) administered to mice exposed to CS and LPS; GO-20 and GO-40: GO (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS; DADS-20 and DADS-40: DADS (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS. Values are expressed as means ± SD (n = 6). Significantly different from NC: ^## (p < 0.01); significantly different from CS + LPS: ** (p < 0.01).

Figure 5

GO and DADS inhibited MMP-9 expression in the lung tissues of the mice, which was induced in the CS + LPS group. Protein expression was analyzed by immunohistochemistry, and values of protein expression were determined using IMT i-Solution software (IMT i-Solution Inc.). Scale bar = 50 µm. (A) Representative figure showing protein expression in lung tissue; (B) value of MMP-9 expression. NC: normal control; CS + LPS: mice exposed to CS and LPS; ROF: ROF (10 mg/kg) administered to mice exposed to CS and LPS; GO-20 and GO-40: GO (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS; DADS-20 and DADS-40: DADS (20 and 40 mg/kg, respectively) administered to mice exposed to CS and LPS. Values are expressed as means ± SD (n = 6). Significantly different from NC: ^## (p < 0.01); significantly different from CS + LPS: *, ** (p < 0.05 and < 0.01, respectively).