Red ginseng extract blocks histamine-dependent itch by inhibition of H1R/TRPV1 pathway in sensory neurons

Abstract

Background: Korean Red Ginseng-a steamed root of Panax ginseng Meyer-has long been used as a traditional medicine in Asian countries. Its antipruritic effect was recently found, but no molecular mechanisms were revealed. Thus, the current study focused on determining the underlying molecular mechanism of Korean Red Ginseng extract (RGE) against histamine-induced itch at the peripheral sensory neuronal level.

Methods: To examine the antipruritic effect of RGE, we performed in vivo scratching behavior test in mice, as well as in vitro calcium imaging and whole-cell patch clamp experiments to elucidate underlying molecular mechanisms.

Results: The results of our in vivo study confirmed that RGE indeed has an antipruritic effect on histamine-induced scratching in mice. In addition, RGE showed a significant inhibitory effect on histamine-induced responses in primary cultures of mouse dorsal root ganglia, suggesting that RGE has a direct inhibitory effect on sensory neuronal level. Results of further experiments showed that RGE inhibits histamine-induced responses on cells expressing both histamine receptor subtype 1 and TRPV1 ion channel, indicating that RGE blocks the histamine receptor type 1/TRPV1 pathway in sensory neurons, which is responsible for histamine-dependent itch sensation.

Conclusion: The current study found for the first time that RGE effectively blocks histamine-induced itch in peripheral sensory neurons. We believe that the current results will provide an insight on itch transmission and will be helpful in understanding how RGE exerts its antipruritic effects.

Keywords: H1R; Korean Red Ginseng extract; TRPV1; histamine; itch.

Fig. 1

Korean Red Ginseng extract (RGE) inhibits histamine-induced scratching in mice. RGE was intraperitoneally injected 30 minutes prior to histamine application, and 5 μg/site of histamine was administered subcutaneously into the dorsal neck of 6–8-week-old ICR mice. (A) A time-course graph for histamine-induced scratching between mice that did not receive pretreatment (His, filled circle, n = 6) versus mice pretreated with 50 mg/animal RGE (+RGE 50 mg, empty square, n = 7) for 1 hour. (B) Total bouts of histamine-induced scratching (5 μg/site) among different doses (5-, 10-, and 50-mg/animal) of RGE pretreatment. ANOVA with Tukey's post hoc test was applied for statistical analysis. Error bars indicate standard error mean. * p < 0.05. ANOVA, analysis of variance; ICR, Institute of Cancer Research strain.

Fig. 2

RGE blocks histamine-induced responses in primary culture of mouse dorsal root ganglia (DRG). (A) A representative current induced by 100μM histamine (His) in mouse DRG with whole-cell configuration. (B) When RGE is added during histamine application, the current started to recover to the basal levels faster. (C) Summary of peak currents induced by 100μM histamine alone (His, white, n = 7) or RGE-added cells (+RGE, black, n = 7). *** p < 0.001 with Student t test. (D) Summary of recovery time between the two groups. Recovery time was defined as time that elapsed until currents reverted to 95% of the basal level. *** p < 0.001 with Student t test. (E) Representative ratiometric calcium imaging data showing the effect of RGE (10 μg/mL and 100 μg/mL) over histamine-induced responses. Donut-shaped diagrams indicate how many cells were responsive to the stimuli. Cells were regarded as responsive when F/F₀ is > 1.8. Notice that 100 μg/mL of RGE significantly blocked histamine-induced Ca²⁺ influx. Pseudocolors represent F/F₀, where F indicates the fluorescence intensity of calcium-specific dye and F₀ indicates the initial fluorescence intensity. Scale bar = 100 μm. (F) Time-course traces of calcium imaging with histamine with or without RGE pretreatment up to 2 minutes. RGE, red ginseng extract.

Fig. 3

RGE inhibits histamine-induced H1R/TRPV1 pathway. (A) Traces of Ca²⁺ influx induced by histamine in the absence (empty circle, n = 1873) or presence (10 μg/mL: gray, n = 918, 100 μg/mL: black, n = 1123) of RGE for up to 120 seconds in HEK293T cells transiently coexpressing H1R and TRPV1 (H1R/TRPV1). (B) Summary of peak Ca²⁺ influx (F/F₀) induced by either histamine alone or histamine pretreated with RGE. *** p < 0.001, ANOVA with Tukey's post hoc test. (C) Representative ratiometric calcium imaging data of histamine-induced Ca²⁺ influx before and after treatment of 10 μg/mL or 100 μg/mL RGE at 60 seconds. Scale bar = 100 μm. ANOVA, analysis of variance; H1R, histamine receptor subtype 1; RGE, red ginseng extract.

Fig. 4

RGE shows mixed effects on capsaicin-induced Ca²⁺ influx. (A) Traces of Ca²⁺ influx induced by capsaicin in the absence (open circle, n = 250) or presence (10 μg/mL: gray, n = 194, 100 μg/mL: black, n = 338) of RGE for 120 seconds in HEK293T cells expressing TRPV1 alone. (B) Summary of peak Ca²⁺ influx (F/F₀) induced by either capsaicin alone or capsaicin pretreated with RGE. Notice the different effects induced by different RGE concentrations. *** p < 0.001, ANOVA with Tukey's post hoc test. (C) Representative ratiometric calcium imaging data of capsaicin-induced Ca²⁺ influx before and after treatment of 10 μg/mL or 100 μg/mL RGE at 60 seconds. Scale bar = 20 μm. ANOVA, analysis of variance; RGE, red ginseng extract.