Bone marrow stromal cell-derived exosomal circular RNA improves diabetic foot ulcer wound healing by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and inhibiting ferroptosis

Background: Diabetic foot ulcer (DFU) remains a serious chronic diabetic complication that can lead to disability. CircRNA-itchy E3 ubiquitin protein ligase (circ-ITCH) was observed to be downregulated in diabetic retinopathy and diabetic nephropathy, and overexpression of circ-ITCH could inhibit the processes of these diseases. However, the detailed physiological and pathological functions of circ-ITCH in wound healing of DFU remain undetermined.

Methods: Exosomes derived from bone marrow stromal cells (BMSCs) were isolated and identified. Cell viability and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by cell counting kit-8 (CCK-8) and tube formation assays, respectively. The interplays of circ-ITCH, TATA-Box binding protein associated factor 15 (TAF15), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA were analyzed by RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH) combined immunofluorescent staining, and RNA pull-down assays. qRT-PCR, western blot or immunohistochemistry (IHC) were used to measure the expression of circ-ITCH, TAF15, Nrf2, vascular endothelial growth factor (VEGFR), and ferroptosis-related makers. The mice DFU model was established to verify the in vitro results.

Results: Circ-ITCH was downregulated in in vitro and in vivo models of DFU. Deferoxamine (DFO), an iron chelating agent, improved the viability and angiogenic ability of high glucose (HG)-treated HUVECs. Overexpression of circ-ITCH or co-cultured with exosomal circ-ITCH from BMSCs could alleviate HG-induced Ferroptosis and improve the angiogenesis ability of HUVECs. Circ-ITCH in HUVECs recruited TAF15 protein to stabilize Nrf2 mRNA, thus activating the Nrf2 signaling pathway and suppressing Ferroptosis. Exosomal circ-ITCH from BMSCs also accelerated the wound healing process through inhibiting Ferroptosis in the DFU mice in a time-dependent manner.

Conclusion: Exosomal circ-ITCH from BMSCs inhibited Ferroptosis and improved the angiogenesis of HUVECs through activation of Nrf2 signaling pathway by recruiting TAF15 protein, ultimately accelerating the wound healing process in DFU.

Angiogenesis; BMSCs; Circ-ITCH; Diabetic foot ulcer; Exosomes; Ferroptosis; HUVECs; Nrf2 pathway; TAF15; Wound healing.