*3.1. Cytoprotective Properties of Urolithin A in Neuro-2a Cells*

3.1.1. Urolithin A Improves Mitochondrial Activity in Neuro-2a Cells Subjected to Oxidative Stress (MTT Assay)

The viability of Neuro-2a cells was evaluated by the MTT assay. In this case, different physiological concentrations of urolithin (0.5–50 μM) were tested in neurons for 24 h (Figure 2A). Results were as expected because this range of concentration was non-toxic, as mitochondrial activity was not significantly reduced.

The next purpose was to evaluate the protective effects of urolithin A on Neuro-2a cells using hydrogen peroxide as a neurotoxic insult. Different conditions (100 μM to 1000 μM of H2O2) and exposure times (15, 30, 45, 60 min) determined that incubation of hydrogen peroxide for 45 min at 250 μM was the most appropriate time period for inducing oxidative stress in N2a cells. Figure 2B shows how urolithin A improves mitochondrial activity against hydrogen peroxide (250 μM) in this cell line.

**Figure 2.** Mitochondrial activity in Neuro-2a cells culture (MTT assay). (**A**) Cytotoxicity of Neuro-2a cells after exposure to different concentrations of urolithin A. (**B**) Cytoprotective effects of urolithin A versus hydrogen peroxide (250 μM). Note: \* *p* < 0.05 versus H2O2; ## *p* < 0.01 versus control.

3.1.2. Urolithin A Decreases Intracellular ROS Production in Neuro-2a Cells Subjected to Oxidative Stress (DCFHA-DA Assay)

Figure 3 shows the intracellular ROS production for 90 min. After 40 min of exposure, intracellular ROS reached its highest formation (165%) for cells treated with hydrogen peroxide, whereas control cells (non-treated) maintained a regular level close to 100%; cells stressed with hydrogen peroxide and treated with urolithin A at different concentrations showed lower values for ROS production. Next, the transition (50 min) showed a slight decrease in ROS formation for every treatment and maintained a similar level for the rest of the experiment.

3.1.3. Urolithin A Decreases Lipid Peroxidation in Neuro-2a Cells Subjected to Oxidative Stress (Thiobarbituric Acid Reactive Species, TBARS)

Figure 4 shows how thiobarbituric acid reactive species (TBARS) are generated. The results are exhibited as a percentage over the control (100%). As expected, lipid peroxidation generated by hydrogen peroxide was higher (147%) compared to control cells. Urolithin A response to lipid peroxidation was more effective at lower concentrations (0.5 and 1 μM).

**Figure 3.** ROS production in Neuro-2a cells subjected to oxidative stress by hydrogen peroxide (250 μM) and treatments with urolithin A (0.5–4 μM). Data are expressed as percentage over control cells and the assay was carried out for 90 min in order to measure intracellular ROS production. Note: # *p* < 0.005 versus control; ## *p* < 0.001 versus control. Significant differences appeared at the starting point for H2O2-N2a cells over control cells (*p* < 0.001). However, pre-treatments with 0.5 and 2 μM urolithin A at 0 and 10 min were associated with significant differences (*p* < 0.01). After 20 min, significant differences were reached at 1 μM (*p* < 0.01). Finally, 4 μM of the antioxidant exhibited significant differences (*p* < 0.01) between 40 and 60 min. Urolithin A (2 μM) displayed a greater mitochondrial response than any other co-treatment.

**Figure 4.** Thiobarbituric acid reactive species (TBARS) formation in Neuro-2a cells. Note: \* *p* < 0.05 versus H2O2.

μ 3.1.4. Urolithin A Enhanced the Activity of Antioxidant Enzymes in Neuro-2a Cells Subjected to Oxidative Stress (CAT, SOD, GR, GPx)

μ Results obtained after three replications in three different lysates showed a dose-dependent tendency of catalase for the treatments with urolithin A (Figure 5A). Surprisingly, the activity of the enzyme for the lowest concentration was even higher than the control. Therefore, the in vitro antioxidant effect observed for this metabolite may be more effective at lower doses. When the cells were treated only with hydrogen peroxide, the response of catalase decreased compared to urolithin A treatments, detecting significant differences against basal cells (*p* < 0.005) and the metabolite (*p* < 0.05; 0.01; 0.001).

**Figure 5.** Neuro-2a cell culture redox status. (**A**) Catalase activity. (**B**) Superoxide dismutase activity. (**C**) Glutathione reductase activity. (**D**) Glutathione peroxidase activity. Note: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.005; \*\*\*\* *p* < 0.001 versus H2O2; # *p* < 0.05 versus Control; ### *p* < 0.005 versus Control.

Different outcomes were found for the superoxide dismutase (SOD) enzyme (Figure 5B); a dose-dependent tendency appeared up to 2 μM for SOD activity. Similar activity was observed at the highest concentration as the lowest. Significant differences appeared at 2 μM (*p* < 0.05).

Comparable effects were obtained for GR and GPx activities (Figure 5C,D). Activities for 0.5 μM urolithin A looked similar to control activities for both enzymes. Each concentration of the antioxidant compound exerted significant differences for GR activity (*p* < 0.01; *p* < 0.005). However, significant differences in GPx activity were only found at 2 μM urolithin A (*p* < 0.05).
