Daikenchuto (Da-Jian-Zhong-Tang) ameliorates intestinal fibrosis by activating myofibroblast transient receptor potential ankyrin 1 channel

Figure 3 Daikenchuto-induced Ca²⁺ influx via transient receptor potential ankyrin 1 and anti-fibrosis effects in intestinal myofibroblasts. A: Method for ethanol-extraction of DKT and its components, i.e., ginger, Japanese pepper, and ginseng; B: Representative traces of whole-cell currents recorded from InMyoFibs cells at a holding potential of -60 mV. TRPA1 nonselective cation currents are activated by AITC (10 μmol/L) and inhibited by HC-030031 (10 μmol/L); C: Representative [Ca²⁺]_i response in InMyoFibs cells. Cells were exposed to DKT extracts (0.1%), which evoked prominent increases in [Ca²⁺]_i. The TRPA1 antagonist HC-030031 (10 μmol/L) was added to examine whether the [Ca²⁺]_i response involved the activation of TRPA1 channels. Respective data points represent means ± SEM from > 30 cells; D: Immunostaining images of InMyoFibs with anti-α-SMA (green) antibody and DAPI (blue) in untreated controls, 24 h TGF-β1 (5 ng/mL) treatment, or 24 h TGF-β1 (5 ng/mL) + DKT extracts (0.1%) treatment; E and F: DKT extracts (0.001%, 0.01%, and 0.1%) were co-administered with TGF-β1, and acta2 (α-SMA), myocd (myocardin), and col1a1 mRNA expression levels were quantified by real-time PCR; G: Concentrations of Type I collagen in the culture medium were measured by ELISA from the control and TGF-β1-treated cells. ^aP < 0.05 vs TGF-β1-treated cells (n = 4). DKT extracts (0.001%, 0.01%, and 0.1%) were co-administered with TGF-β1. DKT: Daikenchuto; InMyoFibs: Intestinal myofibroblasts; TRPA1: Transient receptor potential ankyrin 1; AITC: Allyl isothiocyanate; α-SMA: α-Smooth muscle actin; DAPI: 4',6-diamidino-2-phenylindole; TGF-β1: Transforming growth factor-β1.