Torilis japonica Extract Suppresses the Induction of Atopic Inflammation

Abstract

As one of the major intractable allergic disorders, atopic inflammation is commonly accompanied by itching, dry skin, and inflammation. Atopic inflammation deteriorates the quality of life and has no fundamental cure, so it is crucial to urgently explore and develop natural resources for long-term treatment without any side effects. This study aimed to verify Torilis japonica extract (TJE)'s relieving effect and mechanism against atopic inflammation using skin cells and skin equivalent models, as well as to investigate torilin's effect (obtained from TJE) and other unknown components as marker compounds. Torilin concentration was verified in TJE using high-performance liquid chromatography and analyzed the unknown components using nuclear magnetic resonance spectroscopy. Furthermore, TJE's cytotoxicity, regenerative effect, and cell cycle regulation effects were confirmed using skin cells with atopic inflammation (human dermal fibroblasts and HaCaT keratinocytes) by using TNF-α and IFN-γ treatments. Consequently, TJE was demonstrated to regulate TARC and CTACK expressions as chemokines and those of interleukin-4, -5, and -13 as cytokines related to atopic inflammation. TJE was further confirmed to affect the matrix metalloproteinase-1, -2, and -9 expressions, which are essential in skin damage. Lastly, this study confirmed TJE's relieving effect against atopic inflammation through a 3D skin model and RhCE model using human dermal fibroblasts and HaCaT keratinocytes. These findings on atopic inflammation verified torilin's relieving effects and TJE's other components.

Keywords: 3D skin model; HaCaT keratinocytes; RhCE model; Torilis japonica; atopic inflammation.

Figure 1

(A) HPLC profile of TJE, torilin, and osthol. Torilin was detected, but osthol was not detected in TJE (A: the unknown substance). (B) NMR profile of the unknown peak (substance) A.

Figure 2

(A) Cell viability was measured by MTT assay. HaCaT keratinocytes were treated with variable concentrations of TJE (0–150 μg/mL) and certain cytokine (TNF-α (10 ng/mL) and IFN-γ (10 ng/mL)) for 24–72 h. (B) Cell viability was measured by MTT assay. Dermal fibroblasts were treated with variable concentrations of TJE (0–150 μg/mL) and certain cytokine (TNF-α (10 ng/mL) and IFN-γ (10 ng/mL)) for 24–72 h. (C) Cell viability assay using flow cytometry. The statistical analysis of the data was carried out by use of a t-test. *^,# p < 0.05 (each experiment, n = 3). N.S.; not significant. N: negative control, C: positive control (only TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) treated group).

Figure 3

(A,B) TJE occurs in cell cycle progression. G2/M phase ratio increased compared with to the positive control. Cell cycle arrest effect was measured by flow cytometry. (C) Cell motility was measured using a wound-healing assay. Representative images of the cell migration were captured. (D) The graph of a wound-healing area using a wound-healing assay. The statistical analysis of the data was carried out by use of a t-test. ^b p < 0.01 and ^a,c,d,e p < 0.001 (each experiment, n = 3). N.S.; not significant. N: negative control, C: positive control (only TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) treated group).

Figure 4

(A–C) Measurement of TARC, CTACK, and IgE through ELISA assay. TJE regulated TARC, CTACK, and IgE in HaCaT Keratinocytes. The statistical analysis of the data was carried out by use of a t-test. *^,# p < 0.05, **^,## p < 0.01 and ***^,### p < 0.001 (each experiment, n = 3). N.S.: not significant. N: negative control, C: positive control (only TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) treated group).

Figure 5

(A,B) The levels of IL-4, -5, -13 gene and MMP-1, -2, -9 gene were determined by RT-PCR. The β-actin probe served as protein-loading control. The statistical analysis of the data was carried out by use of a t-test. ^a p < 0.01 and ^b p < 0.001 vs. con (N). ^c p < 0.01 and ^d p < 0.001 vs. con (C) (each experiment, n = 3). N.S.: not significant. N: negative control, C: positive control (only TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) treated group).

Figure 6

(A) The organotypic three-dimensional models using HaCaT keratinocytes and dermal fibroblasts were stained for H&E as red and pink areas and represented by a microscope (Carl Zeiss, USA). The photographs were taken at a magnification of ×200. (B) Measurement of the keratin layer. The layer was counted from randomly selected fields of each section using the ImageJ program. (C) In vitro RhCE assay (eye irritation test) was tested for TJE (100 μg/mL and 200 μg/mL) using the OCL 200 EIT. Methylactase was used as positive control. The statistical analysis of the data was carried out by use of a t-test. ***^,### p < 0.001 (each experiment, n = 3). N.S.: not significant. N: negative control, C: positive control (only TNF-α (10 ng/mL) and IFN-γ (10 ng/mL) treated group).