Tangtang He, 1 , 2 , 3 Pengfei Sun, 1 , 3 Bo Liu, 1 , 3 Shiwei Wan, 1 , 2 , 3 Penghua Fang, 2 , 3 Jun Chen, 4 Guicheng Huang, 1 , 3 , * and Wen Min
Keywords: diabetic wound ulcer, Puffball spores, oxidative stress, wound healing, angiogenesis
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.
Introduction
Diabetic foot ulcers (DFUs) are a common complication of diabetes. It is a significant health concern worldwide with a challenging clinical treatment (1, 2). This highly preventable diabetes complication is the primary cause of lower-extremity amputations (3). A study found that patients with DFUs had a much 2.5-fold higher risk of death than patients without lower extremity wounds, with an estimated 5-year mortality rate of 42% for patients with DFUs (4). Although various drugs are availabled for DFUs, the ulcers usually remain refractory and impact the quality of life of patients (5). Therefore, it is essential to investigate new strategies to accelerate healing of these cutaneous ulcers.
Wound healing is a complex process,that can be divided into main four progressive stages: hemostasis, inflammation, proliferation, and remodeling (6). Compared with non-diabetic wounds, the diabetic wound healing process has a prolonged the inflammatory stage, with the excessive secretion of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin(IL)-1β, and IL-6. This leads to increased tissue damage, which delays diabetic wound healing by interfering with the proliferation and maturation stages (7). In long-term hyperglycemia, accumulated advanced glycation end products (AGEs) increase reactive oxygen species (ROS) production (8). Excessive free radicals and ROS production in wound tissue can lead to an imbalance between oxidation and antioxidation. ROS, including anion superoxide, hydroxyl radicals, peroxide radicals, and nitric acid radicals, are key signaling molecules contributing to inflammatory disease progressions (9). As a result, wound healing results in a prolonged inflammatory response and a severe angiogenesis disorder. In patients with diabetic wounds, prolonged inflammation and blocked angiogenesis are the main reasons why taking a longer healing time than that for patients with normal wounds. Maintaining oxidative stress balance is favorable for accelerating diabetic wound healing.
Puffball is a traditional Chinese medicine, commomly used to trean several diseases such as hemostasis, throat pain, and cough (10). Puffball is mostly used as wound dressings, because dry, mature spores have a positive effect on wound hemostasis. Although it has been confirmed to possess effective clinical value, its active components and monomers have only recently been separated and identified. Some puffball extracts exert anti-cancer activity (11). Puffball extracts were shown to decrease the formation of nitric oxide (NO) in RAW264.7 macrophages activated with lipopolysaccharide (LPS), eliciting an anti-inflammatory effect (12). In addition, Puffball extracts have also shown scavenging potential, reducing the levels of free radicals and enhancing antioxidative action (13).
Growing evidence indicates that puffball spores have positive effects on diabetic wounds. Nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice with diabetses showed delayed wound healing, accompanied by oxidative damage (14). However, it remains unclear whether puffball spores promote diabetic wound healing by promoting antioxidant enzyme activity and activating the AKT/Nrf2 signaling pathway to enhance antioxidation. Further studies of oxidative stress markers and related signaling pathways are needed to clarify the mechanism underlying the action of these spores. We created a diabetic animal model in vivo. To examine the therapeutic effects of puffball spores on diabetes wound healing and to investigate the machanisms underlying their anti-inflammatory, angiogenesis, and antioxidation mechanisms.