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Hochuekkito exerts the anti-allergic effects via activating regulatory T cells in a murine model of contact hypersensitivity

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Abstract

Hochuekkito (HET) is a Kampo prescription, used for the clinical treatment of skin diseases such as atopic dermatitis (AD), in Japan. Oral administration of HET exerts anti-allergic effects in an experimental dermatitis mice model and in patients with atopic dermatitis; however, the mechanism underlying the anti-allergic effects of HET is still unclear. Therefore, we investigated the immunopharmacological properties of the anti-allergic actions of HET using a 2,4,6-trinitrochlorobenzene (TNCB)-induced murine contact hypersensitivity (CHS) model and adoptive cell transfer experiments. Oral administration of HET (1.4 g/kg) exhibited anti-allergic effects in a TNCB-induced CHS model via activation of Tregs; this activation was observed even without antigen sensitization in donor mice. Activation was dependent on the duration of HET administration and required at least 4 days of dosing. In addition, the anti-allergic effects of HET through the activation of Tregs were not antigen specific. Flow cytometry results indicated that the proportion of CD4+CD25+Foxp3+ cells in the splenic lymphocytes increased after oral administration of HET. Therefore, oral administration of HET induced both inducible regulatory T cells (iTregs) and thymus-derived naturally occurring regulatory T cells (nTregs). Ginseng radix and Bupleuri radix were involved in the anti-allergic actions of HET through the induction and/or activation of Tregs; Bupleuri radix participated in the activation of nTregs. In conclusion, our findings suggest that HET exerts the anti-allergic effects through the induction and/or activation of Tregs. These findings elucidate the usefulness of HET as an immunomodulator.

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Authors and Affiliations

  1. Department of Pharmacognosy, Graduate School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-Ku, Nagoya, Aichi, 468-8503, Japan

    Atsushi Tsuge & Mitsuhiko Nose

  2. Department of Pharmacognosy, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-Ku, Nagoya, Aichi, 468-8503, Japan

    Shunsuke Chiba, Yui Yagura, Mari Okamoto, Satoshi Muto, Shinsuke Hisaka & Mitsuhiko Nose

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  1. Atsushi Tsuge
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  2. Shunsuke Chiba
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  3. Yui Yagura
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  6. Shinsuke Hisaka
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  7. Mitsuhiko Nose
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Correspondence to Mitsuhiko Nose.

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11418_2023_1683_MOESM1_ESM.pdf

Supplementary file1 Fig. S1 3D-HPLC profile of hochuekkito (HET). Fig. S2 Gating strategy for analysis of CD4+CD25+FoxP3+, CD4+CD25+GITR+, and GITR+CD25+Foxp3+ cells in splenic lymphocytes administered by HET. (1) Whole cells were fractionated by side scatter (SSCs) and forward scatter (FSCc) to lymphocytes. (2, 3) Debris and doublets were discriminated. (4) Single cells were then fractionated by Fixable viability stain 575 V into living cells. (5) Living cells were fractionated by CD4. (6) CD4+ populations were then fractionated by Foxp3 and CD25 to analyze the population of CD25+ and Foxp3+ cells. (7) CD4+ populations were then fractionated by GITR and CD25 to analyze the population of GITR+ and CD25+ cells. (8) Alternatively, living cells were fractionated by CD4 and GITR to analyze the population of CD4+ and GITR+ cells. (9) CD4+ and GITR+ cells were fractionated by CD25 and Foxp3 to analyze the population of CD4+CD25+GITR+Foxp3+ cells. Fig. S3 Characterization of regulatory T cells induced and/or activated by oral administration of hots water extracts of Ginseng radix (a) and Bupleuri radix (b) using magnetic-activated cell sorting. Each column represents the mean ± SEM of 3 to 5 mice. *** p < 0.001, * p < 0.05 vs. the control group using ANOVA with Bonferroni correction for multiple comparisons. †† p < 0.01 vs. the control group using ANOVA with Bonferroni correction for the selected two group. See the Materials and Methods section for details. Fig. S4 Effect of different administration periods of hot water extract of Bupleuri radix on the induction and/or activation of regulatory T cells (Tregs) in TNCB-induced CHS response without sensitization with 5% TNCB in donor mice. (a) Experimental protocol. (b) Ear swelling 24 h after the topical application of 1% TNCB on the ear. N, normal group; Vehicle, vehicle-negative control group; CTRL, control group; BR, hot water extract of Bupleuri radix group. Each column represents the mean ± SEM of 3 to 5 mice. ***p < 0.001vs. the control group using ANOVA with Bonferroni correction for multiple comparisons. † p < 0.05 vs. the control group using ANOVA with Bonferroni correction for the selected two group. See the Materials and Methods section for details (PDF 572 KB)

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Tsuge, A., Chiba, S., Yagura, Y. et al. Hochuekkito exerts the anti-allergic effects via activating regulatory T cells in a murine model of contact hypersensitivity. J Nat Med 77, 352–362 (2023). https://doi.org/10.1007/s11418-023-01683-0

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