Allyl methyl trisulfide protected against LPS-induced acute lung injury in mice via inhibition of the NF-κB and MAPK pathways

Abstract

Allyl methyl trisulfide (AMTS) is one major lipid-soluble organosulfur compound of garlic. Previous studies have reported the potential therapeutic effect of garlic on acute lung injury (ALI) or its severe condition acute respiratory distress syndrome (ARDS), but the specific substances that exert the regulatory effects are still unclear. In this study, we investigate the protective effects of AMTS on lipopolysaccharide (LPS)-induced ALI mice and explored the underlying mechanisms. In vivo experiments, ICR mice were pretreated with 25-100 mg/kg AMTS for 7 days and followed by intratracheal instillation of LPS (1.5 mg/kg). The results showed that AMTS significantly attenuated LPS-induced deterioration of lung pathology, demonstrated by ameliorative edema and protein leakage, and improved pulmonary histopathological morphology. Meanwhile, the expression of inflammatory mediators and the infiltration of inflammation-regulation cells induced by LPS were also inhibited. In vitro experiments also revealed that AMTS could alleviate inflammation response and inhibit the exaggeration of macrophage M1 polarization in LPS-induced RAW264.7 cells. Mechanistically, we identified that AMTS treatment could attenuate the LPS-induced elevation of protein expression of p-IκBα, nuclear NF-κB-p65, COX2, iNOS, p-P38, p-ERK1/2, and p-JNK. Collectively, these data suggest that AMTS could attenuate LPS-induced ALI and the molecular mechanisms should be related to the suppression of the NF-κB and MAPKs pathways.

Keywords: NF-κB; acute lung injury; allyl methyl trisulfide; inflammatory mediators; mapks.

FIGURE 1

AMTS pretreatment attenuated LPS-induced ALI in mice. (A) Wet lung weight is normalized by body weight. Protein concentration (B) and total cells (C) in BALF. (D) Representative H and E staining (100×) and lung injury score for histological examination. Data were shown as mean ± S. D (n = 6). *p < 0.05, **p < 0.01, compared with the control group; ^# p < 0.05, ^## p < 0.01, compared with the LPS group.

FIGURE 2

Effects of AMTS on inflammatory mediators. (A)TNF-α (B) IL-1β, (C) IL-6 in BALF. (D) NO in lung tissue. Data were presented as mean ± S. D (n = 6). *p < 0.05, **p < 0.01, compared with the control group; ^# p < 0.05, ^## p < 0.01, compared with the LPS group.

FIGURE 3

Effects of AMTS on the infiltration of inflammation-regulation cells. (A) The activity of MPO was determined to indicate the infiltration of neutrophils. (B) AMTS pretreatment reduced MCP-1, an inflammatory cell recruitment chemokine, which was measured by ELISA. (C) Representative pictures of immune stain for macrophage using F4/80 and CD11 b antibody. The green arrows indicate the infiltration of inflammatory cells. (D)The percentage of positive staining area to the total area for F4/80 and CD68 in lung tissues. *p < 0.05, **p < 0.01, compared with the control group; ^# p < 0.05, ^## p < 0.01, compared with the LPS group.

FIGURE 4

AMTS treatment inhibited the activation of the NF-κB pathway. (A) Representative western blot bands for IκBα, p-IκBα, cytoplasm and nuclear NF-κB p65; (B) quantitative data analyses of the phosphorylation levels of IκB; (C) the expression of cytoplasm and nuclear NFκB p65; (D) the expression of COX2 and iNOS. Data were presented as mean ± SD from at least three independent experiments and expressed as the percentage of the control. *p < 0.05, **p < 0.01, compared with control group; ^# p < 0.05, ^## p < 0.01, compared with LPS group.

FIGURE 5

AMTS treatment inhibited the phosphorylation of Erk1/2, SAPK/NK, and p38MAPK. (A) Representative western blot bands for phospho-Erk1/2, Erk1/2, phospho-SAPK/JNK, SAPK/JNK, phospho-p38MAPK, and p38MAPK; (B) Quantitative data analyses. Data were presented as mean ± SD from at least three independent experiments and expressed as the percentage of the control. *p < 0.05, **p < 0.01, compared with control group; ^# p < 0.05, ^## p < 0.01, compared with LPS group.

FIGURE 6

Effects of AMTS on LPS-induced inflammatory response in RAW264.7 cells. (A) The cytotoxicity of AMTS was assessed by CCK8 assay. The levels of NO (B) and TNF-α (C) in the culture medium were detected by ELISA. (D) Protein levels of the NF-κB pathway and the MAPKs pathway-related proteins were detected by western blotting. (E) Macrophage M1 polarization (F4/80⁺iNOS⁺) was detected by double immunofluorescence. *p < 0.05, **p < 0.01, compared with untreated cells (Control); ^# p < 0.05, ^## p < 0.01, compared with the cells challenged with LPS (LPS).