## 3 Results
### Recovery of Cell Vitality and Anti-Inflammatory Activity  
The role of LMW peptides from breads was further detected by assaying both cell vitality and anti-inflammatory activity on cultured cells stressed with LPS, and the crystal violet method was used to stain live cells that remained attached to culture plates while dead cells were detached and washed away [29]. The percentage of viable cells in the sample treated with LMW peptides and LPS clearly indicates that the presence of peptides has a protective effect on the LPS-induced stress (Figure 4). LMW peptides from breads obtained from the Aubusson breads show same vitality of the negative control, to indicate that peptides reverse the negative effect of LPS. In addition, the peptides from both Rondo and Skorpion breads even increase the number of viable cells, playing a healthy role regarding the LPS-induced effects. Figure 3: Effect of LMW peptides from doughs (**a**) and breads (**b**) on intracellular ROS. Cells were treated with 0.05 mg/mL LMW fractions for 1 h and then co-incubated with 1 mg/mL LPS for 24 h as reported in the material and methods section. NT: negative control, untreated cells; LPS: positive control, cells treated with only LPS; AA: cell treated with 0.05 mM ascorbic acid. Results are the mean (\(\pm\)SD) of three different experiments performed in duplicate. Statistical analysis was performed for each sample vs. the LPS value (* \(p<0.05\); ** \(p<0.01\); *** \(p<0.001\)). The same treatment scheme was also used to unravel the ability of peptides from breads to reverse the LPS-induced inflammation. Upstream markers, either as the ratio between the phosphorylated and total form (pNFkB/NFkB) of the transcription factor NFkB (nuclear factor-kappa B) [23], and the expression of its inhibitor, the IkB factor, were determined for peptides from breads. Moreover, COX2 (cyclooxygenase 2), which remains unexpressed under normal conditions and increases during inflammation, was chosen as a downstream marker of the inflammatory response [25; 26]. Immunoblot analysis was performed using antibodies to detect the expression levels of these factors belonging to the inflammatory pathway. Figure 5 shows the results from blots obtained after cell lysis and detection with the specific antibody. The pNFkB/NFkB ratio (Figure 5a) significantly decreased in macrophages treated with LMW peptides and the percentage of reduction is comparable with that obtained by acetyl salicylic acid treatment and is in agreement with the observation that many anti-inflammatory molecules exert their action by inhibiting nuclear translocation of NFkB, which occurs upon phosphorylation [30]. Results from the IkB detection (Figure 5b) show the same trend: the increase in the IkB value goes together with the decrease in NFkB translocation, as expected for anti-inflammatory compounds. IkB expression values obtained from cells treated with breads are lower than values detected upon ASA treatment; nevertheless, peptides from Aubusson, Rondo, and Skorpion breads are able to increase, in a significant manner, the IkB expression when compared to IkB expression determined for the positive control. Finally, COX2 expression, which is not present in the negative control, is reduced in a significant manner only by peptides from Rondo and Skorpion breads. On the contrary, peptides from the Aubusson sample slightly reduce the expression of COX2, but not significantly (Figure 5c). Figure 4: Recovery of cell viability. RAW 264.7 cells were incubated with LMW peptides (0.05 mg/mL) from the breads for 1 h and then with LPS for 24 h. Cell viability was evaluated using the crystal violet test. The absorbance due to viable cells was read at 595 nm. NT: negative control, untreated cells; LPS: positive control, cells treated with the only LPS; Aubusson, Rondo, and Skorpion indicate cells treated with both LPS and LMW peptides. Data reported in the figure represent the mean (\(\pm\)SD) of three different experiments performed in duplicate. Statistical analysis was performed for each sample vs. LPS.