Galantamine protects against lipopolysaccharide-induced acute lung injury in rats

Abstract

Lipopolysaccharide (LPS)-induced endotoxemia triggers the secretion of proinflammatory cytokines and can cause acute lung injury (ALI). The high mobility group box 1 (HMGB1) protein plays an important role as a late mediator of sepsis and ALI. Galantamine (GAL) is a central acetylcholinesterase inhibitor that inhibits the expression of HMGB1. This study evaluated the effects of GAL by measuring levels of inflammatory mediators and observing histopathological features associated with LPS-induced ALI. Sixty 8-10 week old male Sprague-Dawley rats (200-240 g) were randomized into three groups as follows: control group, LPS group (7.5 mg/kg LPS), and LPS+GAL group (5 mg/kg GAL before LPS administration). Histopathological examination of lung specimens obtained 12 h after LPS administration was performed to analyze changes in wet-to-dry (W/D) weight ratio, myeloperoxidase (MPO) activity, and HMGB1 expression level. Additionally, plasma concentrations of tumor necrosis factor-α, interleukin-6, and HMGB1 were measured using an enzyme-linked immunosorbent assay at 0 (baseline), 3, 6, 9, and 12 h after LPS administration. Mortality in the three groups was recorded at 72 h. LPS-induced ALI was characterized by distortion of pulmonary architecture and elevation of MPO activity, W/D weight ratio, and levels of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, and HMGB1. Pretreatment with GAL significantly reduced the LPS-induced lung pathological changes, W/D weight ratio, levels of pro-inflammatory cytokines and MPO activity (ANOVA). Moreover, GAL treatment significantly decreased the mortality rate (ANOVA). In conclusion, we demonstrated that GAL exerted a protective effect on LPS-induced ALI in rats.

Figure 1. Histopathological changes in lung tissue samples of the three groups. Hematoxylin and eosin stain (×200 magnification). A, Control group (n=5): normal lung structure (bar 50 μm). B, LPS group (n=3): increased alveolar wall thickness, edema, bleeding, and infiltration of inflammatory cells (bar 100 μm). C, LPS+GAL group (n=5): mild structure destruction and inflammatory infiltration. (bar 100 μm). D, comparison of the pulmonary histological scores of the 3 groups. GAL: galantamine; LPS: lipopolysaccharide. ^*P<0.05, LPS group compared to control group; ^#P<0.05, LPS+GAL group compared to LPS group (ANOVA).

Figure 2. Comparison of the wet/dry weight ratio. The extent of pulmonary edema was assessed using the wet/dry ratio at 12 h after lipopolysaccharide (LPS) infusion. Control group: n=5; LPS group: n=3; LPS+galantamine (GAL) group: n=5. Data are reported as the means±SD. ^*P<0.05, LPS group compared to control group; ^#P<0.05, LPS+GAL group compared to LPS group (ANOVA).

Figure 3. Effect of galantamine (GAL) on myeloperoxidase (MPO) activity in rat lungs. Neutrophil infiltration was assessed in terms of MPO activity level at 12 h after lipopolysaccharide (LPS) administration. Control group: n=5; LPS group: n=3; LPS+GAL group: n=5. Data are reported as the means±SD. ^*P<0.05, LPS group compared to control group; ^#P<0.05, LPS+GAL group compared to LPS group (ANOVA).

Figure 4. Immunohistochemical expression of high-mobility group box 1 (HMGB1) protein in rat lungs. A, Control group: n=5 (bar 100 μm). B, lipopolysaccharide (LPS) group: n=3 (bar 50 μm). C, LPS+galantamine (GAL) group: n=5 (bar 100 μm). The arrow indicates cells that stained positive for HMGB1 expression. Representative photomicrographs of lung immunohistochemical analysis (400×) show the increased redistribution of HMGB-1 expression from the nucleus to the cytoplasm and extracellular areas in bronchial epithelial cells, alveolar epithelial cells, and inflammatory cells. D, Scatter plot of HMGB-1-positive (+) cells (%) in lung tissues. ^*P<0.05, LPS and LPS+GAL groups compared to control group; ^#P<0.05, LPS+GAL group compared to LPS group (ANOVA test).

Figure 5. Western blot analysis of high-mobility group box 1 (HMGB1) levels in rat lung tissue at 12 h. A, The concentrations of HMGB1 and β-actin in lung tissue were determined by Western blot analysis at 12 h. Results of a representative experiment are shown. B, Galantamine (GAL) down-regulated the lipopolysaccharide (LPS)-induced elevation of HMGB1 expression. The results show the HMGB1/β-actin ratio obtained from the Western blots. Control group: n=5; LPS group: n=3; LPS+GAL group: n=5. Data are reported as the means±SE. ^*P<0.05, LPS group compared to control group; ^#P<0.05, LPS+GAL group compared to LPS group (ANOVA).

Figure 6. Changes in the levels of pro-inflammatory cytokines: A, tumor necrosis factor (TNF)-α, B, interleukin (IL)-6, and C, high-mobility group box 1 (HMGB1). Control group: n=5 for each time point; LPS group: n=5 (3 and 6 h), n=4 (9 h) and n=3 (12 h); LPS+GAL group: n=5 for each time point. GAL: galantamine; LPS: lipopolysaccharide. Data are reported as the means±SE. ^*P<0.05, LPS and LPS+GAL groups compared to control group; ^#P<0.05, LPS group compared to LPS+GAL group (ANOVA).