Si nrf2 plasmid

The si-Nrf2 plasmid, miR-200a mimic, and their negative controls were procured from GenePharma (Shanghai, China). The RAW264.7 cells were cultured in six-well plates at a density of 1 × 10⁷ cells/ml for 24 h. Lipofectamine 2000 reagent (11668-027, Invitrogen) was used for transfection according to the manufacturer’s instructions. After transfection for 48 h, cells were collected for further researches.

As previously described [36 (link)], MPC5 cells at the logarithmic growth stage were harvested and grouped as follows: (1) blank group: treated with 5 mM glucose; (2) HG group: treated with 25 mM glucose; (3) HG + DMSO group: treated with 25 mM glucose and 0.1% DMSO; (4) HG + TP group: treated with 25 mM glucose and 10 μM TP (the administration method and dosage of TP were referred to the literature [37 (link)]); (5) HG + DAPA group: treated with 25 mM glucose and 2 μM DAPA, with the DAPA dosage referred to the literature [31 (link)]; (6) HG + DMSO + ML385: treated with 25 mM glucose, 0.1% DMSO, and 5 μM ML385 (Nrf2 inhibitor, ab287109, Abcam), with the concentration of ML385 referred to the instruction and reference [38 ]; (7) HG + TP + ML385: treated with 25 mM glucose, 10 μM TP, and 5 μM ML385; (8) HG + TP + si-NC: transfected with si-NC plasmid (GenePharma, Shanghai, China) for 24 h and then treated with 25 mM glucose and 10 μM TP; (9) HG + TP + si-Nrf2: transfected with si-Nrf2 plasmid (GenePharma) for 24 h and next treated with 25 mM glucose and 10 μM TP. After 48 h of treatment, MPC5 cells were collected for subsequent experimentation.