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. 2019 Nov 10;11(11):1045.
doi: 10.3390/v11111045.

Ginsenoside Rg1 Suppresses Type 2 PRRSV Infection via NF-κB Signaling Pathway In Vitro, and Provides Partial Protection against HP-PRRSV in Piglet

Zhi-Qing Yu  1   2 , He-You Yi  1   2 , Jun Ma  1   2 , Ying-Fang Wei  1   2 , Meng-Kai Cai  1   2 , Qi Li  1   2 , Chen-Xiao Qin  1   2 , Yong-Jie Chen  2 , Xiao-Liang Han  1   2 , Ru-Ting Zhong  1   2 , Yao Chen  3 , Guan Liang  1   2 , Qiwei Deng  1   2 , Kegong Tian  4   5 , Heng Wang  1   2 , Gui-Hong Zhang  1   2
Affiliations

Ginsenoside Rg1 Suppresses Type 2 PRRSV Infection via NF-κB Signaling Pathway In Vitro, and Provides Partial Protection against HP-PRRSV in Piglet

Zhi-Qing Yu et al. Viruses. .

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 µM. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1β, IL-8, IL-6 and TNF-α, and decreased NF-κB signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine.

Keywords: NF-κB signaling pathway; antiviral activity; ginsenoside Rg1; porcine reproductive and respiratory syndrome virus; pro-inflammatory factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cytotoxicity and anti-PRRSV activity of Rg1 in Marc-145 cells and PAMs. (A) The chemical structures of ginsenoside Rg1 (Rg1). (B,C) Cytotoxicity of Rg1 in Marc-145 cells (B) and PAMs (C) were analyzed by using the WST-1 assay. Results are showed as the relative cell viability of PAMs or Marc-145 cells cultured without Rg1 (set as 100%). (D) Rg1 affects the proliferation of Marc-145 cells. Cells were seeded and cultured without FBS for 12 h and the medium was replaced with DMEM contains 0, 5, 10, 50, 100, 200 and 400 μM Rg1 respectively. (E,F) PRRSV XH-GD (0.1 MOI) infected Marc-145 cells or PAMs for 1 h at 37 °C, and then cells were cultured in DMEM or RPMI 1640 supplemented with 2% FBS and indicated concentrations of Rg1. The samples were collected at 48 hpi to analyze PRRSV Nsp9 mRNA levels in different groups by RT-PCR (E). N protein expression levels in cells treated with different concentrations of Rg1 were detected by western blot (F). (G) Marc-145 cells and PAMs infected with PRRSV XH-GD (0.1 MOI) for 1 h at 37 °C and then cultured in fresh medium supplemented with 10 or 50 μM Rg1. The expression levels of PRRSV Nsp9 in Marc-145 cells and PAMs were detected by RT-PCR analysis at the indicated time points. Each data represents results of three independent experiments (means ± SD). Significant differences compared with the control group are denoted by * (p < 0.05), ** (p < 0.01), *** (p < 0.001) and **** (p < 0.0001).
Figure 2
Figure 2
Inhibitory effects on the virus lifecycle of Rg1 in Marc-145 cells. In the Pre-treatment assay, Marc-145 cells were pretreated with DMEM supplemented with 10 or 50 μM Rg1 for 2 h, then cells were washed twice with PBS before being infected with type 2 PRRSV XH-GD (0.1 MOI), and then samples were collected at 48 h.p.i. For the attachment and internalization assay, Marc-145 cells were pre-cultivated at 4 °C for 1 h and then infected with virus (0.1 MOI) at 4 °C for 2 h. During virus attachment upon PRRSV infection, cells were cultured with DMEM or DMEM containing 10 or 50 μM Rg1 to analyze PRRSV Nsp9 mRNA level. Marc-145 cells were infected with XH-GD at 4 °C for 2 h and then cultured with or without Rg1 for 3 h at 37 °C. To avoid interference of other steps of viral lifecycle on replication assay, Marc-145 cells were infected with XH-GD for 6 h and then incubated with DMEM with or without 10 or 50 μM Rg1 at 37 °C, and samples were collected at 4 h.p.i. In all of the trials, GAPDH was used as a housekeeping gene for normalization, and cells treated with 0.4% DMSO was used as a reference control. (A) The effect of Rg1 on viral attachment, internalization, replication, and Rg1 pretreatment was analyzed by evaluating Nsp9 mRNA expression levels. (B) The effect of Rg1 on PRRSV release was detected by the ratio of Nsp9 RNA copy numbers in the supernatant and the cell lysate detected by qPCR. The analysis above was performed in triplicate. Statistical significance is denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 3
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
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.
Figure 5
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.
Figure 6
Figure 6
Rg1 exhibits anti-PRRSV activity in 4-week-piglet. (A) Daily rectal temperature of the pigs in the PRRSV, PRRSV+Rg1, Rg1 and mock groups. Rectal temperature reach or beyond 40 °C was defined as fever. (B) The mortality of each group was recorded daily and calculated as survival rate until 14 dpi. (C) The body weight gain of different groups during the experiment. (D) Severe lung lesions in the PRRSV group characterized by swelling, congestion, fibrosis, and inflammatory cell aggregates; however, in the PRRSV+Rg1 group, these index were moderate. Due to no piglet survived at 14 dpi in the challenge control group (PRRSV), the lung of the deceased pigs at 10 dpi was used to perform pathological analysis. (E) The anti-PRRSV antibody levels in serum at different time-points. The value of S/p ratio ≥ 0.4 was considered antibody positive. (F) The level of PRRSV mRNA in the serum was measured by real-time PCR. (G) The expression level of PRRSV mRNA level in lungs, lymph node and thymus was measured by qRT-PCR. Each tissue sample was measured three times, and the error bars represent the standard deviations of samples.
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