The protective effect of alpha-lipoic Acid in lipopolysaccharide-induced acute lung injury is mediated by heme oxygenase-1

Abstract

Alpha-lipoic acid (ALA), occurring naturally in human food, is known to possess antioxidative and anti-inflammatory activities. Induction of heme oxygenase-1 (HO-1) has been reported to exhibit a therapeutic effect in several inflammatory diseases. The aim of study was to test the hypothesis that the protection of ALA against lipopolysaccharide-(LPS-) induced acute lung injury (ALI) is mediated by HO-1. Pre- or posttreatment with ALA significantly inhibited LPS-induced histological alterations of ALI, lung tissue edema, and production of proinflammatory cytokine, cytokine inducible neutrophil chemoattractant-3, and nitrite/nitrate in bronchoalveolar lavage fluid. In addition, the inflammatory responses including elevation of superoxide formation, myeloperoxidase activity, polymorphonuclear neutrophils infiltration, nitrotyrosine, inducible nitric oxide synthase expression and nuclear factor-kappa B (NF- κ B) activation in lung tissues of LPS-instilled rats were also markedly reduced by ALA. Interestingly, treatment with ALA significantly increased nuclear factor-erythroid 2-related factor 2 (Nrf2) activation and HO-1 expression in lungs of ALI. However, blocking HO-1 activity by tin protoporphyrin IX (SnPP), an HO-1 inhibitor, markedly abolished these beneficial effects of ALA in LPS-induced ALI. These results suggest that the protection mechanism of ALA may be through HO-1 induction and in turn suppressing NF- κ B-mediated inflammatory responses.

Figure 1

The structure of ALA.

Figure 2

Effect of ALA on HO-1 expression and Nrf2 translocation in lungs instilled by LPS. Rats were treated with ALA (100 mg/kg, i.p.) 1 h before or 3 h after intratracheal instillation of LPS (5 mg/kg) for 6 h. Lungs were harvested for HO-1 protein expression by Western blot (a), and Nrf2 translocation assays with immunofluorescence staining (b). Rats received with vehicle (i.p.) and intratracheal instillation of PBS alone acted as control group. Data were represented as means ± SEM (n = 5). *P < 0.05; **P < 0.01 versus LPS-instilled alone group.

Figure 3

Effect of SnPP on the inhibition of ALA on LPS-induced neutrophil accumulation in BALF and lung MPO activity. Rats were intraperitoneally injected with ALA 1 h before or 3 h after intratracheal instillation of LPS (5 mg/kg). To investigate the role of HO-1, SnPP (50 mg/kg, i.p.) was administered 2 h and ALA 1 h before LPS instillation. The total leukocyte counts in BALF (a) and lung MPO activity (b) were measured at 6 h after LPS instillation. Data were presented as means ± SEM (n = 5). *P < 0.05; **P < 0.01 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 4

Effects of SnPP on the attenuation of ALA on LPS-induced protein accumulation in BALF and lung wet/dry weight ratio. Rats were intraperitoneally injected with ALA 1 h before or 3 h after intratracheal instillation of LPS (5 mg/kg). In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. The protein level in BALF (a) and pulmonary edema determined by the wet/dry weight ratio (b) were measured at 6 h after LPS instillation. Data were presented as means ± SEM (n = 5). *P < 0.05; **P < 0.01 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 5

Effect of SnPP on the improvement of ALA on LPS-induced lung histopathological changes. Rats were intraperitoneally injected with ALA 1 h before or 3 h after intratracheal instillation of LPS. In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. The histopathological assays (200× magnification) in lungs were performed at 6 h after LPS instillation in control group (a), LPS-instilled alone group (b), ALA (posttreatment)+LPS group (c), ALA (pretreatment)+LPS group (d), SnPP+ALA (pretreatment)+LPS group (e). The lung injury score was also determined (f). Data were represented as means ± SEM (n = 5). *P < 0.05 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 6

Effect of SnPP on the inhibition of ALA on LPS-induced proinflammatory cytokine and CINC-3 production in BALF. Rats were pretreated with ALA 1 h before intratracheal instillation of LPS for 6 h. In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. Data were presented as means ± SEM (n = 5). *P < 0.05; **P < 0.01 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 7

Effect of SnPP on the suppression of ALA on LPS-induced O₂ ⁻ formation in lungs. Rats were pretreated with ALA 1 h before intratracheal instillation of LPS for 6 h. In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. The lung O₂ ⁻ formation was measured by lucigenin chemiluminescence (a) and DHE immunofluorescent staining (red) (b). Data were represented as means ± SEM (n = 5). *P < 0.05 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 8

Effects of SnPP on the inhibition of ALA on LPS-induced NO_x level in BALF, iNOS and nitrotyrosine expression in lung tissues. Rats were pretreated with ALA 1 h before intratracheal instillation of LPS for 6 h. In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. The NO_x level in BALF (a), iNOS (b) and nitrotyrosine expression (c) in lungs were measured. Data were represented as means ± SEM (n = 5). *P < 0.05 versus LPS-instilled alone group; ^# P < 0.05 versus LPS+ALA group.

Figure 9

Effect of SnPP on the reduction of ALA on LPS-induced NF-κB activation in lungs. Rats were pretreated with ALA 1 h before intratracheal instillation of LPS for 6 h. In some rats, SnPP was administered 2 h and ALA 1 h before LPS instillation. The expression of phospho-NFκB-p65 in nucleus of lungs with immunofluorescence stains was determined (a) and semiquantified (b) at 6 h after LPS instillation. Data were represented as means ± SEM (n = 5). *P < 0.05 versus LPS-instilled alone group.