Tokishakuyakusan ameliorates lowered body temperature after immersion in cold water through the early recovery of blood flow in rats

'Cold feeling' is a subjective feeling of unusual coldness that aggravates fatigue, stiffness, and other symptoms, thereby reducing quality of life. Tokishakuyakusan (TSS) is a Kampo medicine reported to improve cold feeling and is used to treat symptoms aggravated by cold feeling. However, the mechanism of action of TSS is unclear. Cold feeling may involve reduced blood flow and subsequent inhibition of heat transport. Therefore, elucidating the effects of TSS on blood flow is one of the most important research topics for clarifying the mechanism of action of TSS.We aimed to evaluate the effect of TSS on recovery from lowered body temperature by the immersion of rats in cold water and to clarify the involvement of blood flow in the action of TSS.After female Wistar rats underwent 9 days of low room temperature stress loading (i.e. room temperature of 18 °C), they were subjected to immersion in cold water (15 °C) for 15 min. Body surface temperature, rectal temperature, and plantar temperature were measured before and after immersion in cold water. Blood flow was measured before and after immersion in cold water without low room temperature stress loading. TSS (0.5 g/kg or 1 g/kg) or the vehicle (i.e. distilled water) was orally administered once daily for 10 days for the measurement of body temperature or once 30 min before immersion in cold water for the measurement of blood flow. In addition, we examined the effect of TSS on calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) cells, the effect of TSS ingredients on transient receptor potential (TRP) channels, and the effect of TSS ingredients on the membrane potential of vascular smooth muscle cells and evaluated the mechanism of the effects of TSS on blood flow.Body temperature and blood flow decreased after immersion in cold water and then recovered over time. A comparison of body temperature at each timepoint or area under the curve showed that TSS (1 g/kg) accelerated the recovery of body surface temperature, rectal temperature, and blood flow. TSS significantly increased CGRP release from DRG cells, which disappeared after pretreatment with HC-030031 (a transient receptor potential ankyrin 1 [TRPA1] antagonist). The effects of seven TSS ingredients on TRP channels were examined. The agonistic effect on TRPA1 was observed for atractylodin, atractylodin carboxylic acid and levistolide A. Among the TSS ingredients, atractylodin carboxylic acid had significant hyperpolarising effects.The mechanism by which TSS accelerates the recovery of lowered body temperature in rats after immersion in cold water may involve the acceleration of the recovery of lowered blood flow. Increased CGRP release from DRG cells by TSS, TRPA1 activation by TSS ingredients, and membrane potential changes in vascular smooth muscle cells caused by TSS ingredients are part of the mechanism of action of TSS. These findings may partly contribute to the interpretation of the beneficial effects of TSS on cold feeling.