Fig. 2

HMH inhibits LPS-induced NO and PGE₂ production. RAW 264.7 macrophages (1 × 10⁵ cells/mL) were treated with HMH (0–10 mg/mL) for 2 h prior to LPS (200 ng/mL) treatment. (A) Total RNA was extracted 6 h after LPS treatment, and RT-PCR was performed to evaluate the expression of iNOS and COX-2. iNOS and COX-2 expression was normalized to GAPDH expression. (B) In a parallel experiment, total proteins were extracted at 12 h, and western blotting was performed to evaluate the expression of iNOS and COX-2. iNOS and COX-2 expression was normalized to β-actin expression. (C) Cells were stained using 1 μM DAF-FMDA, and the images were captured using a CELENA S Digital Imaging System. Cell-free culture media were collected 24 h after LPS treatment. (D) Griess reagent assay was performed to quantify NO production, and (E) PGE₂ levels were quantified using an ELISA assay. ∗∗∗p < 0.001 vs. untreated cells; ^#p < 0.05 and ^###p < 0.001 vs LPS-treated cells.