Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Abstract

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Fig. 1

HPLC chromatograms of alcohol extract of EPA: (A) lipophilic alkamides and ketones detected with UV absorbance at 210 nm (upper line) and 260 nm (lower line), and (B) water soluble caffeic acid derivatives detected with UV absorbance at 330 nm. HPLC analysis was performed as described previously by Wu et al. (2004) and Lalone et al. (2007).

Fig. 2

Effects of RST and EPA on body weight loss. A sterile 3.5 mm punch biopsy was used to make duplicate wounds on the dorsum on day 0. The stressed animals were administrated three cycles of 12-h interval restraint (RST) stress prior to wounding and 4 cycles post wounding. The non-stressed mice were deprived of water and food (FWD) when the stressed mice were under restraint cycles except the food ad lib group, which had free access to food and water throughout the experimental procedure. Mice were gavaged with three doses of alcohol extract of EPA (130 mg/kg) prior to and four doses post wounding, one dose per day or an equal volume of vehicle. Body weight was recorded on days as indicated and then was adjusted by the weight at the start of the experiment to reflect change in body weight. Data represent the mean ± SE of each treatment group (n= 6–7 mice/group).

Fig. 3

Effects of RST and EPA on wound closure of (A) the non-restraint stressed groups and (B) the stressed groups with the non-stressed FWD group. The animals were treated with RST and EPA as described in Fig. 1. Wounds were photographed immediately after wounding and then daily until healing. Individual wound size was calculated as a ratio in comparison to a standard-sized dot placed next to the wound and expressed as the percentage of the original size. Data represent the mean ± SE of each treatment group (n= 6–7 mice/group).

Fig. 4

Effects of RST and EPA on healing time. Healing, assessed daily by application of 3% hydrogen peroxide beginning 7 days after wounding, was defined as the absence of enzymatic reaction. Data represents the mean ± SE of each treatment group (n=6–7 mice/group). The healing time of each individual animal is the average of its two wounds. *p<0.001 vs the Food ad lib and FWD groups; # p<0.001 vs the RST + EPA group.

Fig. 5

Effects of RST and EPA on (A) spleen/body weight ratio, (B) proliferation of splenocytes treated with LPS (1 µg/ml) and corticosterone, and plasma concentrations of (C) corticosterone and (D) IL-6. Animals were administrated four consecutive 12-h interval restraint stress cycles (during light phase) and at the same time treated with an alcohol extract of EPA (130 mg/kg) body weight once daily for 4 doses or an equal volume of vehicle. Two hours after the last stress cycle, animals were euthanized and body weight and spleen weight were recorded. Corticosterone resistance, plasma corticosterone and IL-6 were measured as described in the Material and methods. Dashed line in B indicates cell viability of splenocytes of each treatment group stimulated with LPS alone and referred to 100%. Data represent mean ± standard error of the mean of 3–4 mice. *p<0.05; ** p<0.001.