Skin Wound-Healing Potential of Polysaccharides from Medicinal Mushroom Auricularia auricula-judae (Bull.)

Abstract

Auricularia auricula-judae, a nutrient-rich mushroom used in traditional medicine, is a macrofungi that exhibits various biological properties. In this study, we have reported on the mechanisms that promote the wound-healing effects of a water-soluble polysaccharide-rich extract obtained from A. auricula-judae (AAP). AAP contained high amounts of polysaccharides (349.83 ± 5.00 mg/g extract) with a molecular weight of 158 kDa. The main sugar composition of AAP includes mannose, galactose, and glucose. AAP displayed antioxidant activity in vitro and was able to abort UVB-induced intracellular ROS production in human fibroblasts in cellulo. AAP significantly promoted both fibroblast and keratinocyte proliferation, migration, and invasion, along with augmentation of the wound-healing process by increasing collagen synthesis and decreasing E-cadherin expression (All p < 0.05). Specifically, the AAP significantly accelerated the wound closure in a mice skin wound-healing model on day 9 (2.5%AAP, p = 0.031 vs. control) and day 12 (1% and 2.5%AAP with p = 0.009 and p < 0.001 vs. control, respectively). Overall, our results indicate that the wound-healing activities of AAP can be applied in an AAP-based product for wound management.

Keywords: Auricularia auricula-judae; collagen synthesis; fibroblasts proliferation; in vivo skin wound healing; keratinocytes proliferation; medicinal mushroom; polysaccharide-rich extract; wound healing.

Figure 1

Polysaccharide characteristics of A. auricula-judae (AAP). The molecular weight of the polysaccharides was determined by Sephacryl S-400 gel filtration chromatography. (a) AAP was eluted with 0.2 M ammonium bicarbonate at a flow rate of 25 mL/h. The fractions were assayed for hexose sugars (A490 nm), and the standard proteins are represented by arrows to indicate molecular size (kDa). (b) The molecular weight was calculated using the calibration logMW curve of standard proteins (ranging from 1.3–670 kDa.).

Figure 2

Antioxidant activities of AAP. The antioxidant activities of AAP were determined by ABTS assay (a) and DCF-DA fluorescent assay (b). For ABTS assay, the AAP (0–400 μg/mL), and Vit E at 15 μg/mL was used as a positive control. The intracellular ROS production was calculated as the percent of inhibition. For DCF-DA antioxidant activity, primary human skin fibroblasts were exposed to UVB at 15 mJ/cm² using ultraviolet crosslinker. The intracellular ROS after UVB irradiation in fibroblasts was determined by DCF-DA dye. Vit C at 25 µg/mL was used as positive control. Data are represented as mean ± S.D. values of three independent experiments, ** p < 0.01 and *** p < 0.001 vs. the control.

Figure 3

AAP induced fibroblast and keratinocyte cell proliferation. The effect of AAP on human fibroblasts and keratinocyte cell proliferation was determined by MTT assay (a,b) and trypan blue cell counting (c,d). The cells were treated with increasing concentrations of AAP for 24 and 48 h and cell proliferation was determined by MTT assay or trypan blue cell counting method. Data are represented as mean ± S.D. values of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control.

Figure 4

AAP promoted healing activity via facilitating migration and invasion in both human fibroblast and keratinocyte cells. The effects of AAP on human fibroblast and keratinocyte cells wound healing were determined by scratch assay ((a); fibroblasts and (b); HaCat cells and the wound-healing activity at 48 h was calculated as shown in the histogram inserts). Transwell assay was used to investigate human fibroblasts (c) and Hacat (d) cells migration (cell migration at 48 h was calculated as shown in the histogram inserts) and invasion ((e); fibroblast and (f); Hacat cells and cell invasion was calculated as shown in the histogram inserts). Data are presented as mean ± S.D. values of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control.

Figure 5

AAP decreased E-cadherin expression in keratinocytes and enhanced collagen synthesis in fibroblasts. The E-cadherin expression (a) in keratinocyte cells after AAP treatment for 24 h was determined by Western blot analysis. The bands were normalized to β-actin as a loading control. The quantification level of protein expression was determined by IMAGE J software. Collagen synthesis (b) was determined using a Sirius Red Collagen Detection kit. Human fibroblasts were starved in serum-free medium for 24 h. Cells were then treated with various concentrations of AAP (0–25 µg/mL) for 48 h. Data are presented as mean ± S.D. values of three independent experiments, ** p < 0.01 and *** p < 0.001 vs. control.

Figure 6

AAP accelerated wound closure and wound healing in vivo. (a) Photographs of the full thickness excision wounds in BALB/C mice in each group on day 0, 3, 6, 9, and 12 of the experiment. (b) The ratio of wound closure was expressed as relative wound area compared with that which was present on each day of the experiment. (c) H&E (upper panel) and Masson’s Trichrome (lower panel) stained wound tissues after 12 days of treatment with 0.9% sterilized normal saline solution (left), 1%w/v AAP (middle), and 2.5%w/v AAP (right). Data are expressed as Mean ± SE values, n = 7 for each group. *p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle control group.