Xanthohumol Interferes with the Activation of TGF-beta Signaling in the Process Leading to Intestinal Fibrosis

Fibrosis has various biological processes and affects almost every organ, especially in patients with inflammatory bowel disease, including Crohn's disease, who experience discomfort caused by intestinal fibrosis, which is a problem that needs to be resolved. TGF-beta signaling is known to act as a key regulator of intestinal fibrosis, and its modulation could be an excellent candidate for fibrosis therapy. Xanthohumol (XN) has various effects, including anti-inflammation and anti-cancer; however, the detailed mechanism of TGF-beta signaling has not yet been studied. The purpose of this study was to investigate the mechanism underlying the anti-fibrotic effect of XN on TGF-beta 1-induced intestinal fibrosis using primary human intestinal fibroblasts (HIFs). In this study, to check the anti-fibrotic effects of XN on intestinal fibrosis, we assessed the expression of fibrosis-related genes in TGF-beta 1-stimulated HIFs by qPCR, immunoblotting, and immunofluorescence staining. As a result, XN showed the ability to reduce the expression of fibrosis-associated genes increased by TGF-beta 1 treatment in HIFs and restored the cell shape altered by TGF-beta 1. In particular, XN repressed both NF-kappa B- and Smad-binding regions in the alpha-SMA promoter, which is important in fibrosis. In addition, XN inhibited NF-kappa B signaling, including phosphorylated-IkB alpha and cyclooxygenase-2 expression, and TNF-alpha-stimulated transcriptional activity of NF-kappa B. XN attenuated TGF-beta 1-induced phosphorylation of Smad2 and Smad3, and the transcriptional activity of CAGA. Particularly, XN interfered with the binding of TGF-Receptor I (T beta RI) and Smad3 by binding to the kinase domain of the L45 loop of T beta RI, thereby confirming that the fibrosis mechanism did not proceed further. In conclusion, XN has an inhibitory effect on TGF-beta 1-induced intestinal fibrosis in HIFs, significantly affecting TGF-beta/Smad signaling.