*3.3. SEP Co-Treatment Enhances Antioxidant Defenses*

As our results showed a higher neuroprotective activity of SEP co-treatment (1 μM SF, 2.5 μM EGCG, and 0.5 μM PB) compared to the single treatments of 1 μM SF, 2.5, μM EGCG, or 0.5 μM PB, we investigated the ability of SEP co-treatment to modulate the cellular redox state by evaluating GSH levels with an MCB assay. The effect of the different treatments after 24 h on GSH levels is reported in Figure 5. All the treatments were able to significantly increase GSH levels in respect to control cells. In agreemen<sup>t</sup> with the viability data, we observed the most effective increase of GSH levels after SEP co-treatment (1 μM SF, 2.5 μM EGCG, and 0.5 μM PB) in comparison to the single treatment of 1 μM SF, 2.5 μM EGCG, or 0.5 μM PB.

**Figure 5.** Antioxidant activity of SF, EGCG, and PB compounds on SH-SY5Y cells. Cells were treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB, and after 24 h GSH levels were evaluated with an monochlorobimane (MCB) assay as reported in Materials and Methods. Data are expressed as a percentage of untreated cells (CTRL). Each bar represents mean ± SEM of three independent experiments. Data were analyzed with a one-way ANOVA followed by the Fisher's test. \* *p* < 0.05 vs. untreated cells; § *p* < 0.05 vs. SEP co-treatment.

#### *3.4. SEP Co-Treatment Modulates Genes Involved in Oxidative Stress Control*

As the previous data showed that SEP co-treatment was significantly more effective compared to the single treatments, we decided to study its ability to modulate cellular antioxidant status. Real-time PCR analysis was employed to investigate the ability of SEP co-treatment to modulate the mRNA level of different antioxidant enzymes. The cDNA was obtained from 3D SH-SY5Y cultures that were co-treated (1 μM SF, 2.5 μM EGCG, and 0.5 μM PB) for 6 h. The 3D cultures were then exposed to 700 μM H2O2 for 1 h prior to lysis (Figure 6). Importantly, SEP co-treatment induced a significant and marked upregulation of heme oxygenase 1 (HO1), NADPH: quinone oxidoreductase 1 (NQO1), glutathione reductase (GR), and thioredoxin reductase (TR) in 3D cultures although with different levels of upregulation (Figure 6). Moreover, SEP co-treatment in the presence of oxidative stress induced a significant upregulation of all tested genes in respect to H2O2-treated cells.

NADPH oxidase (NOX) enzymes have been shown to be a major source of ROS in the brain and to be involved in several neurological diseases [35]. On this basis, we studied the modulatory effect of SEP co-treatment on NOX1 and NOX2 expression using real-time PCR analysis (Figure 7). In the absence of oxidative stress, SEP co-treatment had a strong effect on these enzymes as it significantly reduced NOX1 and NOX2 expression compared to untreated cells. In the presence of oxidative stress (700 μM H2O2), SEP co-treatment significantly reduced NOX1 and NOX2 expression compared to H2O2-treated cells. Of note, SEP co-treatment before peroxide exposure maintained NOX1 levels at a value comparable to control cells.

**Figure 6.** Effect of SEP co-treatment on antioxidant enzyme expression. Cells were co-treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB for 6 h. Oxidative stress was induced with 700 μM H2O2 for 1 h prior to lysis. Real time-PCR was performed to detect heme oxygenase 1 (HO1), NADPH: quinone oxidoreductase 1 (NQO1), glutathione reductase (GR), and thioredoxin reductase (TR) mRNA levels. Data are expressed as relative abundance compared to untreated cells. Each bar represents mean ± SEM of three independent experiments. Data were analyzed with a one-way ANOVA followed by the Fisher's test. \* *p* < 0.05 vs. untreated cells, ◦ *p* < 0.05 vs. H2O2.

**Figure 7.** Effect of SEP co-treatment on NADPH oxidase 1 (NOX1) and NADPH oxidase 2 (NOX2). Cells were co-treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB for 6 h. Oxidative stress was induced with 700 μM H2O2 for 1 h prior to lysis. Real time-PCR was performed to detect NOX1 and NOX2 mRNA levels. Data are expressed as relative abundance compared to untreated cells. Each bar represents mean ± SEM of three independent experiments. Data were analyzed using a one-way ANOVA followed by the Fisher's test. \* *p* < 0.05 vs. untreated cells, ◦ *p* < 0.05 vs. H2O2.

#### *3.5. SEP Co-Treatment is able to Modulate Insulin-Degrading Enzyme (IDE) Gene Expression*

To investigate if SEP co-treatment had other neuroprotective activities besides the antioxidant one, we studied its effect on insulin-degrading enzyme (IDE) expression. IDE plays a significant role in Aβ degradation [36], which is one of the main hallmarks of Alzheimer's disease. Moreover, recent studies have demonstrated that increasing Aβ degradation as opposed to inhibiting synthesis is a more effective strategy for preventing Aβ build-up [37]. In our 3D SH-SY5Y cultures, IDE mRNA levels were downregulated by oxidative stress, but, interestingly, SEP co-treatment (1 μM SF, 2.5 μM EGCG, and 0.5 μM PB) was able to upregulate its expression at levels comparable to untreated cells (Figure 8).

**Figure 8.** Effect of SEP co-treatment on insulin-degrading enzyme (IDE). Cells were co-treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB for 6 h. Oxidative stress was induced with 700 μM H2O2 for 1 h prior to lysis. Real time-PCR was performed to detect IDE mRNA levels. Data are expressed as relative abundance compared to untreated cells. Each bar represents mean ± SEM of three independent experiments. Data were analyzed with a one-way ANOVA followed by the Fisher's test. \* *p* < 0.05 vs. untreated cells, ◦ *p* < 0.05 vs. H2O2.
