## 3 Results
### Effects of Curcumin and PEA on LPS-Induced TG2 Expression Levels in BV2 Cells
#### 3.2.1 Effects of A\(\beta\)1-42 on TG2 Protein Expression in BV2 Cells  
In order to establish the best concentration of A\(\beta\)1-42 capable of stimulating TG2 protein expression in BV2 cells, we conducted a dose-dependent analysis of the impact of different A\(\beta\)1-42 concentrations on TG2 protein expression in BV2 cells. As shown in Figure 5, we found the highest TG2 protein expression in BV2 cells when they were treated with 1 \(\upmu\)M A\(\beta\)1-42 for 48 h.  
2.2 Effects of Curcumin and PEA on A\(\beta\)1-42-Induced TG2, Inflammatory or Neuroprotective Markers RNA Expression Levels in BV2 Cells  
In order to evaluate whether curcumin and PEA could modulate TG2 (Figure 6D), inflammatory (Figure 6A) or neuroprotective (Figure 6B,C) cytokines' expression levels in BV2 cells under a different inflammatory condition, the BV2 cells were treated with 1 \(\upmu\)M A\(\beta\)1-42 peptide in the absence or presence of curcumin or PEA, and the RNA levels were evaluated using a RT-PCR (Figure 6).  
Figure 3: Panel (**A**): Western blot for TG2 protein expression. BV2 cells were cultured in medium supplemented or not (NT) with 10 \(\upmu\)M of PEA and Cu or in presence of 1 \(\upmu\)g/mL LPS for 48 h. Cells were treated also with 10 \(\upmu\)L of ethanol (ETOH) as a negative control or with 10 \(\upmu\)M retinoic acid (RA) as a positive control. SDS-PAGE was blotted with an antibody against TG2. GAPDH protein expression was used as loading control. The experiments were performed at least 3 different times and the results were always similar; Panel (**B**): graph shows the densitometric intensity of TG2/GAPDH bands ratio. # \(p\) value \(<\) 0.05 compared with control group (NT). #_p_\(<\) 0.05 compared with LPS group.  
2.3 TG2 Protein Levels in BV2 Cells after Treatment with Neuronutraceuticalels and A\(\beta\)1-42  
To evaluate the anti-inflammatory effects of the neuronutraceuticalels under study on the TG2 protein expression levels in a BV2 cell model, this cell type has been treated with A\(\beta\)1-42 1 \(\upmu\)M in the absence or presence of the tested nutraceuticalels at a concentration of 10 \(\upmu\)M for PEA and curcumin. The Western blot results on the BV2 microglia cell line have confirmed the regulation of TG2 under these treatments (Figure 7). In fact, the TG2 protein levels increased under A\(\beta\)1-42-derived pro-inflammatory stimulus and decreased when the tested nutraceuticalels were added.  
Figure 4: Real Time-PCR was performed to measure the mRNA expression. Murine primary microglial cells were treated with the indicated concentrations of PEA followed by treatment with 1 \(\upmu\)g/mL LPS for 24 h. Data are shown as the mean \(\pm\) SD of three independent experiments. # \(p\) value \(<\) 0.05 compared with control group. # \(p\)\(<\) 0.05 compared with LPS group.  
Figure 5: Panel (**A**): Western blot for TG2 expression in BV2 cells after treatment with different concentrations of A\(\beta\)1-42 for 48 h. SDS-PAGE blotted with antibody against TG2. GAPDH protein expression was used as loading control. The experiments were performed at least 3 different times and the results were always similar; Panel (**B**): graph shows the densitometric intensity of TG2/GAPDH bands ratio. The intensity of signals is expressed as a percentage in respect to the control (CTRL). #_p_ value < 0.05; #_p_ value < 0.01.  
Figure 6: Real Time-PCR was performed to measure the RNA expression levels of TG2 (**D**), inflammatory (**A**) or neuroprotective (**B**,**C**) markers after 24 h treatment. BV2 cells were pre-treated with the indicated concentrations of PEA or curcumin (Cu), followed by treatment with A\(\beta\)1-42 (1 uM for 24 h). Data are shown as the mean \(\pm\) SD of three independent experiments. #_p_ value < 0.05 compared with control group. #_p_ < 0.05 compared with A\(\beta\)1-42 group.