Background: The annual incidence of diabetic foot ulcers (DFUs) has been reported to vary from 0.2% to 11% in diabetes-specific clinical settings and less than 0.1% to 8% in community- and population-based cohorts. According to the International Diabetes Foundation, approximately 40 million to 60 million people worldwide are affected by DFUs, and a recent meta-analysis indicates a global prevalence of 6.3% among adults with diabetes, or about 33 million individuals. The cost of diabetes care is significant, amounting to $273 billion in direct and $90 billion in indirect expenses annually, in America. Foot complications in diabetes care excess annual expenditures ranging from 50% to 200% above the baseline cost of diabetes-related care. The cost of advanced-stage ulcers can be more than $50,000 per wound episode, and the direct expenses of major amputation are even higher. DFUs can be treated using various methods, including wound dressings, antibiotics, pressure-off loading, skin substitutes, stem cells, debridement, topical oxygen therapy, gene therapy and growth factors. For severe DFUs patients are at risk of amputation if treatment is not timely or appropriate. Amputating limbs not only causes physical pain to patients, but also brings economic burden due to lost productivity, and decreased employment linked to DFUs. Currently, long-term use of local antibiotics in clinical practice is prone to induce drug resistance, while growth factors do not effectively inhibit bacterial growth and control inflammation in wounds. Stem cell and gene therapies are still in the experimental stage. The method of local debridement combined with negative pressure therapy is expensive. Therefore, we urgently need an affordable, non-surgical method to treat diabetic ulcers. Extracts of bark of Bauhinia purpurea, Paeoniae rubrae, Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., Acorus calamus L, and Radix Angelicae biseratae have been used as traditional remedies to treat inflammation-related diseases and cutaneous wounds due to their anti-inflammatory properties and their ability to promote vascular renewal. However, there have been few studies on the mixture of these five herbal extracts on diabetic wound healing.

Purpose: This study was designed to assess the healing effect of a mixture of five aforementioned herbal extracts on diabetic ulcer wounds in rats, and to reveal the potential mechanisms behind any potential wound healing using transcriptomics and proteomics.

Study design: We designed the experiment to explore the effects of five herbal extracts on diabetic wound healing process through in vivo experiments and to investigate the underlying mechanisms through proteomics and transcriptomics.

Methods: We used a mixture of five aforementioned herbal extract to treat rat model of diabetic established by intraperitoneal injection of streptozotocin, and a 2 × 2 cm round full-thickness skin defect was created on the back of the rat. Staphylococcus aureus (1 ml of 1.5 × 10⁹ cfu/ml) was evenly applied to the wound. The wound was then observed for 72 h. The infected ulcer model of diabetic rats was considered to be successfully established if the wound was found to be infected with S. aureus. According to different medications, the rats were divided into three groups, namely mixture of herbal extract (MHE), Kangfuxin solution (KFS) and control (Ctrl). The effects of the medicine on wound healing were observed. HE staining and Masson staining were performed to evaluate the histopathological changes and collagen synthesis. IHC staining was used to assess the neovascularization, and M2 macrophage proliferation was determined by immunofluorescence staining. Proteomic and transcriptomic studies were performed to explore potential mechanism of five herbal extracts to promote wound healing. UHPLC-QE-MS was performed to identify the chemical composition of mixture of herbal extract.

Results: The study show that the mixed herbal extract promotes angiogenesis, proliferation of M2 macrophages, and collagen synthesis. Transcriptomics showed that rno-miR-1298, rno-miR-144-5p, and rno-miR-92a-1-5p are vital miRNAs which also play a significant role in role in regulating wound healing. Proteomics results showed that the following proteins were important in wounds treated with MHE: Rack1, LOC100362366, Cops2, Cops6, Eif4e, Eif3c, Rpl12, Srp54, Rpl13 and Lsm7. Autophagy, PI3-Akt and mTOR signaling pathways were enriched after treatment with MHE compared to other groups.

Conclusion: Herein, we have shown that MHE containing extracts of bark of Bauhinia purpurea, P. rubrae, A. dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., A. calamus L, and R. A. biseratae has significant wound healing effects in the diabetic ulcer wound rat model. These results suggest that local application of MHE in diabetic wounds can accelerate the wound healing process. Moreover, in vivo experiments revealed that the diabetic wound healing process was primarily mediated by angiogenesis and M2 macrophage transition. Therefore, this study may provide a promising and non-surgical therapeutic strategy to accelerate diabetic wound healing, thereby decreasing the number of limb amputations in diabetic patients.