USP28 participates in high glucose-mediated endothelial dysfunction via deubiquitinating SIRT1 protein in diabetic foot ulcers.

BACKGROUND: Silent information regulator sirtuin 1 (SIRT1) protects and improves diabetic wound healing, but SIRT1 undergoes ubiquitination degradation in various cellular environments. The research aims to reveal a mechanism related to SIRT1 deubiquitination to attenuate HG-induced injury in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs treated with high glucose (HG) were utilized to simulate hyperglycemic conditions in vitro. Cell viability, proliferation, apoptosis, invasion, and angiogenesis were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, flow cytometry, transwell, and tube formation assays, respectively. Ferroptosis was analyzed by analyzing Fe(2+) levels, reactive oxygen species production, and glutathione activity. Endoplasmic reticulum stress (ERS) was evaluated by detecting CHOP and GRP78 protein levels. The interaction between SIRT1 and ubiquitin-specific peptidase 28 (USP28) was determined by co-immunoprecipitation analysis and ubiquitination assays. RESULTS: Serum SIRT1 mRNA levels were lower in patients with DFUs. SIRT1 overexpression impaired HG-induced injury, ERS, and ferroptosis in HUVECs. USP28 deubiquitinates and stabilizes SIRT1 protein. USP28 overexpression eased HG-induced injury, ERS, and ferroptosis in HUVECs, but the USP28 inhibitor AZ1 counteracted the function of USP28 overexpression. Furthermore, both SIRT1 knockdown and the SIRT1 inhibitor EX-527 undercut USP28 overexpression-mediated protective effect on HUVEC injury, ERS, and ferroptosis under HG stimulation. Additionally, USP28 regulated the NRF2/HO-1 pathway by deubiquitinating SIRT1 in HG-stimulated HUVECs. CONCLUSION: USP28 weakens HG-mediated endothelial dysfunction via activating the NRF2/HO-1 pathway through stabilizing SIRT1 protein, indicating that targeting USP28 is the direction for developing clinical strategies for diabetic wound healing.