Korean J Physiol Pharmacol.  2015 May;19(3):211-218. 10.4196/kjpp.2015.19.3.211.

Silymarin Inhibits Morphological Changes in LPS-Stimulated Macrophages by Blocking NF-kappaB Pathway

• Kim EJ
• Lee MY
• Jeon YJ

• KMID: 1791421
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.211

Abstract

The present study showed that silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), inhibited lipopolysaccharide (LPS)-induced morphological changes in the mouse RAW264.7 macrophage cell line. We also showed that silymarin inhibited the nuclear translocation and transactivation activities of nuclear factor-kappa B (NF-kappaB), which is important for macrophage activation-associated changes in cell morphology and gene expression of inflammatory cytokines. BAY-11-7085, an NF-kappaB inhibitor, abrogated LPS-induced morphological changes and NO production, similar to silymarin. Treatment of RAW264.7 cells with silymarin also inhibited LPS-stimulated activation of mitogen-activated protein kinases (MAPKs). Collectively, these experiments demonstrated that silymarin inhibited LPS-induced morphological changes in the RAW264.7 mouse macrophage cell line. Our findings indicated that the most likely mechanism underlying this biological effect involved inhibition of the MAPK pathway and NF-kappaB activity. Inhibition of these activities by silymarin is a potentially useful strategy for the treatment of inflammation because of the critical roles played by MAPK and NF-kappaB in mediating inflammatory responses in macrophages.

Keyword

iNOS; Macrophages; MAPK; NF-kappaB

MeSH Terms

Animals
Cell Line
Cytokines
Gene Expression
Inflammation
Macrophages*
Mice
Milk Thistle
Mitogen-Activated Protein Kinases
Negotiating
NF-kappa B*
Silymarin*
Transcriptional Activation
Cytokines
Mitogen-Activated Protein Kinases
NF-kappa B
Silymarin

Figure

• 

  Fig. 1 Inhibition of macrophage activation by silymarin in LPS-stimulated macrophages. RAW264.7 cells were treated with the indicated concentrations of silymarin in the presence of LPS (200 ng/ml) for 3 h, 6 h, or 12 h on cover slides in 12-well plates. Cells were then subjected to microscopic analysis. (A) Representative photographs. (B) In 12 h LPS treatment samples, fully expanded cells were counted and expressed as a percentage of the total number of cells.

• 

  Fig. 2 Inhibition of morphological change and adhesion activity by silymarin in LPS-stimulated macrophages. (A) RAW264.7 cells were treated with the indicated concentrations of silymarin in the presence of LPS (200 ng/ml) for 3 h, 6 h, or 18 h on cover slides in 12-well plates. Cells were then Giemsa-stained and analyzed for morphological change. (B) RAW264.7 cells were treated with silymarin (50 μg/ml) in the presence of LPS for 24 h. Cells were harvested, washed, plated in 6-well plates (5×105/ml) for 30 min. Cells were then washed with PBS and analyzed for cell attachment using microscopy. Attached cells were counted after washing. (C) Representative photographs of attached cells before and after washing. Each column shows the mean±SD of triplicate determinations. *p<0.05 compared to the control group, as determined by Student's two-tailed t test.

• 

  Fig. 3 Inhibition of the lamelipodia and filopodia formation by silymarin in LPS-stimulated macrophages. RAW 264.7 cells were treated with the silymarin (50 μg/ml) in the presence of LPS for 18 h on cover slides in 12-well plates. Cells were then subjected to immunofluorescence staining of F-actin and DAPI staining.

• 

  Fig. 4 Inhibition of nuclear factor-kappa B (NF-κB) activation and p65 nuclear translocation by silymarin in LPS-stimulated macrophages. (A) RAW264.7 cells were transfected with pNF-κB-Luc plasmid. Cells were incubated for 24 h and then treated with silymarin (50 μg/ml) in the presence of LPS for 18 h. Cell extracts were prepared and luciferase activity was analyzed to indicate NF-κB transactivation activity. (B) RAW264.7 cells were treated with silymarin (50 ±g/ml) in the presence of LPS for 2 h on cover slides in 12-well plates. Cells were then subjected to immunofluorescence staining of p65 and DAPI staining. (C) RAW264.7 cells were treated with silymarin (50 μg/ml) in the presence of LPS for 2 h. Nuclear extracts were then prepared and subjected to Western immunoblot analysis using anti-p65 antibody. Each column shows the mean±SD of triplicate determinations. *p<0.05 compared to the control group, as determined by Student's two-tailed t test.

• 

  Fig. 5 Effects of nuclear factor-kappa B (NF-κB) inhibitor on macrophage activation induced by LPS. (A) RAW264.7 cells were treated with BAY-11-7085 (5 μM) in the presence of LPS for 2 h on cover slides in 12-well plates. Cells were then subjected to immunofluorescence staining of p65 and DAPI staining. (B) RAW 264.7 cells were treated with the indicated concentrations of BAY-11-7085 in the presence of LPS for 24 h. Supernatants were subsequently isolated and analyzed for nitrite. Each column shows the mean±SD of triplicate determinations. *p<0.05 compared to the control group, as determined by Student's two-tailed t test. (C) RAW264.7 cells were treated with the indicated concentrations of BAY-11-7085 in the presence of LPS (200 ng/ml) for 6 h on cover slides in 12-well plates. Cells were then subjected to microscopic analysis.

• 

  Fig. 6 Inhibition of mitogen-activated protein kinase (MAPK) phosphorylation by silymarin in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with silymarin. RAW264.7 cells were pretreated with silymarin for 1 h and incubated for 30 min in the presence of LPS. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and phospho-c-Jun N-terminal kinase (pJNK) was analyzed by Western blot. The relative band densities of ERK1/2 (B) and p38 and pJNK (C) were analyzed using the Image J program.

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