Silybin attenuates LPS-induced lung injury in mice by inhibiting NF-κB signaling and NLRP3 activation

Abstract

Silybin is one of the main flavonoids produced by milk thistle, which has been used in the treatment of liver diseases. In this study, we examined the protective effects and possible mechanisms of action of silybin in lipopolysaccharide (LPS)‑induced lung injury and inflammation. Pre-treatment of mice with silybin significantly inhibited LPS-induced airway inflammatory cell recruitment, including macrophages, T cells and neutrophils. The production of cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in bronchoalveolar fluid and serum was also decreased following treatment with silybin. Elevated cytokine mRNA levels induced by LPS in lung tissue were all suppressed by silybin and lung histological alterations were also improved. In addition, experiments using cells indicated that silybin significantly decreased the mRNA levels and secretion of IL-1β and TNF-α in THP-1 cells. Moreover, the mechanisms responsible for these effects were attributed to the inhibitory effect of silybin on nuclear factor-κB (NF-κB) signaling and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. The data form our study thus support the utility of silybin as a potential medicine for the treatment of acute lung injury‑associated inflammation and pathological changes. Silybin exerts protective effects against lung injury by regulating NF-κB signaling and the NLRP3 inflammasome activation.

Figure 1

Silybin treatment ameliorates lipopolysaccharide (LPS)-induced acute lung injury in mice. Mice were orally administered silybin (50 or 100 mg/kg) once per day for 3 consecutive days prior to LPS sensitization as described in the Materials and methods. (A) Lung tissue were fixed in 4% formalin and subjected to hematoxylin and eosin (H&E) staining. (B) Bronchoalveolar lavage fluid (BALF) from each group was collected and the number of cells in BALF was determined. The cells were then stained with CD3-FITC, CD11b-PE, Gr1-APC and analyzed by FACS. Values were shown as the means ± SD of 8 mice. ^*P<0.05 and ^**P<0.01 vs. mice exposed to LPS alone.

Figure 2

Silybin treatment prevents lipopolysaccharide (LPS)-induced lung inflammation in mice. (A) Cytokine levels in BALF supernatant and serum from each group of mice were detected by enzyme-linked immunosorbent assay (ELISA). Values were shown as the means ± SD of 8 mice. (B) RNA was extracted from the lung tissue of the mice. (C) The mRNA expression of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β and IL-17 was examined by RT-qPCR. Values were shown as the means ± SD of 8 mice. ^*P<0.05 and ^**P<0.01 vs. mice exposed to LPS alone.

Figure 3

Silybin treatment inhibits the phosphorylation of nuclear factor-κB (NF-κB). (A) Lung sections from each group were subjected to immunohistochemical staining for p-NF-κB (p-p65). Representative DAB-stained tissue specimens from each group are shown (×200 magnification). (B) Lung proteins were extracted and subjected to western blot analysis for p-NF-κB (p-p65).

Figure 4

Silybin inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) production in macrophages. RAW264.7 cells were treated with silybin in the absence or presence of 500 µg/ml LPS for 24 h. (A) The mRNA levels of IL-1β and TNF-α in the cell culture medium were determined 24 h following LPS stimulation by RT-qPCR. (B) The protein levels of IL-1β and TNF-α were determined by western blot analysis 8 h after LPS stimulation. Data are shown as the means ± SD of 3 independent experiments. ^*P<0.05 and ^**P<0.01 vs. LPS group.

Figure 5

Silybin inhibits the phosphorylation of nuclear factor-κB (NF-κB) and its nuclear translocation. (A) RAW264.7 cells were treated with silybin for 3 h and stimulated with 500 µg/ml lipopolysaccharide (LPS) for 30 min. The levels of phosphorylated (p-)NF-κB (p65) were determined by western blot analysis. (B) Cytoplasmic and nuclear proteins were extracted and assayed by western blot analysis. The expression levels of actin and histone 3 were used as loading controls. (C) RAW264.7 cells were treated with silybin for 3 h and stimulated with LPS for 30 min. The localization of NF-κB (p65) was analyzed by immnuofluorescence staining.

Figure 6

Silybin inhibits NLRP3 inflammasome activation in THP-1 cells. Lipopolysaccharide (LPS)-primed THP-1 cells were treated with various concentrations of silybin for 1 h, and then incubated with 5 mM ATP for 1 h. (A) Protein levels of pro-caspase-1, cleaved caspase-1, ASC and NRLP3 were determined by western blot analysis. (B) NLRP3 inflammasome complex assembly was analyzed by immunoprecipitation. (C) Measurement of intracellular ROS production by DCF fluorescence.