Intracellular Ca2+-Elevating Effects of Angelicae acutilobae Radix,Paeoniae Radix, and Moutan Cortex Extracts in Keratinocytes and theirPartial Inhibition by Ca2+-Permeable Channel Modulators

Introduction/Background: Cold disorder is a subjective symptom reported in East Asian populations, particularly among women. Kampo formulas, including Tangkuei and Peony Powder (TPP) and Cinnamon Twig and Poria Pill (CTPP), are used to alleviate cold disorder symptoms. TPP contains Angelicae acutilobae Radix (AAR) and Paeoniae Radix (PR), whereas CTPP contains PR and Moutan Cortex (MC). Keratinocytes, recognized as contributors to thermosensation, may mediate some effects of these crude drugs through Ca2+-dependent signaling. However, the mechanisms remain unclear. To compare the effects of AAR, PR, and MC extracts on intracellular Ca²⁷ dynamics and phosphorylation of proline-rich tyrosine kinase 2 (p-Pyk2) in fetal rat skin keratinocytes (FRSK). Method: Intracellular Ca2+ levels were measured using Fluo-4. Phosphorylation of Pyk2 was assessed by Western blotting. The effects of 2-aminoethoxydiphenyl borate (2-APB) and NK-4 on Ca2+ responses were assessed. Expression of TRPM2, TRPM7, and TRPV2 was analyzed by RT-qPCR. Results: All three extracts elevated intracellular Ca2+ and induced p-Pyk2 activation. AAR responses were inhibited by 2-APB and NK-4, whereas PR and MC responses were partially suppressed by 2-APB. TRPM2, TRPM7, and TRPV2 transcripts were detected, with TRPM7 showing the highest expression Discussion: These findings suggest that AAR, PR, and MC modulate keratinocyte Ca²⁷ signaling through multiple Ca²⁷-permeable pathways. Further studies are warranted to clarify the contributions of TRP channels, intracellular Ca²⁷ stores, and other signaling routes. Conclusion: This study establishes a comparative framework under standardized conditions linking Kampo crude drug exposure to Ca²⁷-dependent signaling in keratinocytes, supporting future mechanistic investigations of their physiological relevance.