title: Amelioration of IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by berberine via suppression of MLCK-MLC phosphorylation signaling pathway.
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.
authors.affiliation: State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Min
authors.initials: M
authors.lastname: Cao
authors.forename: Pei
authors.initials: P
authors.lastname: Wang
authors.forename: Chunhong
authors.initials: C
authors.lastname: Sun
authors.forename: Wen
authors.initials: W
authors.lastname: He
authors.forename: Fengjun
authors.initials: F
authors.lastname: Wang
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001599:Berberine / Q000494:pharmacology*; D018938:Caco-2 Cells; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000378:metabolism; D007371:Interferon-gamma / Q000502:physiology*; D007413:Intestinal Mucosa / Q000166:cytology; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010539:Permeability; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D000893:Anti-Inflammatory Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018994:Myosin Light Chains; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D001599:Berberine; D007371:Interferon-gamma; D009219:Myosin-Light-Chain Kinase