Figure 7

Xanthohumol upregulates Nrf2–HMOX1 to inhibit PRRSV proliferation. A Marc-145 cells were treated with 10 µM Xn for different times over 24 h, followed by qRT-PCR for HMOX1 mRNA. ***P < 0.001; **P < 0.01; *P < 0.05 vs DMSO-treated cells. B–D PRRSV replication in Marc-145 cells transfected with the indicated doses of pCAGGS-HMOX1 for 24 h and then infected with PRRSV (0.01 MOI) for 48 h was evaluated by Western blotting, TCID₅₀ and qPCR. E Marc-145 cells were treated with Xn or DMSO for 1 h, then infected with PRRSV (0.1 MOI) for 1 h at 37 °C and finally cultured in medium containing 10 µM Xn or DMSO. Total RNA was extracted from lysates of cells at 24 hpi, and the mRNA levels of PRRSV ORF7 and a set of Nrf2-regulated antioxidant genes were detected by qRT-PCR. ^###P < 0.001; ^##P < 0.01; ^#P < 0.05 vs DMSO-treated cells. ***P < 0.001; **P < 0.01; *P < 0.05 vs DMSO + PRRSV cells. F Cells were lysed and samples were prepared for Western blotting. G qRT-PCR and H Western blot analysis were performed on Marc-145 cells transfected with 50 pmol of siNC or Nrf2 siRNAs (siNrf2-a and siNrf2-b) using 5 µL of Lipofectamine RNAiMAX transfection reagent for 24 h. Cells were then infected with PRRSV (0.01 MOI), and after 36 h, cells were harvested for assays. I qRT-PCR and J Western blot analysis were performed on Marc-145 cells transfected with siNrf2-a and siNC. After 24 h, cells were treated with 10 µM Xn or DMSO for 1 h and then infected with PRRSV (0.01 MOI). After 36 h, cells were harvested for assays. All results are means ± standard deviations from three independent experiments performed in triplicate. ***P < 0.001; **P < 0.01; *P < 0.05 compared with the control group