2.3. Establishment of the H2O2-Induced Oxidative Stress Model

HEK293 cells were treated with H₂O₂ at different concentrations for 4 h to establish an oxidative stress model. The appropriate amount of H₂O₂ was added to medium deprived of serum and antibiotics, and the final concentration of H₂O₂ was maintained at 100, 200, 300, 400, 500, 600, 700, or 800 μM. Complete medium without cells served as a blank group, and HEK293 cells without H₂O₂ were used as a control group. Cell viability was measured using the MTS assay. MTS (20 μL) was added to the cells, which were then incubated at 37°C for 4 h. The 96-well plate was placed in a multimode microplate reader (Tecan, Infinite M200 Pro) to measure the absorbance at 490 nm. Six parallel experiments with cells in each treatment group were performed, and the results are expressed as average values. All the assays were repeated three times. The concentration of H₂O₂ (producing a cell viability of approximately 50%) used to establish the oxidative stress model was selected for subsequent experiments. Cell viability was calculated using the following equation: