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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 attenuates intestinal epithelial barrier dysfunction induced by IFN-γ and TNF-α.A. Caco-2 monolayers were incubated without or with 10 ng/ml IFN-γ and 10 ng/ml TNF-α in the absence or presence of 100 µM berberine for 48 h. Berberine significantly inhibited TER reduction induced by IFN-γ and TNF-α treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 10. B. Caco-2 monolayers were treated as described in panel A. The IFN-γ and TNF-α-induced increase of paracellular permeability to 4 kDa FITC-dextran was significantly lowered by berberine treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 7.
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pone-0061944-g001: Berberine attenuates intestinal epithelial barrier dysfunction induced by IFN-γ and TNF-α.A. Caco-2 monolayers were incubated without or with 10 ng/ml IFN-γ and 10 ng/ml TNF-α in the absence or presence of 100 µM berberine for 48 h. Berberine significantly inhibited TER reduction induced by IFN-γ and TNF-α treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 10. B. Caco-2 monolayers were treated as described in panel A. The IFN-γ and TNF-α-induced increase of paracellular permeability to 4 kDa FITC-dextran was significantly lowered by berberine treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 7.

Mentions: We, along with other investigators, have demonstrated that IFN-γ and TNF-α in combination disrupt intestinal barrier function in vitro, as evidenced by the decreased TER and the increased paracellular permeability [9], [33]–[35]. Thus, in order to investigate the effect of berberine on intestinal barrier function, we adopted an in vitro model in which human colonic Caco-2 epithelial cell monolayers were treated with simultaneous IFN-γ and TNF-α for 48 hours [33], [34]. Both TER, an indicator of epithelial paracellular permeability to ionic solutes, and dextran flux, an indicator of epithelial paracellular permeability to uncharged macromolecules, were employed to assess barrier function. As shown in Fig. 1A and B, berberine alone just caused a small increase in TER, but had no effect on paracellular permeability to 4 kDa FITC-dextran in control Caco-2 monolayers. The TER of Caco-2 monolayer treated with simultaneously IFN-γ and TNF-α for 48 hours was significantly lower than that of control monolayers (Fig. 1A), indicating that IFN-γ and TNF-α treatment increase the paracellular permeability to ionic solutes. However, berberine treatment significantly dampened the TER drop elicited by simultaneously IFN-γ and TNF-α (Fig. 1A).


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 attenuates intestinal epithelial barrier dysfunction induced by IFN-γ and TNF-α.A. Caco-2 monolayers were incubated without or with 10 ng/ml IFN-γ and 10 ng/ml TNF-α in the absence or presence of 100 µM berberine for 48 h. Berberine significantly inhibited TER reduction induced by IFN-γ and TNF-α treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 10. B. Caco-2 monolayers were treated as described in panel A. The IFN-γ and TNF-α-induced increase of paracellular permeability to 4 kDa FITC-dextran was significantly lowered by berberine treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 7.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643960&req=5

pone-0061944-g001: Berberine attenuates intestinal epithelial barrier dysfunction induced by IFN-γ and TNF-α.A. Caco-2 monolayers were incubated without or with 10 ng/ml IFN-γ and 10 ng/ml TNF-α in the absence or presence of 100 µM berberine for 48 h. Berberine significantly inhibited TER reduction induced by IFN-γ and TNF-α treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 10. B. Caco-2 monolayers were treated as described in panel A. The IFN-γ and TNF-α-induced increase of paracellular permeability to 4 kDa FITC-dextran was significantly lowered by berberine treatment. *p<0.05, compared with control, #p<0.05, compared with IFN-γ/TNF-α. n = 7.
Mentions: We, along with other investigators, have demonstrated that IFN-γ and TNF-α in combination disrupt intestinal barrier function in vitro, as evidenced by the decreased TER and the increased paracellular permeability [9], [33]–[35]. Thus, in order to investigate the effect of berberine on intestinal barrier function, we adopted an in vitro model in which human colonic Caco-2 epithelial cell monolayers were treated with simultaneous IFN-γ and TNF-α for 48 hours [33], [34]. Both TER, an indicator of epithelial paracellular permeability to ionic solutes, and dextran flux, an indicator of epithelial paracellular permeability to uncharged macromolecules, were employed to assess barrier function. As shown in Fig. 1A and B, berberine alone just caused a small increase in TER, but had no effect on paracellular permeability to 4 kDa FITC-dextran in control Caco-2 monolayers. The TER of Caco-2 monolayer treated with simultaneously IFN-γ and TNF-α for 48 hours was significantly lower than that of control monolayers (Fig. 1A), indicating that IFN-γ and TNF-α treatment increase the paracellular permeability to ionic solutes. However, berberine treatment significantly dampened the TER drop elicited by simultaneously IFN-γ and TNF-α (Fig. 1A).

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