Fig. 7

HMH protects against LPS-induced oxidative stress via the Nrf2/HO-1 pathway. RAW264.7 macrophages (1 × 10⁵ cells/mL) were treated with 0–10 mg/mL HMH for 2 h prior to LPS (200 ng/mL) treatment for 24 h. (A) Total RNA was extracted at 6 h, and RT-PCR was performed to evaluate Nrf2 and HO-1 expression. (B) In a parallel experiment, nuclear and cytosolic proteins were extracted at 24 h, and western blotting was performed to evaluate Nrf2 and HO-1 expression. The relative density of Nrf2 and HO-1 expression was shown relative to each control. (C) Zebrafish larvae at 3 dpf were microinjected with LPS (0.5 mg/mL, 2 nL per larva) and treated with HMH (5 mg/mL) or ZnPP (10 μM) for 18 h. Total RNA was extracted, and RT-PCR was performed to evaluate iNOS and COX-2a expression. The relative density of iNOS and COX-2a expression was shown relative to β-actin. (D) In a parallel experiment, zebrafish larvae were stained using 5 μM DAF-FMDA, and the images were captured using a CELENA S Digital System. ∗∗∗p < 0.001 vs untreated group, ^###p < 0.001 vs LPS-treated group, and ^&&&p < 0.001 vs. LPS/HMH-treated group.