Diabetic patients often suffer from delayed wound healing due to factors such as hyperglycemia, neuropathy, and impaired immune function. This not only increases the risk of infections but also may lead to severe consequences like amputation. Traditional treatments have limited efficacy in addressing the complex pathological mechanisms of diabetic wounds.

Cold atmospheric plasma (CAP) is a partially ionized gas operating at room temperature. It generates reactive oxygen and nitrogen species (RONS) and exerts multiple biological effects, including antimicrobial activity, promotion of tissue proliferation, and regulation of inflammatory responses. Previous studies have confirmed CAP’s potential in wound healing, but its specific molecular mechanisms in diabetic wound treatment—especially the regulatory roles of key signaling pathways—remain unclear.

Objectives

This study aims to explore the therapeutic effect of CAP on diabetic wounds and clarify its underlying mechanisms. It focuses on how CAP regulates core signaling pathways (such as Hippo/YAP/TAZ, TGFβ, and β-catenin) to accelerate diabetic wound healing, providing scientific evidence for the clinical application of CAP in treating diabetic foot ulcers and other related conditions.

2. Experimental Methods

（1）Animal Model Establishment: Streptozotocin (STZ) was used to induce type 1 diabetes in SKH1 mice. Full-thickness skin wounds were created on the ears of the mice to construct a diabetic wound model.

（2）CAP Treatment Protocol: An argon plasma jet (kINPen MED) was employed for wound treatment. Each session lasted 10 seconds, with treatments administered once every 3 days. Two treatment groups were set: 4 sessions (Day 9) and 6 sessions (Day 20), with an untreated group as the control.

（3）Detection Techniques: Multiple methods were used to evaluate wound healing and molecular mechanisms, including transcriptome sequencing, quantitative polymerase chain reaction (qPCR), Western blot, immunohistochemistry, and tissue staining (Hematoxylin-Eosin staining, Picrosirius Red staining). These techniques were used to detect wound healing indicators (re-epithelialization rate, epidermal/dermal thickness) and the expression and localization of signaling pathway-related molecules.

3. Main Results

（1）Accelerated Wound Healing: CAP treatment significantly improved the re-epithelialization rate of diabetic wounds. The healing speed was noticeably faster than that of the control group starting from Day 3, and the wounds were nearly fully healed by Day 20. Histological results showed increased epidermal and dermal thickness, enhanced granulation tissue formation, and higher collagen fiber density in the CAP-treated group.

（2）Activation of Hippo/YAP/TAZ Signaling Pathway: In the early stage of healing (Day 9), CAP upregulated the mRNA and protein expression of YAP/TAZ and promoted their nuclear localization. This led to increased expression of downstream target genes (e.g., CTGF, Cyr61, FGF1), which promote cell proliferation, migration, and extracellular matrix (ECM) synthesis. In the late stage (Day 20), YAP phosphorylation levels increased, inhibiting excessive cell proliferation to maintain tissue homeostasis.

（3）Regulation of TGFβ Signaling Pathway: In the early stage of healing (Day 9), CAP upregulated the expression of TGFβ1 and its downstream molecules (e.g., SMAD2, CDKN1A). This promoted collagen (Type I and Type III) synthesis and myofibroblast activation, accelerating ECM remodeling. Collagen fibers gradually matured as the healing process progressed.

（4）Modulation of β-Catenin and Antioxidant Pathways: CAP increased the expression of β-catenin (a key factor in the Wnt pathway) and E-cadherin, enhancing cell-cell junctions to promote epithelialization. Additionally, CAP activated the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway, upregulating the expression of downstream antioxidant genes (e.g., HO-1, SOD1, CAT) and reducing oxidative stress-induced damage to wounds.

（5）Reduction of Bacterial Load: CAP exhibited potent antimicrobial activity by generating RONS, significantly reducing the bacterial load in diabetic wounds. This avoided infection-induced delays in wound healing and improved the local microenvironment for healing.

4. Conclusion

[1] Mirpour S, Fathollah S, Mansouri P, Larijani B, Ghoranneviss M, Mohajeri Tehrani M, Amini MR. Cold atmospheric plasma as an effective method to treat diabetic foot ulcers: a randomized clinical trial[J]. Sci Rep. 2020;10:10440.