*3.5. PRRSV-Infection Triggered NF-*κ*B Activation Was Inhibited by Rg1 Treatment in Marc-145 Cells*

In the results above, Rg1 decreases the PRRSV-triggered mRNA level of pro-inflammatory cytokine. It is known that NF-κB is one of the key transcription factors regulating the production of pro-inflammatory factors, such as IL-6 and IL-8 [29]. PRRSV is proven to activate the NF-κB pathway to enhance viral replication [30]. Therefore, the effect of Rg1 treatment on PRRSV mediated NF-κB activation was analyzed by western blotting and immunofluorescence staining, and LPS stimulation, which activates NF-κB, was used as a control to show whether it associated with virus replication or indirectly through cellular response. We find that the increased phosphorylation of p65 induced by PRRSV infection or LPS is weakened by Rg1 treatment, and it also inhibits IκB degradation (Figure 5A). Although the phosphorylation of IκB-α, which is related to IκB degradation, was not significantly enhanced by PRRSV infection compared with mock, it was reduced by Rg1 treatment (Figure 5A). Moreover, the cellular localization of NF-κB was determined by p65 immunofluorescence staining (red) in Marc-145 cells, and the nuclei were stained with DAPI (blue) (Figure 5B). The results are consistent with the phosphorylation level of p65 by western blotting. In the Mock and Rg1 groups, P65 is mainly

localized in cytoplasm and the phosphorylation level is lower. PRRSV triggers P65 phosphorylation to facilitate its translocation into nuclear, and this process is significantly reduced after Rg1 treatment. Taken together, these data indicate that Rg1 treatment decreases PRRSV-mediated NF-κB activation and IκB degradation, and a schematic of this event is presented in Figure 5C.

**Figure 5.** Rg1 inhibits the NF-κB pathway activated by PRRSV infection. (**A**) The expression and phosphorylation level of proteins involved in NF-κB pathway are analyzed in uninfected (Mock) and PRRSV XH-GD (0.1 MOI) infected Marc-145 cells treated with or without Rg1 (200 μM), samples were collected at 24 h.p.i. As a positive control, cells were cultured in DMEM and supplemented with LPS (2.5 μg/mL) at 37 ◦C for 6 h, and then the medium was changed to medium containing 0 or 200 μM Rg1 for 18 h. The western blotting data of each target protein represents three independent experiments with similar results. (**B**) Marc-145 cells were grown on glass cover slips and cultured in medium at 37 ◦C for 24 h, and then infected with PRRSV XH-GD (0.1 MOI). After virus infection, cells were incubated in fresh DMEM supplemented with or without 200 μM Rg1 for 24 h. Cells were washed twice with PBS and performed immunostaining by using anti-P65 antibody and red-fluorescent Alexa Fluor 594-conjugated goat anti-mouse IgG antibody. Nuclei were counterstained with DAPI. P65 protein deposited in the nucleus was indicated by yellow arrow. (**C**) Schematic model of Rg1 affect NF-κB signaling pathway upon PRRSV infection.
