Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn’s disease intestinal fibrosis

Figure 1 Effect of total flavone of Abelmoschus manihot at different concentrations on IEC-6 cell viability. IEC-6 cell viability after treatment with TFA at increasing concentrations was examined by CCK-8 at 24, 48 and 72 h. The results were expressed as the mean ± SD of three independent experiments, and each was performed in triplicate. ^aP < 0.05, ^bP < 0.01 compared with that of non-TFA treated group. TFA: Total flavone of Abelmoschus manihot.

Figure 2 Total flavone of Abelmoschus manihot inhibited transforming growth factor-β1 induced migration and invasion of IEC-6 cells. A: The inhibitory effect of TFA (5, 10 and 15 μg/mL) on TGF-β1-induced morphological changes at 48 h was observed by phase contrast microscopy; B: The representative image of the wound healing assay; C: The representative image of transwell assay. The tendency is concomitant with the wound healing assay. TGF-β1 induced the migration and invasion of IEC-6 cells, whereas TFA suppressed this phenomenon. TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.

Figure 3 Total flavone of Abelmoschus manihot inhibited transforming growth factor-β1-induced epithelial-mesenchymal transition. A and B: The EMT-related label proteins were measured by immunocytochemistry (A) and western blotting (B) assays when IEC-6 cells were treated with TGF-β1 (10 ng/mL) alone or in combination with TFA (5, 10 and 15 μg/mL) for 48 h; C: The inhibitory effect of TFA (5, 10 and 15 μg/mL) on TGF-β1-induced EMT-related mRNAs was detected by qRT-PCR assay. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with thef non-TFA treated group, ^dP < 0.05, ^eP < 0.01 compared with the TGF-β1 (10 ng/mL) treated group. EMT: Epithelial-mesenchymal transition; TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.

Figure 4 Total flavone of Abelmoschus manihot inhibited transforming growth factor-β1-induced activation of the Smad signaling pathway. A: The effects of TFA on TGF-β1-induced activation of Smad signaling were evaluated by western blotting; B: The expression of Smad signaling was examined by western blotting after transfection of si-Smad2/3; C and D: The effect of si-Smad or TFA combined with si-Smad2/3 on TGF-β1-induced activation of Smad signaling was evaluated by western blotting (C) and qRT-PCR (D) assays. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with that of non-TFA treated group, ^eP < 0.01, ^fP < 0.001 compared with that of TGF-β1 (10 ng/mL) treated group. TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.

Figure 5 Total flavone of Abelmoschus manihot (TFA) combined with si-Smad2/3 had synergistic role in inhibiting transforming growth factor-β1-induced migration and invasion of IEC-6 cells. A: The inhibitory effect of si-Smad or TFA (15 μg/mL) combined with si-Smad2/3 on TGF-β1-induced morphological changes at 48 h was observed by phase contrast microscopy; B and C: The migration and invasion abilities of si-Smad or TFA (15 μg/mL) combined with si-Smad2/3 were examined by wound healing (B) and transwell (C) assays. TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.a

Figure 6 Total flavone of Abelmoschus manihot inhibited transforming growth factor-β1-induced activation of MAPK signaling pathway. A: The effect of TFA on TGF-β1-induced activation of MAPK signaling was evaluated by western blotting; B: The expression of MAPK signaling was examined by western blotting after transfection with MAPK inhibitors; B and C: The effects of MAPK inhibitors or TFA combined with MAPK inhibitors on TGF-β1-induced activation of Smad signaling were evaluated by western blotting (B) and qRT-PCR (C) assays. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with that of non-TFA treated group, ^dP < 0.05, ^eP < 0.01 compared with that of TGF-β1 (10 ng/mL) treated group. TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.

Figure 7 Total flavone of Abelmoschus manihot combined with MAPK inhibitors had synergistic role in inhibiting transforming growth factor-β1 -induced migration and invasion of IEC-6 cells. A: The inhibitory effect of MAPK inhibitors or TFA (15 μg/mL) combined with MAPK inhibitors on TGF-β1-induced morphological changes at 48 h was observed by phase contrast microscopy; B and C: The migration and invasion abilities of MAPK inhibitors or TFA (15 μg/mL) combined with MAPK inhibitors were examined by wound healing (B) and transwell (C) assays. TFA: Total flavone of Abelmoschus manihot; TGF: Transforming growth factor.