*2.4. Comparative Effects of T. lutea F&M-M36 Extract and FX on PGE2 Production and COX-2 Protein Expression*

As shown in Figure 3, the methanolic extract of *T. lutea* F&M-M36 (1–100 μg/mL) significantly decreased the LPS-induced production of PGE2 in a concentration-dependent manner, whereas FX was significantly effective (*p* < 0.05) only at the highest concentration tested (470 ng/mL). When we compared directly the PGE2 levels measured in the media

from cells treated with the methanolic extract and those of the cells treated with FX at equivalent concentrations, we observed a clear and significant reduction at all concentrations (*p* < 0.001).

**Figure 3.** Effect of *T. lutea* F&M-M36 extract and FX on PGE2 production in RAW 264.7 stimulated with LPS for 18 h. ### *p* < 0.001 vs. unstimulated RAW 264.7 macrophages (CTRL); \*\*\* *p* < 0.001 vs. LPS ˆˆˆ *p* < 0.001 vs. FX by ANOVA test and Dunnett's Multiple Comparison test. Data are expressed as the mean ± SEM of four replicates.

Immunofluorescent staining for COX-2 protein expression (Figure 4 Panels A–D) and dot blot analyses (Panel F) demonstrated that the methanolic extract from *T. lutea* F&M-M36 significantly counteracted LPS induced COX-2 protein expression (*p* < 0.001) as it did in FX alone, although to a lower extent (*p* < 0.05) (Figure 4 panel E). Similar to the results on PGE2, *T. lutea* F&M-M36 extract also significantly decreased COX-2 protein expression compared to FX alone (*p* < 0.01).

The effects on COX-2 were much more evident at the protein level than gene level, since COX-2 mRNA expression was not significantly decreased neither by *T. lutea* F&M-M36 extract nor by FX compared to LPS-treated cells; in this regard, however, it should be highlighted that when directly compared, the mRNA expression of COX-2 was significantly decreased by *T. lutea* F&M-M36 extract compared to FX (*p* < 0.05).
