Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease

Lin Hai Kurahara; Keizo Hiraishi; Yaopeng Hu; Kaori Koga; Miki Onitsuka; Mayumi Doi; Kunihiko Aoyagi; Hidetoshi Takedatsu; Daibo Kojima; Yoshitaka Fujihara; Yuwen Jian; Ryuji Inoue

doi:10.1016/j.jcmgh.2017.12.005

Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease

Cell Mol Gastroenterol Hepatol. 2017 Dec 21;5(3):299-318. doi: 10.1016/j.jcmgh.2017.12.005. eCollection 2018 Mar.

Authors

Lin Hai Kurahara¹, Keizo Hiraishi¹, Yaopeng Hu¹, Kaori Koga², Miki Onitsuka², Mayumi Doi^{1

3}, Kunihiko Aoyagi⁴, Hidetoshi Takedatsu⁵, Daibo Kojima⁶, Yoshitaka Fujihara⁷, Yuwen Jian⁸, Ryuji Inoue¹

Affiliations

¹ Department of Physiology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
² Department of Pathology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
³ Department of Clinical Pharmacology and Therapeutics, Faculty of Medicine, Oita University, Oita, Japan.
⁴ Department of Gastroenterology, Japanese Red Cross Fukuoka Hospital, Fukuoka, Japan.
⁵ Department of Gastroenterology and Medicine, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
⁶ Department of Gastroenterological Surgery, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
⁷ Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
⁸ College of Letters and Science, University of California-Davis, Davis, California.

Abstract

Background & aims: The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling.

Methods: An intestinal myofibroblast cell line (InMyoFibs) was stimulated by transforming growth factor-β1 to induce in vitro fibrosis. Trpa1 knockout mice were generated using the Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system. A murine chronic colitis model was established by weekly intrarectal trinitrobenzene sulfonic acid (TNBS) administration. Samples from the intestines of Crohn's disease (CD) patients were used for pathologic staining and quantitative analyses.

Results: In InMyoFibs, TRPA1 showed the highest expression among TRP family members. In TNBS chronic colitis model mice, the extents of inflammation and fibrotic changes were more prominent in TRPA1^-/- knockout than in wild-type mice. One-week enema administration of prednisolone suppressed fibrotic lesions in wild-type mice, but not in TRPA1 knockout mice. Steroids and pirfenidone induced Ca²⁺ influx in InMyoFibs, which was antagonized by the selective TRPA1 channel blocker HC-030031. Steroids and pirfenidone counteracted transforming growth factor-β1-induced expression of heat shock protein 47, type 1 collagen, and α-smooth muscle actin, and reduced Smad-2 phosphorylation and myocardin expression in InMyoFibs. In stenotic intestinal regions of CD patients, TRPA1 expression was increased significantly. TRPA1/heat shock protein 47 double-positive cells accumulated in the stenotic intestinal regions of both CD patients and TNBS-treated mice.

Conclusions: TRPA1, in addition to its anti-inflammatory actions, may protect against intestinal fibrosis, thus being a novel therapeutic target for highly incurable inflammatory/fibrotic disorders.

Keywords: AITC, allyl isothiocyanate; CD, Crohn’s disease; Crohn’s Disease; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; HSP47, heat shock protein 47; InMyoFib, intestinal myofibroblast cell line; Intestinal Fibrosis; KO, knockout; MT, Masson trichrome; Myofibroblast; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT-PCR, reverse-transcription polymerase chain reaction; TGF, transforming growth factor; TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; TRP, transient receptor potential; TRPA1, transient receptor potential ankyrin 1; TRPC, transient receptor potential canonical; Transient Receptor Potential Ankyrin 1; WT, wild-type; mRNA, messenger RNA; sgRNA, single-guide RNA; siRNA, small interfering RNA; α-SMA, α smooth muscle actin.