*3.4. Rg1 Treatment Significantly Reduced the Pro-Inflammatory Cytokine mRNA Levels Induced by PRRSV Infection in Both Marc-145 Cells and PAMs*

In view of the pro-inflammatory cytokines triggered by PRRSV, this contributes to its pathogenicity, and the role Rg1 plays in relieving inflammatory responses [19]. RT-PCR was used to determine whether Rg1 treatment alleviates the expression of pro-inflammatory factors, including IL-1β, IL-6, IL-8 and TNF-α, induced by PRRSV infection. The data indicates that Rg1 treatment significantly (*p* < 0.05) reduces the mRNA levels of pro-inflammatory factors increased by PRRSV infection both in Marc-145 cells and PAMs. In Marc-145 cells, mRNA level of IL-1β, IL-6 and TNF-α are significantly reduced by Rg1 from 12 to 24 h upon PRRSV infection, while, IL-8 is decreased by Rg1 at 18 and 24 h (Figure 4A). Further, pro-inflammatory cytokines assay was performed in PAMs and the results showed that IL-6, IL-8 and TNF-α triggered by PRRSV lowered by Rg1 pronouncedly from 12 to 24 h, while the reduction of mRNA level of IL-1β was seen at 18 and 24 h (Figure 4B). Although the inhibitory effect of Rg1 on each pro-inflammatory factor in Marc-145 cells and PAMs was not strictly consistent, the decline trend was both obvious at 18 h and 24 h. The results demonstrate that Rg1 could relieve the inflammatory responses caused by PRRSV infection via decreasing the mRNA expression of pro-inflammatory cytokines

**Figure 3.** The antiviral activity of Rg1 against different lineages of type 2 PRRSV. (**A**) Antiviral activity of Rg1 against PRRSV strains (HP-PRRSV XH-GD and JXA1, classical VR2332 and NADC30-like strain HNLY) was determined in Marc-145 cells by IFA. Marc-145 cells were seeded in 12-well plates and infected with four type 2 PRRSV strain (0.1 MOI) respectively, and then incubated with DMEM supplemented with indicated concentration of Rg1. N protein was used as indicator of PRRSV infection, and the IFA detection of it was performed at 48 h.p.i. by using mouse anti-N protein antibody and goat anti-mouse IgG Alexa Fluor. Nuclei were counterstained with DAPI (blue). These images above

represent three independent IFA trials with similar results. Magnification, 100 ×. (**B**,**C**) The inhibitory effect of Rg1 on PRRSV replication in Marc-145 cells (**B**), and PAMs (**C**). PRRSV replication was analyzed by virus growth curve. Marc-145 cells and PAMs were seeded in 6-well plates and infected with four PRRSV strain (0.1 MOI) for 1 h at 37 ◦C respectively and then cultured with DMEM or RPMI 1640 supplemented with 10 or 50 μM Rg1 or DMSO. Cell supernatants (200 μL) of each well were collected at indicated hours of post-infection. Growth assays for each group were performed in triplicate, and the resulting titers were determined as TCID50/mL (the 50% tissue culture infectious dose per mL) and the data are shown as the means ± SD. T-test was applied to perform statistical analysis. Statistical significance between 10 μM Rg1 and DMSO is denoted by \* *p* < 0.05 and \*\* *p* < 0.01, and significance between 50 μM Rg1 and DMSO is denoted by # *p* < 0.05 and ## *p* < 0.01.

**Figure 4.** Rg1 suppresses inflammatory cytokines mRNA expression in infected PAMs and Marc-145 cells. (**A**) For PRRSV+Rg1 group, the XH-GD strain (0.1 MOI) infected Marc-145 cells for 1 h and then cultured in DMEM supplemented with Rg1 (200 μM), and cells infected with virus or treated with Rg1 (200 μM) were termed as PRRSV group and Rg1 group, respectively. Cells in the mock group were grown in DMEM containing 0.4% DMSO. Cell samples were collected to extract total RNA at 12, 18, and 24 h.p.i. The relative expression of IL-6, IL-8, IL-1β and TNF-α was analyzed by RT-PCR. GAPDH was used as internal control to normalize values. (**B**) PAMs were cultured with RPMI 1640 and treated as described above. The data of each trial represents three independent experiments and the values are shown as the means ± SD. T-test was applied to perform statistical analysis and the significance was indicated by asterisk in the graphs. Statistical significance is denoted by \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.
