Non-pharmacological interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy promote diabetic wound healing.

OBJECTIVE: This study aims to conduct an unbiased assessment of the synergistic effects of non-pharmacological Interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy on the facilitation of diabetic wound healing, while also exploring the underlying mechanisms. The findings of this research will provide a theoretical framework and innovative strategy for unconventional therapeutic interventions aimed at enhancing the healing process of diabetes-related wounds. METHODS: In vivo experiments involved the induction of diabetic models in C57 mice through streptozotocin injection. To simulate a combined therapeutic approach, diabetic mice underwent fasting on days 2 and 6, accompanied by twice daily PRFE applications for 8 days. In vitro experiments were conducted using a serum-free culture medium to replicate fasting conditions. The investigation encompassed wound healing rate, proliferation, migration, angiogenesis, oxidative stress, fibrogenesis, and sensory nerve growth through histological analysis and functional assessments in vivo. Additionally, this study utilized quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting (WB), and immunofluorescence staining techniques to elucidate the potential mechanisms underlying the effects of intermittent Fasting and PRFE combination therapy in diabetic wound healing, both in vitro and in vivo. RESULTS: The intermittent fasting and PRFE combination therapy demonstrated superior efficacy in enhancing diabetic wound healing compared to either treatment alone. It harnessed the respective strengths of individual therapies, fostering migration, mitigating oxidative stress, and enhancing fibrogenesis. Furthermore, the combination therapy manifested a synergistic effect in promoting proliferation, tube formation, angiogenesis, and sensory nerve growth. CONCLUSION: This study demonstrates that intermittent fasting and PRFE combination therapy enhance diabetic wound healing, effectively leveraging the strengths of both therapies and even yielding synergistic benefits. Moreover, it indicates the potential engagement of the P75/HIF1A/VEGFA axis in mediating these effects.

Upregulation of rate-limiting enzymes in cholesterol metabolism by PKCδ mediates endothelial apoptosis in diabetic wound healing.

Diabetic foot ulcer (DFU) is a prevalent complication of diabetes that poses significant challenges in terms of treatment and management. It is characterized by heightened endothelial apoptosis and impaired angiogenesis. In this study, we aimed to investigate the role of protein kinase Cδ (PKCδ) in regulating endothelial apoptosis in diabetic wounds by promoting cholesterol biosynthesis. The expression of PKCδ was increased in human umbilical vascular endothelial cells (HUVECs) cultivated in high glucose medium and skin tissue isolated from diabetic mice. High glucose-induced HUVECs apoptosis was reduced by PKCδ inhibition with siRNA or rottlerin. RNA-seq identified two enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), as the downstream of PKCδ. PKCδ knockdown or inhibition suppressed the expression of HMGCS1 and HMGCR and lowered free cholesterol (FC) levels. Cholesterol restored high glucose-induced apoptosis in siRNA- or rottlerin-treated HUVECs. In vivo use of rosuvastatin calcium, an inhibitor of HMGCR, downregulated free cholesterol levels and accelerated the wound healing process. In conclusion, PKCδ expression in endothelial cells was activated by high glucose, which subsequently upregulates the expression of two enzymes catalyzing cholesterol biosynthesis, HMGCS1 and HMGCR. Enhanced cholesterol biosynthesis raises free cholesterol levels, promotes endothelial apoptosis, and finally delays wound healing.

KRT17 From Keratinocytes With High Glucose Stimulation Inhibit Dermal Fibroblasts Migration Through Integrin α11.

Objective: To investigate the effects of overexpressed keratin 17 (KRT17) on the biology of human dermal fibroblasts (HDFs) and to explore the mechanism of KRT17 in diabetic wound healing. Methods: KRT17 expression was tested in diabetic keratinocytes, animal models, and patient skin tissues (Huazhong University of Science and Technology Ethics Committee, [2022] No. 3110). Subsequently, HDFs were stimulated with different concentrations of KRT17 in vitro. Changes in the proliferation and migration of HDFs were observed. Then, identification of KRT17-induced changes in dermal fibroblast of RNA sequencing-based transcriptome analysis was performed. Results: KRT17 expression was upregulated under pathological conditions. In vitro stimulation of HDFs with different concentrations of KRT17 inhibited cell migration. RNA-seq data showed that enriched GO terms were extracellular matrix components and their regulation. KEGG analysis revealed that the highest number of enriched genes was PI3K-Akt, in which integrin alpha-11 (ITGA11) mRNA, a key molecule that regulates cell migration, was significantly downregulated. Decreased ITGA11 expression was observed after stimulation of HDFs with KRT17 in vitro. Conclusion: Increased expression of KRT17 in diabetic pathological surroundings inhibits fibroblast migration by downregulating the expression of ITGA11. Thus, KRT17 may be a molecular target for the treatment of diabetic wounds.

High glucose-induced IL-7/IL-7R upregulation of dermal fibroblasts inhibits angiogenesis in a paracrine way in delayed diabetic wound healing.

It is widely acknowledged that diabetes leads to slow wound healing and ulceration, and severe serious diabetic foot ulceration may result in amputation. In recent years, much emphasis has been placed on exploring diabetic wound healing to protect patients from adverse events. We recently found interleukin-7 (IL-7), a growth factor for B-cells and T-cells, and its receptor was significantly upregulated in high glucose-induced fibroblasts and skin of diabetic mice. Moreover, IL-7 stimulated fibroblasts secreted ANGPTL4, which inhibited angiogenesis of endothelial cells resulting in delayed wound healing. In our previous study, fibroblasts, endothelial cells and keratinocytes were exposed to normal glucose (5.5 mM) or high glucose (30 mM) medium for 24 h, and RNA sequencing showed that IL-7 and IL-7R were significantly upregulated in fibroblasts. To remove the effect of high glucose and explore the influence of IL-7, exogenous rMuIL-7 used to treat normal mice led to delayed wound healing by inhibiting angiogenesis. Vitro experiments revealed that IL-7-induced fibroblasts inhibited endothelial cell proliferation, migration and angiogenesis. Further experiments showed that fibroblast angiopoietin-like-4 (ANGPTL4) secretion exhibited the inhibitory effect which was blocked by culture with the corresponding neutralizing antibody. Overall, our study revealed signaling pathways associated with diabetic wound healing and provided the foothold for further studies on delayed wound healing in this patient population. Mechanism that high glucose activates IL-7-IL-7R-ANGPTL4 signal pathway in delayed wound healing. High glucose upregulates IL-7 and IL-7R in dermal fibroblasts. IL-7 stimulates dermal fibroblasts secreting Angptl4 which inhibits proliferation, migration and angiogenesis of endothelial cells in a paracrine way.