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Amelioration of IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by berberine via suppression of MLCK-MLC phosphorylation signaling pathway.

Cao M, Wang P, Sun C, He W, Wang F - PLoS ONE (2013)

Bottom Line: The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α.Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1.Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Intestinal barrier dysfunction occurs in many intestinal diseases, in which proinflammatory cytokines play critical roles. However, researchers are still on the way to defining the underlying mechanisms and to evaluate therapeutic strategies for restoring intestinal barrier function. Berberine, a drug that has clinically been used to treat gastroenteritis and diarrhea for thousands of years, has been shown to protect barrier function in both endothelial and epithelial cells, but the mechanisms are completely unknown. In this study, we investigate the protective actions of berberine on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with IFN-γ and TNF-α. Caco-2 monolayers were treated without or with simultaneous IFN-γ and TNF-α in the absence or presence of berberine. Both transepithelial electrical resistance (TER) and paracellular permeability were measured to evaluate barrier function. The expression and distribution of tight junction proteins ZO-1, occluding, and claudin-1 were respectively analyzed by immunoblot or immunofluorescence. The expressions of phosphorylated myosin light chain (pMLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunoblot. The translocation of NF-κB p65 to nuclei was analyzed by immunofluorescence and immunoblot, respectively. The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α. Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1. The increase of both MLC phosphorylation and MLCK protein expression induced by IFN-γ and TNF-α was significantly inhibited by berberine treatment. Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB. Taken together, it is suggested that berberine attenuates IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway of MLCK-dependent MLC phosphorylation mediated by HIF-1α.

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Berberine inhibits the activation of HIF-1α, but not NF-κB.A. Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. The nuclei were stained with DAPI. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min dramatically induced NF-κB p65 accumulation within the nuclei. Berberine had no obvious effect on IFN-γ and TNF-α-elicited nuclear accumulation of NF-κB p65. Data are representative of three independent experiments. The green stands for NF-κB p65. The blue stands for nuclei. Scale bar = 10 µm. B. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min increased nuclear NF-κB p65 significantly, whereas berberine treatment did not significantly change IFN-γ and TNF-α-induced increase of nuclear NF-κB p65. *p<0.05, compared with control (0 min). Data are representative of three similar experiments. C. Caco-2 monolayers were treated as described in Fig. 1A. Berberine treatment significantly inhibited IFN-γ and TNF-α-induced increase of HIF-1α protein. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.
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pone-0061944-g005: Berberine inhibits the activation of HIF-1α, but not NF-κB.A. Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. The nuclei were stained with DAPI. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min dramatically induced NF-κB p65 accumulation within the nuclei. Berberine had no obvious effect on IFN-γ and TNF-α-elicited nuclear accumulation of NF-κB p65. Data are representative of three independent experiments. The green stands for NF-κB p65. The blue stands for nuclei. Scale bar = 10 µm. B. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min increased nuclear NF-κB p65 significantly, whereas berberine treatment did not significantly change IFN-γ and TNF-α-induced increase of nuclear NF-κB p65. *p<0.05, compared with control (0 min). Data are representative of three similar experiments. C. Caco-2 monolayers were treated as described in Fig. 1A. Berberine treatment significantly inhibited IFN-γ and TNF-α-induced increase of HIF-1α protein. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.

Mentions: Proinflammatory cytokines are known to activate nuclear transcription factor NF-κB. Previous studies have revealed that NF-κB activation is involved in barrier function disruption as well as MLCK up-regulation in proinflammatory cytokine-treated intestinal epithelial cells [11], [40], [41], and that berberine is able to inhibit NF-κB activation [23], [42]. Thus, based on the above results, we further determined whether NF-κB signaling pathway was involved in the protective effects of berberine on IFN-γ and TNF-α-induced intestinal barrier dysfunction, and MLCK up-regulation as well. As illustrated in Fig. 5A and B, treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min elicited NF-κB p65 accumulation within the nuclei. Berberine treatment did not affect IFN-γ and TNF-α-triggered nuclear accumulation of NF-κB p65 in Caco-2 monolayers. This suggests that the protective role of berberine against IFN-γ and TNF-α-induced intestinal barrier dysfunction is NF-κB-independent.


Amelioration of IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by berberine via suppression of MLCK-MLC phosphorylation signaling pathway.

Cao M, Wang P, Sun C, He W, Wang F - PLoS ONE (2013)

Berberine inhibits the activation of HIF-1α, but not NF-κB.A. Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. The nuclei were stained with DAPI. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min dramatically induced NF-κB p65 accumulation within the nuclei. Berberine had no obvious effect on IFN-γ and TNF-α-elicited nuclear accumulation of NF-κB p65. Data are representative of three independent experiments. The green stands for NF-κB p65. The blue stands for nuclei. Scale bar = 10 µm. B. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min increased nuclear NF-κB p65 significantly, whereas berberine treatment did not significantly change IFN-γ and TNF-α-induced increase of nuclear NF-κB p65. *p<0.05, compared with control (0 min). Data are representative of three similar experiments. C. Caco-2 monolayers were treated as described in Fig. 1A. Berberine treatment significantly inhibited IFN-γ and TNF-α-induced increase of HIF-1α protein. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643960&req=5

pone-0061944-g005: Berberine inhibits the activation of HIF-1α, but not NF-κB.A. Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. The nuclei were stained with DAPI. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min dramatically induced NF-κB p65 accumulation within the nuclei. Berberine had no obvious effect on IFN-γ and TNF-α-elicited nuclear accumulation of NF-κB p65. Data are representative of three independent experiments. The green stands for NF-κB p65. The blue stands for nuclei. Scale bar = 10 µm. B. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min increased nuclear NF-κB p65 significantly, whereas berberine treatment did not significantly change IFN-γ and TNF-α-induced increase of nuclear NF-κB p65. *p<0.05, compared with control (0 min). Data are representative of three similar experiments. C. Caco-2 monolayers were treated as described in Fig. 1A. Berberine treatment significantly inhibited IFN-γ and TNF-α-induced increase of HIF-1α protein. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.
Mentions: Proinflammatory cytokines are known to activate nuclear transcription factor NF-κB. Previous studies have revealed that NF-κB activation is involved in barrier function disruption as well as MLCK up-regulation in proinflammatory cytokine-treated intestinal epithelial cells [11], [40], [41], and that berberine is able to inhibit NF-κB activation [23], [42]. Thus, based on the above results, we further determined whether NF-κB signaling pathway was involved in the protective effects of berberine on IFN-γ and TNF-α-induced intestinal barrier dysfunction, and MLCK up-regulation as well. As illustrated in Fig. 5A and B, treatment of Caco-2 monolayers with IFN-γ and TNF-α for 15 or 30 min elicited NF-κB p65 accumulation within the nuclei. Berberine treatment did not affect IFN-γ and TNF-α-triggered nuclear accumulation of NF-κB p65 in Caco-2 monolayers. This suggests that the protective role of berberine against IFN-γ and TNF-α-induced intestinal barrier dysfunction is NF-κB-independent.

Bottom Line: The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α.Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1.Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Intestinal barrier dysfunction occurs in many intestinal diseases, in which proinflammatory cytokines play critical roles. However, researchers are still on the way to defining the underlying mechanisms and to evaluate therapeutic strategies for restoring intestinal barrier function. Berberine, a drug that has clinically been used to treat gastroenteritis and diarrhea for thousands of years, has been shown to protect barrier function in both endothelial and epithelial cells, but the mechanisms are completely unknown. In this study, we investigate the protective actions of berberine on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with IFN-γ and TNF-α. Caco-2 monolayers were treated without or with simultaneous IFN-γ and TNF-α in the absence or presence of berberine. Both transepithelial electrical resistance (TER) and paracellular permeability were measured to evaluate barrier function. The expression and distribution of tight junction proteins ZO-1, occluding, and claudin-1 were respectively analyzed by immunoblot or immunofluorescence. The expressions of phosphorylated myosin light chain (pMLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunoblot. The translocation of NF-κB p65 to nuclei was analyzed by immunofluorescence and immunoblot, respectively. The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α. Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1. The increase of both MLC phosphorylation and MLCK protein expression induced by IFN-γ and TNF-α was significantly inhibited by berberine treatment. Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB. Taken together, it is suggested that berberine attenuates IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway of MLCK-dependent MLC phosphorylation mediated by HIF-1α.

Show MeSH
Related in: MedlinePlus