Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice

Abstract

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages. Here we evaluated matrine's suppressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide- (LPS-) stimulated human lung epithelial A549 cells. Additionally, BALB/c mice were given various matrine doses by intraperitoneal injection, and then lung injury was induced via intratracheal instillation of LPS. In LPS-stimulated A549 cells, matrine inhibited the productions of interleukin-8 (IL-8), monocyte chemotactic protein-1, and IL-6 and decreased COX-2 expression. Matrine treatment also decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells. In vitro results demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nuclear transcription factor kappa-B subunit p65 protein translocation into the nucleus. In vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-α and IL-6 in mouse bronchoalveolar lavage fluid and serum. Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines, chemokines, COX-2, and ICAM-1. Our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells.

Figure 1

Matrine affects cytokine and chemokine productions in LPS-stimulated A549 cells. ELISA was used to measure the levels of IL-6 (a), IL-8 (b), CCL5 (c), and MCP-1 (d). All data are presented as mean ± SEM. ^∗ P < 0.05 compared with the LPS control group. ^∗∗ P < 0.01 compared with the LPS control group.

Figure 2

Matrine affects the LPS-induced productions of COX and ICAM-1 in A549 cells. COX-2 and ICAM-1 protein levels were detected using Western blots with β-actin expression as an internal control (a), and ICAM-1 levels were measured by ELISA (b). (c) Treatment with 100–400 μM matrine (M) inhibited the adherence of neutrophil-like HL-60 cells to active A549 cells. Data are presented as the mean ± SEM. ^∗ P < 0.05 compared to LPS alone.

Figure 3

Matrine inhibits the nuclear translocation of NF-κB in A549 cells. (a) Effects of matrine on the LPS-induced phosphorylation of IκB-α, with total IκB-α levels used as internal controls. (b) To assess the nuclear translocation of NF-κB, cells were pretreated with different matrine concentrations for 1 h and then incubated with LPS (1 μg/mL) for 1 h. The internal controls were lamin B1 in the nucleus and β-actin in the cytosol. (c) Matrine suppressed luciferase activity by the NF-κB promoter assay. Three independent experiments were analyzed and compared with the LPS-treated control group. Data are presented as the mean ± SEM. ^∗ P < 0.05 compared to LPS alone.

Figure 4

Matrine affects LPS-induced phosphorylation of MAPK pathway molecules. Western blots showed the effects of the indicated matrine concentrations on the phosphorylation of ERK (top two rows), p38 (middle two rows), and JNK (bottom two rows). Three independent experiments were analyzed and compared with the LPS-treated group.

Figure 5

Matrine inhibits neutrophil infiltration. Hematoxylin and eosin staining was used for histological examination of airway inflammation in lung tissue (magnification, 400x). N group: normal control mice; LPS group: LPS treatment only; M10 group: 10 mg/kg matrine plus LPS; M20 group: 20 mg/kg matrine plus LPS.

Figure 6

Matrine affects neutrophil counts and cytokine and chemokine levels in BALF. In the different treatment groups, we analyzed neutrophil cell counts (a), and the levels of IL-6 (b) and TNF-α (c) were measured by ELISA. N group: normal control mice; LPS group: LPS treatment only; M10 group: 10 mg/kg matrine plus LPS; M20 group: 20 mg/kg matrine plus LPS. All data are presented as mean ± SEM. ^∗ P < 0.05 compared with the LPS control group. ^∗∗ P < 0.01 compared with the LPS control group.

Figure 7

Matrine affects the expressions of cytokines, chemokines, and inflammatory mediators in the lung. Gene expressions were determined using real-time RT-PCR. Fold expression levels were measured relative to β-actin expression (internal control). Data are presented as mean ± SEM. ^∗ P < 0.05 compared with LPS control mice. ^∗∗ P < 0.01 compared with LPS control mice.

Figure 8

Matrine affects the LPS-induced nuclear phosphorylation of NF-κB and phosphorylation of MAPK pathway molecules. Phosphorylation in lung tissue was analyzed by Western blots. Three independent experiments were analyzed and results were compared with the LPS-treated group.

Figure 9

Matrine affects the serum levels of inflammatory cytokines, including IL-6 (a) and TNF-α (b). All data are presented as means ± SEM. ^∗ P < 0.05 compared with the LPS control group.