Figure 9

Xanthohumol upregulates the Nrf2–HMOX1 antioxidative axis to inhibit PRRSV proliferation in PAMs. A PAMs were pre-treated with DMSO or concentrations of Xn for 1 h and then infected with PRRSV (0.10 MOI) for 1 h at 37 °C. Cells were washed and incubated in fresh medium containing DMSO or Xn. At 24 hpi, cells were harvested for the viral titre. B PAMs 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. C PAMs treated with CoPP (an inducer of HMOX1) for 6 h before PRRSV infection were examined for N-protein and endogenous HMOX1 by Western blotting. D PAMs were treated with an increasing multiplicity of Xn (0–10 µM) or DMSO for 1 h and then infected with PRRSV (MOI 0.01) for 1 h. After washing three times with PBS, culture medium containing Xn (0–10 µM) or DMSO was added back to the wells. At 24 hpi, the levels of HMOX1 and PRRSV N-protein were detected by Western blotting. E PAMs were treated with different doses of Xn for 24 h, and Nrf2 and HMOX1 were detected by Western blotting. F PAMs were treated with 15 µM Xn or DMSO for 1 h and then infected with PRRSV (MOI 0.01) for 1 h. After washing three times with PBS, culture medium containing 15 µM Xn or DMSO was added back to wells. At 24 hpi, the levels of Nrf2, HMOX1 and PRRSV N-protein were detected by Western blotting. G, H PAMs were transfected with 50 pmol of siRNA targeting Nrf2. After 48 h, they were treated with 10 µM Xn or DMSO for 1 h and then infected with PRRSV (0.01 MOI). After 36 hpi, the mRNA levels of Nrf2 and PRRSV ORF7 were detected by qRT-PCR (G). Meanwhile, the levels of Nrf2, HMOX1 and PRRSV N-protein were detected by Western blotting (H). ***P < 0.001; **P < 0.01; *P < 0.05 compared with the control group