SH-SY5Y cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and maintained in a humidified incubator with 5% CO₂. For the OGD/R group, the culture medium was replaced with glucose-free DMEM, and cells were incubated in a hypoxic chamber for 4 h, followed by restoration to normoxic culture conditions. ISO and the positive drug NBP (N-butylphthalide) were administered at the time of reperfusion.

The levels of oxidative stress markers, including SOD (Beyotime, S0101S), MDA (Beyotime, S0131S), GSH (Beyotime, S0053), and H₂O₂ (Beyotime, S0038), were determined using commercial kits from Beyotime Institute of Biotechnology. The experimental procedures were performed strictly in accordance with the manufacturer’s instructions.

Intracellular proteins were extracted using a protein extraction kit. Target proteins were separated by SDS-PAGE and electrotransferred onto PVDF membranes. The membranes were incubated overnight with primary antibodies against Nrf2 (Proteintech, 16396-1-AP), AKT (CST, 9272), p-AKT (CST, 9271), GPX4 (Abmart, T56959S), and COX2 (Abcam, ab179800), respectively. On the following day, the membranes were incubated with the corresponding secondary antibodies. Protein expression was subsequently detected using chemiluminescence, and the results were recorded.

AutoDock was used to simulate the interaction between ISO and AKT (PDB: 3O96). During the protein preparation process, all H₂O were removed.

The cells were collected and repeatedly frozen and thawed using liquid nitrogen. The processed cell supernatant was incubated with ISO and DMSO for 60 min and then denatured at different temperatures, followed by WB detection.

The cell lysates were mixed with ISO and DMSO, respectively and incubated at room temperature for 1 h. Then, protease inhibitor was added for 30 min. The addition of protease inhibitor was to terminate the reaction. After the experiment, WB detection was performed.

Statistical analyses were conducted using SPSS 22.0 software. For comparisons among multiple groups, data were subjected to one-way analysis of variance (ANOVA), followed by either the least significant difference (LSD) test or Dunnett’s T3 test. Results are presented as the mean ± standard deviation (SD). A p-value of less than 0.05 was considered statistically significant.

When cells were treated with different concentrations of ISO, MTT assay results showed that cell viability was significantly reduced at 30 μM ISO, indicating obvious cellular injury (Figure 1(A)). Therefore, ISO concentrations of 5, 10, and 20 μM were used in subsequent experiments. As shown in Figure 1(B), ISO treatment attenuated the OGD/R-induced decrease in SH-SY5Y cell viability in a concentration-dependent manner.

The results of experiments evaluating the ROS scavenging and lipid peroxidation inhibitory effects of ISO demonstrated that ISO significantly reduced the levels of H₂O₂ and MDA (Figure 2).

Assessment of the intracellular antioxidant capacity of ISO revealed that ISO enhanced SOD activity and increased GSH content in OGD/R-injured SH-SY5Y cells (Figure 3).

Western blot results showed that ISO inhibited the expression of Nrf2 in the cytoplasm and increased its expression in the nucleus in a dose-dependent manner, indicating that ISO could promote the nuclear translocation of Nrf2, thereby activating the Nrf2 signaling pathway (Figure 4(A)).

In addition, Western blot analysis was performed to evaluate the regulatory effect of ISO on the expression of ferroptosis-related proteins. The results showed that OGD/R treatment downregulated GPX4 expression and upregulated COX2 expression, while ISO treatment reversed this phenomenon, indicating that ISO could inhibit OGD/R-induced neuronal ferroptosis (Figure 4(B)).

Western blot results indicated that ISO could promote the phosphorylation of AKT, suggesting that ISO is capable of activating the PI3K/AKT signaling pathway (Figure 5(A)). Meanwhile, molecular docking assay results showed that ISO could bind to AKT with a binding energy of −7.713 kcal/mol (Figure 5(B)). The DARTS experimental results indicated that ISO can inhibit the degradation of AKT protein caused by protease, and this effect was concentration dependent (Figure 5(C) & Figure 5(D)). The CETSA results indicated that ISO can improve the thermal stability of AKT (Figure 5(E) & Figure 5(F)).

Collectively, these results suggest that ISO can target AKT to activate the PI3K/AKT signaling pathway, which serves as the upstream regulatory pathway of Nrf2-mediated antioxidant response.