Korean J Physiol Pharmacol.  2017 Jul;21(4):449-456. 10.4196/kjpp.2017.21.4.449.

Beauvericin, a cyclic peptide, inhibits inflammatory responses in macrophages by inhibiting the NF-κB pathway

• Yoo S
• Kim MY
• Cho JY

• KMID: 2384459
• DOI: http://doi.org/10.4196/kjpp.2017.21.4.449

Abstract

Beauvericin (BEA), a cyclic hexadepsipeptide produced by the fungus Beauveria bassiana, is known to have anti-cancer, anti-inflammatory, and anti-microbial actions. However, how BEA suppresses macrophage-induced inflammatory responses has not been fully elucidated. In this study, we explored the anti-inflammatory properties of BEA and the underlying molecular mechanisms using lipopolysaccharide (LPS)-treated macrophage-like RAW264.7 cells. Levels of nitric oxide (NO), mRNA levels of transcription factors and the inflammatory genes inducible NO synthase (iNOS) and interleukin (IL)-1, and protein levels of activated intracellular signaling molecules were determined by Griess assay, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), luciferase reporter gene assay, and immunoblotting analysis. BEA dose-dependently blocked the production of NO in LPS-treated RAW264.7 cells without inducing cell cytotoxicity. BEA also prevented LPS-triggered morphological changes. This compound significantly inhibited nuclear translocation of the NF-κB subunits p65 and p50. Luciferase reporter gene assays demonstrated that BEA suppresses MyD88-dependent NF-κB activation. By analyzing upstream signaling events for NF-κB activation and overexpressing Src and Syk, these two enzymes were revealed to be targets of BEA. Together, these results suggest that BEA suppresses NF-κB-dependent inflammatory responses by suppressing both Src and Syk.

Keyword

Anti-inflammatory activity; Beauvericin; NF-κB; Src; Syk

MeSH Terms

Beauveria
Fungi
Genes, Reporter
Immunoblotting
Interleukins
Luciferases
Macrophages*
Nitric Oxide
Nitric Oxide Synthase
RNA, Messenger
Transcription Factors
Interleukins
Luciferases
Nitric Oxide
Nitric Oxide Synthase
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 Effect of BEA on inflammatory responses of macrophages. (A) Secreted levels of NO in the culture supernatant from LPS-stimulated RAW264.7 cells incubated for 12 h in the presence or absence of BEA (left panel) or L-NAME (right panel) were determined by Griess assay. (B) Morphological changes of RAW264.7 cells treated with LPS (1 µg/ml) for 12 h in the presence or absence of BEA (2 and 4 µM) were observed using a confocal microscope. Actin cytoskeleton and nuclei were stained with rhodamine phalloidin conjugate and Hoechst, respectively (C, D) Cytotoxic effect of BEA on RAW264.7 and HEK293 cells was evaluated according to treatment time using the MTT assay. *p<0.05 and **p<0.01 compared to control or normal groups.

• 

  Fig. 2 Effect of BEA on the transcriptional regulation of inflammatory genes in LPS-treated RAW264.7 cells. (A) mRNA expression levels of IL-1β and iNOS in LPS-stimulated RAW264.7 cells exposed to BEA for 6 h were measured by semi-quantitative RT-PCR, as described in the Materials and Methods section. (B, C) NF-κB-binding promoter activity was explored using the luciferase reporter gene assay. HEK293 cells and LPS-stimulated RAW264.7 cells were co-transfected with NF-κB-Luc plasmid, TRIF, or MyD88 plasmids in the presence or absence of BEA for 9 h. Luciferase activity was determined by a luminometer. (D) Translocation of NF-κB subunits (p65 and p50) to the nucleus was assessed in LPS-stimulated RAW264.7 cells exposed to BEA by immunoblot analysis of nuclear fractions. NF, nuclear fraction. **p<0.01 compared to control groups.

• 

  Fig. 3 Effect of BEA on upstream signaling events regulating NF-κB translocation in LPS-stimulated RAW264.7 cells. (A, B) Levels of phosphorylated and total IκBα, p85/PI3K, AKT, Src, and Syk were upregulated in LPS-stimulated RAW264.7 Protein expression was assessed by immunoblot analysis.

• 

  Fig. 4 Effect of BEA on Src- or Syk-induced phosphorylation of PI3K/p85 in HEK293 cells. (A, B) HEK293 cells transfected with HA-Src and Myc-Syk for 24 h were incubated in the presence or absence of BEA for 9 h. Levels of phosphorylated and total p85/PI3K were measured by immunoblot analysis. (C) PP2- or BEA-treated RAW264.7 cells were stimulated with LPS for 15 min. Total and phosphorylated levels of PI3K/p85 from the lysates of these cells were detected by immunoblot analysis. (D) Secreted levels of NO in the culture supernatant of LPS-stimulated RAW264.7 cells incubated for 12 h in the presence or absence of PP2 or piceatannol were determined by the Griess assay. **p<0.01 compared to the control group.

• 

  Fig. 5 Schematic of the anti-inflammatory pathway affected by BEA in LPS-stimulated macrophages.

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