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. 2024 Feb;14(2):683-691.
doi: 10.5455/OVJ.2024.v14.i2.8. Epub 2024 Feb 29.

Suppressive effect of Yokukansan on glutamate released from canine keratinocytes

Yoichiro Kasuga  1 , Ailing Hu  2 , Zenji Kawakami  2 , Masahiro Tabuchi  2 , Takuji Yamaguchi  2 , Hiroyuki Kobayashi  2 , Shigaku Ikeda  1
Affiliations

Suppressive effect of Yokukansan on glutamate released from canine keratinocytes

Yoichiro Kasuga et al. Open Vet J. 2024 Feb.

Abstract

Background: Canine atopic dermatitis (CAD) is caused by skin barrier dysfunction due to allergen exposure. Excessive glutamate release in the skin is associated with delayed skin barrier function recovery and epidermal thickening and lichenification. Treatment with Yokukansan (YKS), a traditional Japanese medicine, reduces dermatitis severity and scratching behavior in NC/Nga mice by decreasing epidermal glutamate levels. However, the association between canine keratinocytes and glutamate and the mechanism by which YKS inhibits glutamate release from keratinocytes remains unknown.

Aim: We aimed to investigate glutamate release from canine progenitor epidermal keratinocytes (CPEKs) and the inhibitory effect of YKS on this release. We also explored the underlying mechanism of YKS to enable its application in CAD treatment.

Methods: Glutamate produced from CPEKs in the medium at 24 hours was measured. The measurement conditions varied in terms of cell density and YKS concentration. CPEKs were treated with a glutamate receptor antagonist (MK-801), a glutamate transporter antagonist (THA), and a glutamate dehydrogenase inhibitor (epigallocatechin gallate; EGCG), and the inhibitory effect of YKS, YKS + THA, MK-801, and EGCG on this release was determined. MK-801 and glutamate dehydrogenase inhibitor were tested alone, and THA was tested in combination with YKS. Finally, glutamine incorporated into CPEKs at 24 hours was measured using radioisotope labeling.

Results: CPEKs released glutamate in a cell density-dependent manner, inhibited by YKS in a concentration-dependent manner. Moreover, YKS reduced the intracellular uptake of radioisotope-labeled glutamine in a concentration-dependent manner. No involvement of glutamate receptor antagonism or activation of glutamate transporters was found, as suggested by previous studies. In addition, EGCG could inhibit glutamate release from CPEKs.

Conclusion: Our findings indicated that glutamate release from CPEKs could be effectively inhibited by YKS, suggesting the utility of YKS in maintaining skin barrier function during CAD. In addition, CPEKs are appropriate for analyzing the mechanism of YKS. However, we found that the mechanism of action of YKS differs from that reported in previous studies, suggesting that it may have had a similar effect to EGCG in this study. Further research is warranted to understand the exact mechanism and clinical efficacy in treating CAD.

Keywords: Canine atopic dermatitis; Canine progenitor epidermal keratinocyte; Glutamate; Glutamine; Yokukansan.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Fig. 1.
Fig. 1.. Correlation between the cell density of CPEKs and glutamate release. Mean + SEM (n = 6), one-way ANOVA + Tukey’s test, *p < 0.001.
Fig. 2.
Fig. 2.. YKS inhibits measurements by the glutamate assay kit in a concentration-dependent manner. Mean + SEM (n = 6), one-way ANOVA + Tukey’s test, *p < 0.001.
Fig. 3.
Fig. 3.. Effect of YKS on glutamate released from CPEKs under (a) confluent and (b) subconfluent conditions. Glutamate was calculated as the amount released from the cells over 24 hours. Mean + SEM (n = 6), one-way ANOVA + Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 4.
Fig. 4.. Glutamine uptake by CPEKs decreased in a YKS concentration-dependent manner. No significant difference was observed at 125 μg/ml as compared with control; however, there was a significant decrease at 250 and 500 μg/ml. Mean + SEM (n = 6), one-way ANOVA + Tukey’s test, *p < 0.05, **p < 0.001.
Fig. 5.
Fig. 5.. EGCG affects glutamate release. Glutamate levels in the (a) cells and (b) medium were measured via a glutamate assay. No significant difference was observed at 125 μg/ml compared with controls, but there were significant decreases at 250 and 500 μg/ml (a). Mean + SEM (n = 6), one-way ANOVA + Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. S1.
Fig. S1.. Effect of MK-801 on YKS-induced reduction of glutamate in the medium. The effect of the NMDAR antagonist activity was investigated using only MK-801. No statistically significant differences were observed between no MK-801 and with MK-801 (2, 20, and 200 μM). Mean + SEM (n = 6), one-way ANOVA + Dunnett’s test, NS: Not Significant.
Fig. S2.
Fig. S2.. Effect of THA on the YKS-induced reduction of glutamate in the medium. YKS and THA were coadministered to examine the involvement of glutamate transporter. No statistically significant differences were observed between no THA and with THA at 10, 100, and 1,000 μM. Mean + SEM (n = 6), one-way ANOVA + Dunnett’s test, NS: Not Significant.
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