■犬特应性皮炎(CAD)是由过敏原暴露引起的皮肤屏障功能障碍引起的。皮肤中过量的谷氨酸盐释放与延迟的皮肤屏障功能恢复以及表皮增厚和苔藓化有关。用Yokukansan(YKS)治疗,一种传统的日本药物,通过降低表皮谷氨酸水平来降低NC/Nga小鼠的皮炎严重程度和抓挠行为。然而,犬角质形成细胞与谷氨酸之间的关联以及YKS抑制角质形成细胞释放谷氨酸的机制尚不清楚.
■我们旨在研究犬祖细胞表皮角质形成细胞(CPEK)的谷氨酸释放以及YKS对这种释放的抑制作用。我们还探索了YKS的潜在机制,使其能够在CAD治疗中应用。
■测定在24小时时在培养基中由CPEK产生的谷氨酸。测量条件根据细胞密度和YKS浓度而变化。用谷氨酸受体拮抗剂(MK-801)处理CPEK,谷氨酸转运蛋白拮抗剂(THA),和谷氨酸脱氢酶抑制剂(表没食子儿茶素没食子酸酯;EGCG),以及YKS的抑制作用,YKS+THA,确定了MK-801和EGCG对该释放的影响。MK-801和谷氨酸脱氢酶抑制剂单独测试,THA与YKS联合测试。最后,使用放射性同位素标记测量在24小时掺入CPEK中的谷氨酰胺。
■CPEKs以细胞密度依赖性方式释放谷氨酸,以浓度依赖的方式被YKS抑制。此外,YKS以浓度依赖性方式降低了放射性同位素标记的谷氨酰胺的细胞内摄取。未发现谷氨酸受体拮抗作用或谷氨酸转运体的激活,正如以前的研究所建议的那样。此外,EGCG可抑制CPEK的谷氨酸释放。
■我们的发现表明,YKS可以有效抑制CPEK中的谷氨酸释放,提示YKS在CAD期间维持皮肤屏障功能的效用。此外,CPEK适用于分析YKS的机制。然而,我们发现YKS的作用机制与以前的研究不同,这表明在这项研究中它可能与EGCG有类似的作用。需要进一步研究以了解治疗CAD的确切机制和临床疗效。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.