Cell experiment:

RAW 264.7 cells, derived from murine macrophages, are cultured in DMEM supplemented with 10% endotoxin-free, heat-inactivated fetal calf serum, Penicillin (100 units/mL), and Streptomycin (100 μg/mL) in a 5% CO2 atmosphere at 37℃ in a humidified incubator. For all assay, cell is plated at 2×105 cells/cm2 in culture dishes or plates. Treatment with vehicle (0.1% DMSO or 0.1% ethanol), test compounds and/or LPS is carried out under serum-free conditions[1].

Tomatidine acts as an anti-inflammatory agent by blocking NF-κB and JNK signaling.

Tomatidine decreases inducible NO synthase and COX-2 expression through suppression of I-κBα phosphorylation, NF-κB nuclear translocation and JNK activation, which in turn inhibits c-jun phosphorylation and Oct-2 expression. Tomatidine, solasodine and diosgenin (40 μM) show 66%, 22% and 41% inhibition of nitrite production, respectively. The iNOS protein is barely detectable in unstimulated cells but markedly increases after LPS treatment, and Tomatidine causes dose-dependent inhibition of LPS-induced iNOS expression. p65 is the major component of NF-κB in LPS-stimulated macrophages, the effect of Tomatidine on p65 DNA-binding activity is determined. In the presence of Tomatidine at 10-40 μM, the binding activity of NF-κB is suppressed in a dose-dependent manner. Tomatidine inhibits the phosphorylation of I-κB, blocks the I-κB production, and furthermore suppresses p65 NF-κB translocation to the nucleus and modulated binding activity[1].

[1]. Chiu FL, et al. Tomatidine inhibits iNOS and COX-2 through suppression of NF-kappaB and JNK pathways in LPS-stimulated mouse macrophages. FEBS Lett. 2008 Jul 9;582(16):2407-12.