Korean J Physiol Pharmacol.  2016 Jul;20(4):415-424. 10.4196/kjpp.2016.20.4.415.

Activating transcription factor-3 induction is involved in the anti-inflammatory action of berberine in RAW264.7 murine macrophages

• Bae YA
• Cheon HG

• KMID: 2371059
• DOI: http://doi.org/10.4196/kjpp.2016.20.4.415

Abstract

Berberine is an isoquinoline alkaloid found in Rhizoma coptidis, and elicits anti-inflammatory effects through diverse mechanisms. Based on previous reports that activating transcription factor-3 (ATF-3) acts as a negative regulator of LPS signaling, the authors investigated the possible involvement of ATF-3 in the anti-inflammatory effects of berberine. It was found berberine concentration-dependently induced the expressions of ATF-3 at the mRNA and protein levels and concomitantly suppressed the LPS-induced productions of proinflammatory cytokines (TNF-α, IL-6, and IL-1β). In addition, ATF-3 knockdown abolished the inhibitory effects of berberine on LPS-induced proinflammatory cytokine production, and prevented the berberine-induced suppression of MAPK phosphorylation, but had little effect on AMPK phosphorylation. On the other hand, the effects of berberine, that is, ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation, were prevented by AMPK knockdown, suggesting ATF-3 induction occurs downstream of AMPK activation. The in vivo administration of berberine to mice with LPS-induced endotoxemia increased ATF-3 expression and AMPK phosphorylation in spleen and lung tissues, and concomitantly reduced the plasma and tissue levels of proinflammatory cytokines. These results suggest berberine has an anti-inflammatory effect on macrophages and that this effect is attributable, at least in part, to pathways involving AMPK activation and ATF-3 induction.

Keyword

Activating transcription factor-3; Berberine; Inflammation; Lipopolysaccharide; Macrophage

MeSH Terms

Activating Transcription Factor 3*
AMP-Activated Protein Kinases
Animals
Berberine*
Cytokines
Endotoxemia
Hand
Inflammation
Interleukin-6
Lung
Macrophages*
Mice
Phosphorylation
Plasma
RNA, Messenger
Spleen
AMP-Activated Protein Kinases
Activating Transcription Factor 3
Berberine
Cytokines
Interleukin-6
RNA, Messenger

Figure

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  Fig. 1 Effects of berberine on LPS-induced proinflammatory cytokine production and ATF-3 expression.Raw264.7 cells were treated with different concentrations of berberine (10~100 µM) for 20 hr and then with 10 ng/ml of LPS for 4 hr. Cell culture media were collected, and TNF-α, IL-1β, and IL-6 levels were determined by ELISA (A); Proinflammatory cytokine mRNA expressions were determined by RT-PCR (B); and the effect of berberine on ATF-3 expression was determined by RT-PCR and western blot (D). The time dependency of berberine action was examined after incubation of RAW264.7 cells with 20 µM berberine for different incubation times (0~20 hr), followed by 4 hr exposure of LPS (10 ng/ml) (C). Nuclear translocation of ATF-3 after berberine treatment was examined by subcellular fractionation followed by western blot (E). The experiment was repeated twice in triplicate, and results are expressed as means±SEMs. *p<0.05 vs. control. #p<0.05 vs. LPS alone.

• 

  Fig. 2 Effects of ATF-3 knockdown on the berberine-induced suppression of proinflammatory cytokine production by LPS.ATF-3 siRNA (100 nM/well) or control siRNA was transfected into Raw264.7 cells, and extents of ATF-3 knockdown were assessed by RT-PCR and western blot (A). Cells transfected with control siRNA or ATF-3 siRNA were treated with berberine (10 or 20 µM) for 20 hr and then with LPS (10 ng/ml) for 4 hr. Levels of TNF-α, IL-1β, and IL-6 in culture medium were determined by ELISA (B). The experiment was repeated twice in triplicate, and results are expressed as means±SEMs. *p<0.05 vs. control. #p<0.05 vs. LPS alone. †p<0.05 vs. control siRNA.

• 

  Fig. 3 Effects of ATF-3 knockdown on the berberine-induced suppression of MAPK phosphorylation by LPS.Control siRNA or ATF-3 siRNA (100 nM/well) was transfected into Raw264.7 cells, which were then treated with berberine for 20 hr and then with LPS (10 ng/ml) for 30 min. Phosphorylated levels of p38 (Thr180/Tyr182), ERK (Tyr204), JNK (Thr183/Tyr185) (A), and AMPK-α (Thr172) (B) were determined by western blot. Densitometric analysis was carried out using UN-SCAN-IT gel ver. 5.1 software. The experiment was repeated three times and representative results are shown. *p<0.05 vs. control. #p<0.05 vs. LPS alone. †p<0.05 vs. control siRNA.

• 

  Fig. 4 Effects of AMPK knockdown on LPS-induced proinflammatory cytokine production.AMPK siRNA (100 nM/well) or control siRNA was transfected into Raw26.4 7 cells, and extents of AMPK knockdown were determined by RT-PCR and western blot (A). Cells transfected with control or AMPK siRNA were treated with berberine for 20 hr and then with LPS for 4 hr (B) and for 30 min (C). Levels of TNF-α, IL-1β, and IL-6 in culture media were determined by ELISA (B). Phosphorylated levels of p38 (Thr180/Tyr182), ERK (Tyr204), JNK (Thr183/Tyr185), and AMPK-α (Thr172) were determined by western blot, and densitometric analysis was carried out by using UN-SCAN-IT gel ver. 5.1 software (C). The experiment was repeated three times, and representative results are shown. *p<0.05 vs. control. #p<0.05 vs. LPS alone. †p<0.05 vs. control siRNA.

• 

  Fig. 5 In vivo effects of berberine in the LPS-induced septic shock mouse model.C57BL/6J mice (7 weeks old, male) were administered berberine (100 or 200 mg/kg, n=8 per dose) or vehicle (0.5% CMC, n=7) orally for 3 consecutive days, and on the 4th day administered LPS (20 mg/kg, i.p.). One hour after LPS administration, plasma was obtained by cardiac puncture, and plasma levels of TNF-α, IL-1β, and IL-6 were determined by ELISA (A). Lung and spleens were obtained immediately after cardiac puncture and homogenized. The expression patterns of ATF-3, p-AMPK (Thr172), TNF-α, and IL-6 in spleen (B) and lung tissues (C) were determined by western blot; densitometric analysis was carried out using UN-SCAN-IT gel ver. 5.1 software. The experiment was repeated twice and results are expressed as means±SEMs. *p<0.05 vs. control. #p<0.05 vs. LPS alone.

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