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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 IFN-γ and TNF-α-induced increases of MLC phosphorylation and MLCK protein expression.Caco-2 monolayers were treated as described in Fig. 1A. A. Berberine significantly suppressed the increase of phosphorylated MLC expression induced by IFN-γ and TNF-α. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments. B. Berberine abolished the IFN-γ and TNF-α-caused up-regulation of MLCK protein expression. *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-g004: Berberine inhibits IFN-γ and TNF-α-induced increases of MLC phosphorylation and MLCK protein expression.Caco-2 monolayers were treated as described in Fig. 1A. A. Berberine significantly suppressed the increase of phosphorylated MLC expression induced by IFN-γ and TNF-α. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments. B. Berberine abolished the IFN-γ and TNF-α-caused up-regulation of MLCK protein expression. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.

Mentions: It is well recognized that MLCK-mediated phosphorylation of MLC plays a very important role in the physiological and pathophysiological regulation of intestinal epithelial tight junctions and paracellular leak pathways [1], [2]. Based on the above-mentioned remarkable protective effect of berberine on intestinal epithelial barrier function, we asked whether berberine alleviated IFN-γ and TNF-α-induced barrier dysfunction and tight junction disruption by blocking the increase of MLC phosphorylation. As shown in Fig. 4A, berberine alone had no significant effect on MLC phosphorylation in Caco-2 monolayers as compared with control. Treatment of Caco-2 monolayers with IFN-γ and TNF-α induced a significant increase of MLC phosphorylation, without markedly change in total MLC expression. Berberine treatment significantly attenuated the MLC phosphorylation increase elicited by IFN-γ and TNF-α.


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 IFN-γ and TNF-α-induced increases of MLC phosphorylation and MLCK protein expression.Caco-2 monolayers were treated as described in Fig. 1A. A. Berberine significantly suppressed the increase of phosphorylated MLC expression induced by IFN-γ and TNF-α. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments. B. Berberine abolished the IFN-γ and TNF-α-caused up-regulation of MLCK protein expression. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643960&req=5

pone-0061944-g004: Berberine inhibits IFN-γ and TNF-α-induced increases of MLC phosphorylation and MLCK protein expression.Caco-2 monolayers were treated as described in Fig. 1A. A. Berberine significantly suppressed the increase of phosphorylated MLC expression induced by IFN-γ and TNF-α. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments. B. Berberine abolished the IFN-γ and TNF-α-caused up-regulation of MLCK protein expression. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. Data are representative of five similar experiments.
Mentions: It is well recognized that MLCK-mediated phosphorylation of MLC plays a very important role in the physiological and pathophysiological regulation of intestinal epithelial tight junctions and paracellular leak pathways [1], [2]. Based on the above-mentioned remarkable protective effect of berberine on intestinal epithelial barrier function, we asked whether berberine alleviated IFN-γ and TNF-α-induced barrier dysfunction and tight junction disruption by blocking the increase of MLC phosphorylation. As shown in Fig. 4A, berberine alone had no significant effect on MLC phosphorylation in Caco-2 monolayers as compared with control. Treatment of Caco-2 monolayers with IFN-γ and TNF-α induced a significant increase of MLC phosphorylation, without markedly change in total MLC expression. Berberine treatment significantly attenuated the MLC phosphorylation increase elicited by IFN-γ and TNF-α.

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