2.7. Anti-inflammatory experiment design

The in vitro experimental model of LPS-induced inflammation was used in the present study. AR extract concentrations of 25, 50, and 100 μg/mL were selected depending on the result of the cytotoxic test, and the experimental groups were designed as follows: (1) control group (Cont), cells were not treated with an extract or LPS; (2) LPS group (LPS), cells were treated only with LPS (0.1 μg/mL); (3) Low-(E25), medium- (E50), and high- (E100) dose extract groups, cells were pretreated with 25, 50, and 100 μg/mL extracts, respectively, before LPS (0.1 μg/mL) stimulation for several times. The production of nitric oxide (NO), prostaglandin E2 (PGE₂), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 or −1β, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in cells were determined to evaluate the anti-inflammatory activity. The phosphorylation of MAPKs, including p38 MAPK (p38), c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases (ERK) were investigated and the level of IκB-α was determined to clarify the molecular mechanism of anti-inflammation for the co-cultured Echinacea ARs.