Figure 1.
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.
Figure 2.
Berberine does not affect the expression of tight junction proteins.
Caco-2 monolayers were treated as described in Fig. 1A. Cell lysates were analyzed to detect the expression of tight junction proteins ZO-1 (A), occludin (B) and claudin-1 (C) by immunoblot. The total protein expressions of cellular tight junction proteins ZO-1, occludin and claudin-1 were not significantly altered by the treatment of Caco-2 monolayers without or with IFN-γ and TNF-α in the absence or presence of berberine. Data are representative of five similar experiments.
Figure 3.
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.
Figure 4.
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.
Figure 5.
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.