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 Fe2+ 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.