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. 2021 Dec;36(6):1626-1643.
doi: 10.1007/s12250-021-00455-y. Epub 2021 Oct 27.

Andrographolide and Its Derivative Potassium Dehydrographolide Succinate Suppress PRRSV Replication in Primary and Established Cells via Differential Mechanisms of Action

Lizhan Su #  1 , Yarou Gao #  1 , Mingxin Zhang  1 , Zexin Liu  1 , Qisheng Lin  1 , Lang Gong  2 , Jianying Guo  2 , Lixia Chen  3 , Tongqing An  4 , Jianxin Chen  5   6
Affiliations

Andrographolide and Its Derivative Potassium Dehydrographolide Succinate Suppress PRRSV Replication in Primary and Established Cells via Differential Mechanisms of Action

Lizhan Su et al. Virol Sin. 2021 Dec.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently, vaccination strategies provide limited protection against PRRSV infection, and consequently, new antiviral strategies are urgently required. Andrographolide (Andro) and its derivative potassium dehydrographolide succinate (PDS) have been used clinically in China and other Asian countries as therapies for inflammation-related diseases, including bacterial and viral infections, for decades. Here, we demonstrate that Andro and PDS exhibit robust activity against PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages (PAMs). The two compounds exhibited broad-spectrum inhibitory activities in vitro against clinically circulating type 2 PRRSV GD-HD, XH-GD, and NADC30-like HNhx strains in China. The EC50 values of Andro against three tested PRRSV strain infections in Marc-145 cells ranged from 11.7 to 15.3 μmol/L, with selectivity indexes ranging from 8.3 to 10.8, while the EC50 values of PDS ranged from 57.1 to 85.4 μmol/L, with selectivity indexes ranging from 344 to 515. Mechanistically, the anti-PRRSV activity of the two compounds is closely associated with their potent suppression on NF-κB activation and enhanced oxidative stress induced by PRRSV infection. Further mechanistic investigations revealed that PDS, but not Andro, is able to directly interact with PRRSV particles. Taken together, our findings suggest that Andro and PDS are promising PRRSV inhibitors in vitro and deserves further in vivo studies in swine.

Keywords: Andrographolide (Andro); Inhibit; NF-κB signaling pathway; Oxidative stress; Porcine reproductive and respiratory syndrome virus (PRRSV); Potassium dehydrographolide succinate (PDS).

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Andro and PDS inhibit PRRSV replication with minimal cytotoxicity in Marc-145 cells. (A) Chemical structures of andrographolide (Andro) and potassium dehydroandrographolide succinate (PDS). (B) Cellular toxicity of Andro and PDS was examined in Marc-145 cells using an MTT assay. (C and D) Antiviral activity of Andro and PDS against PRRSVs (GD-HD, XH-GD, and NADC30-like HNhx strains) infection in Marc-145 cells was examined using indirect immunofluorescence assay (IFA). Cells grown in 96-well plates were infected with PRRSV (0.05 MOI) for 2 h at 37 °C and then cultured in fresh medium containing various concentrations of Andro or PDS. The IFA for the N protein of PRRSV was performed at 48 hpi using Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (red). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Representative IFA images of three independent experiments are shown in C and D. D and F demonstrate the percentage of inhibition based on the fluorescence optical densities (OD) of the images from three independent experiments. Image J was used to digitize image OD. Scale bar: 250 µm.
Fig. 2
Fig. 2
Confirmation of the anti-PRRSV activity of Andro and PDS in Marc-145 cells. Marc-145 cells were cultured in 12-well plates for 24 h. Cells were infected with 0.05 MOI of PRRSV GD-HD for 2 h and incubated with fresh medium containing different concentrations of Andro or PDS. Samples were collected at 24, 48, and 72 h post infection. The supernatants were used to determine the virus titers using the end-point dilution assay (A and B), and the cells were used for the analysis of relative PRRSV NSP9 mRNA levels by qRT-PCR (C and D) and viral N protein expressions using Western blot (E and F). α-Tubulin was used as a loading control, and DMSO-treated sample (0 μmol/L Andro and PDS) was used as the control. **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control.
Fig. 3
Fig. 3
Cytotoxicity and anti-PRRSV activity of Andro and PDS in PAMs. (A) The cellular toxicity of Andro and PDS was examined in PAMs at 24 h using the MTT assay. PAMs grown in 24-well plates were infected with GD-HD PRRSV (1 MOI) for 2 h at 37 °C and then treated with various concentrations of Andro or PDS for 24 h. The expression of viral N protein was analyzed by IFA (B), and the percentage of inhibition based on the fluorescence optical densities (OD) of the images from three independent experiments are shown (C). Parallel samples were submitted for analysis of the viral titer using the end-point dilution assay (D), viral NSP9 gene mRNA level using qRT-PCR (E), and N protein level using Western blot (F). Scale bar: 250 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control.
Fig. 4
Fig. 4
Andro and PDS suppress PRRSV replication in co- and post-treatment modes. Marc-145 cells were grown in 24-well plates for 24 h and then treated with Andro (45 μmol/L) or PDS (560 μmol/L) for 2–8 h prior to virus infection (pre-treatment), for 2 h during viral infection (co-treatment), or for 2–8 h after virus infection and removal (post-treatment) (A). For all three treatment modes, 0.05 MOI of PRRSV GD-HD was used to infect the cells for 2 h. At 24 hpi, the cells were subjected to viral N protein analysis using IFA (B). The percentages of inhibition based on the fluorescence optical densities (OD) of the images from three independent experiments are shown in C. Scale bar: 250 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control.
Fig. 5
Fig. 5
PDS rather than Andro interacts with PRRSV directly. 100 μL PRRSV GD-HD (2 × 106 TCID50) was mixed with various concentrations of Andro or PDS in essential medium (0.9 mL total volume) for 1 h at 37 °C. Then, the PRRSV and compounds were separated by ultrafiltration, as shown in (A). Recovered PRRSV was resuspended and used to infect Marc-145 cells for 2 h, followed by incubation in fresh medium. At 48 hpi, samples were subjected to IFA for the detection of viral N protein (B and C). The images in C are representative results from the three independent IFA assays shown in B. Parallel samples were subjected for viral titration using the end-point dilution assay (D) and viral mRNA analysis using qRT-PCR (E). Scale bar: 250 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control. NS: No statistically significant difference compared to the control.
Fig. 6
Fig. 6
Electron micrographs of PDS-induced PRRSV aggregation. PRRSV (1 × 106 TCID50/mL) was treated with different concentrations of PDS (0, 280, and 560 μmol/L) for 1 h at 37 °C; 2% PTA was used to stain the viral particles. The morphology of PRRSV virions was observed by transmission electron microscopy (TEM). The yellow arrows represent the virus particles. The white arrows represent the aggregation of virus particles. Scale bars: 200 nm.
Fig. 7
Fig. 7
Andro and PDS inhibit NF-κB activation and subsequent production of pro-inflammatory cytokines in Marc-145 cells and PAMs. Marc-145 cells were infected with PRRSV GD-HD (0.05 MOI) for 2 h at 37 °C and treated with or without Andro (45 μmol/L) or PDS (560 μmol/L) for 24 h. Cells were inoculated with LPS (5 µg/mL) at 37 °C for 6 h. (A) The protein expression of p65, p-p65, IκBα, and p-IκBα in Marc-145 cells treated with Andro or PDS was assessed by Western blot. (B) Confocal microscopy analysis of p65 nuclear translocation. At 24 hpi, cells were fixed and stained with Alexa Fluor 488-conjugated goat anti-rabbit anti-p65 IgG antibody (green). Nuclei were counterstained with DAPI (blue). The white arrows represent p65 nuclear translocation. Scale bar: 50 µm. (C and D) Effects of Andro and PDS on the production of TNF-α, IL-6, and IL-1β in PRRSV-infected and LPS-induced Marc-145 cells (C) and PAMs (D). Cells were collected to extract total RNA at 24 hpi. The relative expression of TNF-α, IL-6, and IL-1β was analyzed by qRT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the mock-infected control.
Fig. 8
Fig. 8
Andro and PDS suppress oxidative stress induced by PRRSV in Marc-145 cells. Marc-145 cells were infected with PRRSV (0.05 MOI) for 2 h at 37 °C and then incubated in fresh medium in the presence or absence of Andro (45 μmol/L) or PDS (560 μmol/L) for 24 h. As a positive control, Marc-145 cells were stimulated with H2O2 (2.5 µmol/L) at 37 °C for 0.5 h. (A and B) The ROS level, reflected by the ratio of positive cells stained with dichlorofluorescein (DCF), was determined using IFA (A) and flow cytometry (B). (C–E) Effects of Andro and PDS on the levels of MDA, SOD, and GSH in Marc-145 cells infected with PRRSV or stimulated with H2O2 were determined using their respective ELISA assay kits. Scale bar: 250 µm. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the DMSO-treated control; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the mock control.
Fig. 9
Fig. 9
Schematic overview of the inhibitory cascade effect of Andro and PDS on PRRSV replication.
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