Puffball spores improve wound healing in a diabetic rat model

Abstract

Persistent chronic oxidative stress is a primary pathogenic characteristics of diabetic foot ulcers. Puffball spores are a traditional Chinese medicine used to treat diabetic foot ulcers infections and bedsores. However, their effects against diabetic wounds and the mechanism underlying these effects remain largely unknown. The present study explored the effectiveness of puffball spores in diabetic wound treatment and the mechanisms underlying their effects. Sprague-Dawley rats with streptozotocin (STZ)-induced diabetes were treated with puffball spores to ascertain whether they accelerated wound healing.Real-time quantitative PCR, western blotting, hematoxylin-eosin and Masson's trichrome staining, immunohistochemistry analysis, and immunofluorescence assays were performed. As indicated by wound and serum histology and biochemical analyses, the puffball spores accelerated wound healing by activating Akt/Nrf2 signaling and promoting the expression of its downstream antioxidant genes, markedly stimulating antioxidant activity and enhanceing angiogenesis and collagen deposition. Our findings showed that puffball spores could accelerate diabetic wound healing, enhance antioxidant ability, promote the expression of vascular markers, and suppress inflammation, thus providing a theoretical basis for the treatment of diabetic and refractory wounds.

Keywords: Puffball spores; angiogenesis; diabetic wound ulcer; oxidative stress; wound healing.

Figure 1

The effects of wound healing by puffball spore treatment in diabetic rats. (A) The wound areas were recorded by a camera on days 0, 3, 7, and 14. (B) Heat map of the wound healed by different treatments over 14 days. (C) Quantitative data of relative wound area at different time points.(Means ± SEM, n = 7; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Model group, ##P < 0.01 vs. positive group.).

Figure 2

Puffball spores improved diabetic foot ulcer recovery in rats. (A) Representative HE staining images of wound tissues, (scale bar = 100 µm; magnification, 200×; n = 3). (B) Representative images of Masson’s trichrome (MT) staining, (scale bar = 100 µm; magnification, 200×; the blue color represents collagen; n = 3). (C) Wound tissue collagen fiber quantification using Image-Pro Plus. (Means ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P<0.001 vs. model group).

Figure 3

Effects of puffball spores on cytokine production in the serum of diabetics rats. (A) CD31 expression in wound tissues was detected by immunohistochemistry (scale bar = 100 µm; magnification, 400×; n = 3). (B) Quantitative data of wound tissue CD31 expression using IPP. (Means ± SEM, n = 3, **P < 0.01, ***P < 0.001 vs. model group). (C) VEGF, (D) IL-1β and (E)TNF-α were assayed by ELISA. (Means  ±  SEM, n  =  6, *P < 0.05, **P < 0.01 vs. model group; #P < 0.05 vs. positive group).

Figure 4

Puffball spores reduced oxidative damage by increasing antioxidant capacity. (A, B) Immunofluorescent staining reveals a decrease in the fluorescence intensity in the samples from the puffball spore-treatments group (scale bar = 100 µm; magnification, 400×; n = 3). (C) 8-oxo-dG, (D) MDA, (E) CAT, (F) GSH-PX, (G) SOD, and (H) T-AOC levels in serum from diabetics rats were determined using relevant kits. (Means ± SEM; n = 6; *P < 0.05, **P < 0.01, ***P < 0.001, ****p<0.0001 vs. Model group; #P < 0.05 vs. positive group).

Figure 5

Puffball spores activates Akt/Nrf2/HO-1 signal transduction to promote wound healing (A, B) Protein levels of AKT, pAKT, Nrf2, NQO1, and HO-1 protein levels in wound tissue. (C) Nrf2, NQO1, GCLC, GPX1, and HO-1 gene expression in wound tissue. (Means ± SEM, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. Model group). (D) Nrf2, pAKT expression in wound tissues was detected by immunohistochemistry (scale bar = 100 µm; magnification, 200×; n = 3).