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. 2016 Jun 7:4:54.
doi: 10.3389/fcell.2016.00054. eCollection 2016.

Efficient Use of a Crude Drug/Herb Library Reveals Ephedra Herb As a Specific Antagonist for TH2-Specific Chemokine Receptors CCR3, CCR4, and CCR8

Kazuhiko Matsuo  1 , Keiichi Koizumi  2 , Mitsugu Fujita  3 , Toshio Morikawa  4 , Michiko Jo  2 , Naotoshi Shibahara  2 , Ikuo Saiki  5 , Osamu Yoshie  3 , Takashi Nakayama  1
Affiliations

Efficient Use of a Crude Drug/Herb Library Reveals Ephedra Herb As a Specific Antagonist for TH2-Specific Chemokine Receptors CCR3, CCR4, and CCR8

Kazuhiko Matsuo et al. Front Cell Dev Biol. .

Abstract

Chemokine receptors CCR3 and CCR4 are preferentially expressed by TH2 cells, mast cells, and/or eosinophils, all of which are involved in the pathogenesis of allergic diseases. Therefore, CCR3 and CCR4 have long been highlighted as potent therapeutic targets for allergic diseases. Japanese traditional herbal medicine Kampo consists of multiple crude drugs/herbs, which further consist of numerous chemical substances. Recent studies have demonstrated that such chemical substances appear to promising sources in the development of novel therapeutic agents. Based on these findings, we hypothesize that Kampo-related crude drugs/herbs would contain chemical substances that inhibit the cell migration mediated by CCR3 and/or CCR4. To test this hypothesis, we screened 80 crude drugs/herbs to identify candidate substances using chemotaxis assay. Among those tested, Ephedra Herb inhibited the chemotaxis mediated by both CCR3 and CCR4, Cornus Fruit inhibited that mediated by CCR3, and Rhubarb inhibited that mediated by CCR4. Furthermore, Ephedra Herb specifically inhibited the chemotaxis mediated by not only CCR3 and CCR4 but CCR8, all of which are selectively expressed by TH2 cells. This result led us to speculate that ephedrine, a major component of Ephedra Herb, would play a central role in the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. However, ephedrine exhibited little effects on the chemotaxis. Therefore, we fractionated Ephedra Herb into four subfractions and examined the inhibitory effects of each subfraction. As the results, ethyl acetate-insoluble fraction exhibited the inhibitory effects on chemotaxis and calcium mobilization mediated by CCR3 and CCR4 most significantly. In contrast, chloroform-soluble fraction exhibited a weak inhibitory effect on the chemotaxis mediated by CCR8. Furthermore, maoto, one of the Kampo formulations containing Ephedra Herb, exhibited the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. Taken together, our data suggest that these crude drugs/herbs might be useful sources to develop new drugs targeting TH2-mediated allergic diseases.

Keywords: CCR3; CCR4; Ephedra Herb; antagonist; chemokine receptor; maoto.

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Figures

Figure 1
Figure 1
Ephedra Herb inhibits the chemotaxis mediated by CCR3, CCR4, and CCR8. (A), Cell migration assay was performed using L1.2 cells stably expressing CCR3 (L1.2-CCR3) and 10 nM CCL11 in the presence of each crude drug/herb extract at 10 μg/ml (open columns) or 100 μg/ml (closed columns). Each experiment was repeated three times. Cell migration activity is shown in a percentage relative to the control (mean ± SE). (B), Cell migration assay was performed using L1.2-CCR4 and 10 nM CCL22 in the presence of each extract at 10 μg/ml (open columns) or 100 μg/ml (closed columns). (C), Cell migration assay was performed using the following cells and corresponding chemokines in the presence of Ephedra Herb at 10 μg/ml (open columns) or 100 μg/ml (closed columns): L1.2-CCR1/CCL5, L1.2-CCR2/CCL2, L1.2-CCR3/CCL11, L1.2-CCR4/CCL22, L1.2-CCR5/CCL5, and L1.2-CCR8/CCL1. Each chemokine was used at 10 nM. P-values were based on ANOVA with Holm's post-hoc test (A,B) and Student's t-test (C). *P < 0.05 and **P < 0.01 compared with the controls.
Figure 2
Figure 2
Ethyl acetate (EtOAc)-insoluble fraction of Ephedra Herb inhibits the chemotaxis mediated by CCR3 and CCR4. (A), Cell migration assay was performed using the following cells and corresponding chemokines in the presence of ephedrine at the indicated concentrations: L1.2-CCR3/CCL11, L1.2-CCR4/CCL22, and L1.2-CCR8/CCL1. Unfractionated Ephedra Herb was used as a control. Each chemokine was used at 10 nM. Each experiment was repeated three times. Cell migration activity is shown in a percentage relative to the control (mean ± SE). (B), Cell migration assay was performed using L1.2-CCR3/CCL11, L1.2-CCR4/CCL22, and L1.2-CCR8/CCL1 in the presence of each fraction of Ephedra Herb at 100 μg/ml. (C), Cell migration assay was performed as described in the panel (B) in the presence of Ephedra Herb and its EtOAC-insoluble fraction at the indicated concentrations. EC50 was also calculated. (D), Cell migration assay was performed using the following cells and corresponding chemokines in the presence of EtOAC-insoluble fraction of Ephedra Herb at 0 μg/ml (open column), 10 μg/ml (closed columns), or 100 μg/ml (gray columns): L1.2-CCR1/CCL5, L1.2-CCR2/CCL2, L1.2-CCR3/CCL11, L1.2-CCR4/CCL22, L1.2-CCR5/CCL5, and L1.2-CCR8/CCL1. Each chemokine was used at 10 nM. (E), Calcium mobilization assay was performed using L1.2-CCR3/CCL11 and L1.2-CCR4/CCL22 in the presence of Ephedra Herb and its EtOAC-insoluble fraction. The cells were loaded with fura 2-AM and stimulated with the corresponding chemokines at 10 nM with or without Ephedra Herb and its EtOAC-insoluble fraction at 100 μg/ml. Intracellular calcium mobilization was measured on a fluorescence spectrophotometer. Each experiment was repeated three times; representative results are presented. P-values were based on ANOVA with Holm's post-hoc test (B) and Student's t-test (A,D). *P < 0.05 and **P < 0.01 compared with the controls.
Figure 3
Figure 3
EtOAc-insoluble fraction of Ephedra Herb inhibits all ligand-induced chemotaxis mediated by CCR3 and CCR4. (A), Cell migration assay was performed using L1.2-CCR3 and the following chemokines in the presence of the EtOAC-insoluble fraction of Ephedra Herb at 0 μg/ml (open column), 10 μg/ml (closed column), or 100 μg/ml (gray column): CCL11, CCL24, CCL26, CCL13, and CCL5. Each chemokine was used at 10 nM. (B), Cell migration assay was performed using L1.2-CCR4, CCL22, and CCL17 in the presence of the EtOAC-insoluble fraction of Ephedra Herb at 0 μg/ml (open columns), 10 μg/ml (closed columns), or 100 μg/ml (gray columns). Both chemokines were used at 10 nM. Each experiment was repeated three times; representative results are presented. Cell migration activity is shown in a percentage relative to the control (mean ± SE). P-values were based on Student's t-test. *P < 0.05 and **P < 0.01 compared with the controls.
Figure 4
Figure 4
Maoto inhibits the chemotaxis mediated by CCR3, CCR4, and CCR8. Cell migration assay was performed using the following cells and corresponding chemokines in the presence of maoto at the indicated concentrations: L1.2-CCR3/CCL11 (A), L1.2-CCR4/CCL22 (B), and L1.2-CCR8/CCL1 (C). Each chemokine was used at 10 nM. Each experiment was repeated three times; representative results are presented. Cell migration activity is shown in a percentage relative to the control (mean ± SE). P-values were based on Student's t-test. *P < 0.05 and **P < 0.01 compared with the controls.
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