Protective Effects of Kirenol against Lipopolysaccharide-Induced Acute Lung Injury through the Modulation of the Proinflammatory NFκB Pathway and the AMPK2-/Nrf2-Mediated HO-1/AOE Pathway

Abstract

Acute lung injury (ALI) is an acute and life-threatening inflammatory disease of the lung parenchyma that is associated with high mortality worldwide. No therapeutic strategies have been developed for the mitigation of the proinflammatory response that characterizes ALI. Kirenol has anti-inflammatory, antiarthritic, and immunoregulatory effects. In the present study, we investigated the protective effects of kirenol against lipopolysaccharides (LPS)-induced ALI in mice. Kirenol reduced the LPS-induced histopathology changes involving edema and thickening of the interstitial or alveolar walls, infiltration of leukocytes, formation of hyaline membrane. Pretreatment with kirenol reduced leukocytes infiltration in bronchoalveolar lavage fluid (BALF), the alveolar-capillary barrier disruption and lipid peroxidation in lung tissues induced by LPS. Kirenol significantly inhibited the secretion of cytokines, IL-1β, IL6, and TNFα, into the BALF of the mice with LPS-induced ALI through NFκB activation. Moreover, kirenol attenuated the downregulation of the antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and catalase that was induced by LPS. HO-1 expression and the phosphorylation of Nrf2 and AMPK2 were also induced by kirenol. The results indicate that kirenol can be developed as a treatment strategy for ALI, and its effects are induced through the inhibition of the NF-κB proinflammatory pathway and promotion of AMPK2/Nrf2-mediated HO-1 and antioxidant enzymes (AOE) activation.

Keywords: AMPK2/Nrf2-mediated HO-1; AOE pathway; NF-κB pathway; acute lung injury; kirenol; lipopolysaccharide.

Figure 1

Kirenol protected against histopathological changes in lung tissues in lipopolysaccharide (LPS)-induced Acute lung injury (ALI) mice. (A) Control group also named Group I; (B) LPS group also named Group II; (C) 30 mg/kg kirenol + LPS group also named Group III; (D) 50 mg/kg kirenol + LPS group also named Group IV; (E) 100 mg/kg kirenol + LPS group also named Group V; (F) 1 mg/kg dexamethasone + LPS group also named Group VI. Histopathological changes were observed using Hematoxylin-eosin staining of lung sections under light microscopy. The magnification of the enlargements are 100 times. The scale bars represent 100 μm. Green arrow indicates neutrophil infiltration; black arrow indicates hyaline membrane formation; orange arrow indicates alveolar wall thickness. (G) The histologic scores are presented for the lung tissues. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 2

Kirenol protected against LPS-induced alveolar-capillary barrier disruption. The contents of protein in bronchoalveolar lavage fluid (BALF) were measured by Bradford protein analysis. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 3

Kirenol protected against leukocyte infiltration and lipid peroxidation. (A) Leukocytes infiltration was determined by cell counter assay activity in BALF. (B) Lipid peroxidation was determined by thiobarbituric acid (TBA) reactive substance assay in the lungs. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 4

Kirenol protected against the phosphorylation of NF-κB p65 and degradation of IκB induced by LPS. The levels of NF-κB p65 phosphorylation and IκB degradation in the lung tissues were analyzed by Western blotting analysis. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 5

Kirenol protected against LPS-induced generation of proinflammatory cytokines in BALF. Proinflammatory cytokines, including TNFα, IL-1β, and IL-6, were determined by the ELISA assay. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 6

Kirenol attenuated the LPS-reduced the activities of superoxide dismutase (SOD), catalase, GPx. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 7

Kirenol enhanced the phosphorylation of Nrf2 and expression of HO-1 induced by LPS. The levels of Nrf2 phosphorylation and HO-1 expression in the lung tissues were analyzed by Western blotting analysis. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with the Group II (p < 0.05).

Figure 8

Kirenol attenuated the LPS-reduced the phosphorylation of AMPK2. The levels of AMPK2 phosphorylation in the lung tissues were analyzed by Western blotting analysis. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group I (p < 0.05); * represents the significant difference compared with Group II (p < 0.05).

Figure 9

Kirenol suppressed the leukocyte infiltration and alveolar-capillary barrier disruption. (A) Leukocyte infiltration was determined by cell counter assay activity in BALF. (B) The contents of protein in BALF were measured by Bradford protein analysis. Values are shown the mean ± S.D. of 3–4 mice per group. ^# represents the significant difference compared with the Group A (p < 0.05); * represents the significant difference compared with Group B (p < 0.05).

Figure 10

Scheme of the mechanisms in the protective effect of kirenol on LPS-induced ALI.