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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 prevents morphological disruption of tight junction induced by IFN-γ and TNF-α.Caco-2 monolayers were treated as described in Fig. 1A. Tight junction proteins ZO-1, occludin and claudin-1 were stained by immunofluorescence. Berberine dramatically prevented the IFN-γ and TNF-α-induced morphological disruption of tight junction proteins ZO-1, occludin and claudin-1 in Caco-2 monolayers. Data are representative of four independent experiments. Scale bar = 10 µm.
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pone-0061944-g003: Berberine prevents morphological disruption of tight junction induced by IFN-γ and TNF-α.Caco-2 monolayers were treated as described in Fig. 1A. Tight junction proteins ZO-1, occludin and claudin-1 were stained by immunofluorescence. Berberine dramatically prevented the IFN-γ and TNF-α-induced morphological disruption of tight junction proteins ZO-1, occludin and claudin-1 in Caco-2 monolayers. Data are representative of four independent experiments. Scale bar = 10 µm.

Mentions: The intestinal epithelial barrier function is regulated by tight junction structure. Previous studies have shown that intestinal barrier dysfunction induced by proinflammatory cytokines is associated with the morphological tight junction disruption and the relocalization of tight junction proteins [9], [10], [15], [33]–[35]. Thus, we next determined whether berberine affected the morphological localization of tight junction proteins in Caco-2 monolayers treated with or without IFN-γ and TNF-α. As illustrated in Fig. 3, in control Caco-2 monolayers, the tight junction proteins ZO-1, occludin and claudin-1 were respectively localized to the intercellular tight junctions, along the edge of the cells. These regular distributions were not obviously changed in Caco-2 monolayers treated with berberine alone for 48 hours. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 48 hours induced pronounced reorganization of tight junction proteins ZO-1, occludin and claudin-1 such that the distribution profiles became irregular and discontinuous. In addition, both occludin and claudin-1 were partially internalized into cytoplasmic vesicles, but ZO-1 internalization was not obviously seen. In contrast, berberine treatment largely attenuated the IFN-γ and TNF-α-caused reorganization of ZO-1, occludin and claudin-1 in Caco-2 monolayers. These indicate that berberine could prevent the reorganization of tight junction proteins induced by proinflammatory cytokines in intestinal epithelia.


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 prevents morphological disruption of tight junction induced by IFN-γ and TNF-α.Caco-2 monolayers were treated as described in Fig. 1A. Tight junction proteins ZO-1, occludin and claudin-1 were stained by immunofluorescence. Berberine dramatically prevented the IFN-γ and TNF-α-induced morphological disruption of tight junction proteins ZO-1, occludin and claudin-1 in Caco-2 monolayers. Data are representative of four independent experiments. Scale bar = 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643960&req=5

pone-0061944-g003: Berberine prevents morphological disruption of tight junction induced by IFN-γ and TNF-α.Caco-2 monolayers were treated as described in Fig. 1A. Tight junction proteins ZO-1, occludin and claudin-1 were stained by immunofluorescence. Berberine dramatically prevented the IFN-γ and TNF-α-induced morphological disruption of tight junction proteins ZO-1, occludin and claudin-1 in Caco-2 monolayers. Data are representative of four independent experiments. Scale bar = 10 µm.
Mentions: The intestinal epithelial barrier function is regulated by tight junction structure. Previous studies have shown that intestinal barrier dysfunction induced by proinflammatory cytokines is associated with the morphological tight junction disruption and the relocalization of tight junction proteins [9], [10], [15], [33]–[35]. Thus, we next determined whether berberine affected the morphological localization of tight junction proteins in Caco-2 monolayers treated with or without IFN-γ and TNF-α. As illustrated in Fig. 3, in control Caco-2 monolayers, the tight junction proteins ZO-1, occludin and claudin-1 were respectively localized to the intercellular tight junctions, along the edge of the cells. These regular distributions were not obviously changed in Caco-2 monolayers treated with berberine alone for 48 hours. Treatment of Caco-2 monolayers with IFN-γ and TNF-α for 48 hours induced pronounced reorganization of tight junction proteins ZO-1, occludin and claudin-1 such that the distribution profiles became irregular and discontinuous. In addition, both occludin and claudin-1 were partially internalized into cytoplasmic vesicles, but ZO-1 internalization was not obviously seen. In contrast, berberine treatment largely attenuated the IFN-γ and TNF-α-caused reorganization of ZO-1, occludin and claudin-1 in Caco-2 monolayers. These indicate that berberine could prevent the reorganization of tight junction proteins induced by proinflammatory cytokines in intestinal epithelia.

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