Magnoflorine Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Suppressing NF-κB and MAPK Activation

Abstract

Acute lung injury (ALI) which is featured by a strong pulmonary inflammation, is a major cause of morbidity and mortality in critically ill patients. Magnoflorine, a quaternary alkaloid isolated from Chinese herb Magnolia or Aristolochia, has been reported to have potent anti-inflammatory properties. However, the effect of magnoflorine on lipopolysaccharide (LPS)-induced ALI in mice has not been reported. The purpose of the present study is to investigate the anti-inflammatory effect of magnoflorine on LPS-induced ALI and elucidate its possible molecular mechanisms in RAW264.7 cells. The results of histopathological changes as well as the myeloperoxidase (MPO) activity indicated that magnoflorine significantly alleviated the lung injury induced by LPS. In addition, qPCR results showed that magnoflorine dose-dependently decreased the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. Immunofluorescence assay also confirmed that the level of Toll-like receptor 4 (TLR4) induced by LPS was inhibited by magnoflorine treatment. Further experiments were performed using Western blotting to detect the expression of related proteins in the NF-κB and MAPK signaling pathways. The results showed that magnoflorine suppressed the levels of phosphorylated p65, IκBα, p38, ERK, and JNK. In conclusion, all data indicate that magnoflorine could protect against LPS-induced inflammation in ALI at least partially by inhibiting TLR4-mediated NF-κB and MAPK signaling pathways.

Keywords: ALI; LPS; MAPK; NF-κB; anti-inflammation; magnoflorine.

FIGURE 1

(A) Chemical structure of magnoflorine. (B) HPLC chromatogram of magnoflorine.

FIGURE 2

Effects of magnoflorine on LPS-induced lung injury. (A) Morphology of the lung. (B) Control group. (C) LPS group. (D–F) Magnoflorine (5, 10, and 20 mg/kg) groups. (G). MPO activity assay. CG is the control group. LPS is the LPS-stimulated group. The values are presented as means ± S.E.M. of three independent experiments. ANOVA, p < 0.0001, post hoc ^#p < 0.05 vs. control group, ^∗p < 0.05 vs. LPS group.

FIGURE 3

The effects of magnoflorine on cell viability. RAW 264.7 cells were cultured with LPS (1 μg/mL) and different concentrations of magnoflorine (25, 50, and 100 μg/mL) for 12 h, and then the cell viability was measured using the CCK-8 assay. The values are presented as means ± S.E.M. of three independent experiments.

FIGURE 4

Effects of magnoflorine on the production of cytokines. (A) The expression of TNF-α, IL-1β, and IL-6 mRNA in lung tissues were measured by qPCR. (B) The expression of TNF-α, IL-1β, and IL-6 mRNA in RAW264.7cells were measured by qPCR. GAPDH was used as a control. CG is the control group. LPS is the LPS-stimulated group. The data are presented as the mean ± S.E.M. of three independent experiments. ANOVA, p < 0.0001, post hoc ^#p < 0.05 vs. control group, ^∗p < 0.05 vs. LPS group.

FIGURE 5

Effects of magnoflorine on the expression level of TLR4 protein. Immunofluorescence staining was performed to identify the expression of TLR4 (×200), scale bar = 100 μm. Blue spots represent cell nuclei, and green spots indicate TLR4 staining. CG is the control group. LPS is the LPS-stimulated group.

FIGURE 6

The effects of magnoflorine on the NF-κB pathway activation. (A) The expression levels of IκBα and p65 proteins were analyzed using specific antibodies in lung tissues. (B) The expression levels of IκBα and p65 proteins in RAW264.7 cells. β-actin was used as the control. CG is the control group. LPS is the LPS-stimulated group. The data represent the mean ± S.E.M. ANOVA, p < 0.0001, post hoc ^#p < 0.05 vs. control group, ^∗p < 0.05 vs. LPS group.

FIGURE 7

Effects of magnoflorine on p65 translocation into the nucleus. Translocation of the p65 subunit from the cytoplasm into the nucleus was assessed by immunofluorescence staining (×200), scale bar = 100 μm. Blue spots represent cell nuclei, and red spots indicate p-p65 staining. CG is the control group. LPS is the LPS-stimulated group.

FIGURE 8

Effects of magnoflorine on the MAPK pathway activation. (A) The expression levels of p38, ERK, and JNK proteins in lung tissues. (B) The expression levels of p38, ERK, and JNK proteins in RAW264.7 cells. β-actin was used as the control. CG is the control group. LPS is the LPS-stimulated group. The data represent the mean ± S.E.M. ANOVA, p < 0.0001, post hoc ^#p < 0.05 vs. control group, ^∗p < 0.05 vs. LPS group.

FIGURE 9

NF-κB and MAPK pathways in LPS-stimulated ALI.