## 3 Results
### Antioxidant Cytoprotective Activity  
The antioxidant power of beer, beer with spirulina 0.25% \(w/v\) and spirulina at the corresponding concentrations in the beer (0.25% \(w/v\)) was also evaluated in a biological system with the pro-oxidant damage induced by tBOOH. Specifically, the H69 cells were exposed to a 24 h pre-treatment with the beer samples, followed by a 3 h co-treatment in the presence of tBOOH. Under our experimental conditions, the beer samples were nontoxic up to the concentration of 3 \(\mu\)L/mL (Figure 5A) and induced no signs of oxidative stress (Figure 6A), while cytotoxic effects were registered at higher concentrations (data not shown). Therefore, the cytoprotective properties of the tested samples towards tBOOH were evaluated up to 3 \(\mu\)L/mL. As displayed in Figure 6B, tBOOH significantly affected the  
Figure 5: Effect of beer, beer with spirulina 0.25% \(w/v\) and spirulina 0.25% \(w/v\) at the corresponding concentrations in the beer on the H69 cell viability in the absence (**A**) and presence (**B**) of the prooxidant agent tBOOH. Data represent the average and standard error of at least three independent experiments (\(n=3\)). *** \(p<0.001\), significant difference with respect to control (ANOVA followed by [PERSON]’s multiple comparison post test). \({}^{000}\)\(p<0.01\), significant difference with respect to tBOOH (ANOVA followed by [PERSON]’s multiple comparison post test). *** \(p<0.001\), significant difference of beer + spirulina 0.25% \(w/v\) with respect to beer (Student’s \(t\)-test). Figure 6: Effect of beer, beer with spirulina 0.25% \(w/v\) and spirulina 0.25% \(w/v\) at the corresponding concentrations in the beer on the levels of reactive oxygen species (ROS) in the absence (**A**) and presence (**B**) of the prooxidant agent tBOOH in H69 cells. Data represent the average and standard error of at least three independent experiments (\(n=3\)). *** \(p<0.001\), significant difference with respect to control (ANOVA followed by [PERSON]’s multiple comparison post test). \({}^{000}\)\(p<0.01\), significant difference with respect to tBOOH (ANOVA followed by [PERSON]’s multiple comparison post test). *** \(p<0.001\), significant difference of beer with spirulina 0.25% \(w/v\) with respect to beer (Student’s \(t\)-test). cell viability, lowering it by almost 45% with respect to the control; accordingly, the ROS levels were doubled by the treatment (Figure 6B). The sample of beer did not counteract the tBOOH damage, despite significant cytoprotective effects of the beer with spirulina 0.25% \(w/v\), which achieved a maximum of a 20% increase in the cell viability with respect to tBOOH at the highest tested concentrations. Similarly, the intracellular ROS levels were reduced by up to 1.5 times with respect to tBOOH, although without achieving the basal levels. A slight increase in cell viability, albeit without a significant ROS reduction, was also induced by the highest concentrations of spirulina, thus suggesting a possible contribution of the extract to the beer's antioxidant activity (Figures 5B and 6B). Finally, we explored the ability of the tested samples to modulate the cytoplasmic expression of Nrf2, a known intracellular factor activated during stress conditions in order to activate the cellular defense responses [31]. After 30 min exposure, the samples of beer (3 \(\upmu\)L/mL) did not affect the basal levels of Nrf2, despite a significant increase induced by the corresponding concentration of spirulina (7.5 \(\upmu\)g/mL); conversely, tBOOH induced a significantly reduced expression of Nrf2, which was restored and also increased (up to 20% with respect to tBOOH) by beer with 0.25% \(w/v\) spirulina compared to the control (Figure 7A,B). Surprisingly, the beer and spirulina alone were unable to counteract the damage induced by tBOOH, with the Nrf2 expression being comparable to that of tBOOH.