An ethyl acetate fraction of flavonoids from Polygonum hydropiper L. exhibits an anti-inflammatory activity in PCV2-infected porcine alveolar macrophages via PI3K/Akt and NF-κB pathways

Abstract

Porcine circovirus type 2 (PCV2) widely exists in swine production systems causing porcine circovirus diseases (PCVD) which is associated with significant economic losses. Polygonum hydropiper L. was used as a traditional Chinese medicine to treat a variety of diseases. This study was carried out to investigate anti-inflammatory activity of the ethyl acetate fraction of flavonoids from Polygonum hydropiper L. (FEA) in PCV2-induced porcine alveolar macrophages (3D4/2 cell line). The production of oxygen species (ROS) and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-8 (IL-8) were detected to evaluate the anti-inflammatory activities of FEA. The translocation of nuclear factor-kappa B (NF-κB) and the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathways were investigated to document the potential anti-inflammatory mechanisms. In PCV2 induced 3D4/2 cells, FEA treatment significantly reduced the production of ROS, and sharply down-regulated the levels of TNF-α, IL-1β and IL-8 in both secretion and mRNA expression level. The FEA also decreased the mRNA expression of Akt and NF-κB p65, reduced the transfer of p65 to nuclear, and inhibited the activation of PI3K/Akt signaling pathway. The findings suggest that FEA exhibited an anti-inflammatory activity in vitro and could be used as a candidate in treatment of inflammation induced by PCV2 infection.

Keywords: 3D4/2 cell; Anti-inflammatory activity; Polygonum hydropiper L.; Porcine circovirus type 2.

Fig. 1

Preparation of FEA and PCV2. A) HPLC analysis of FEA. 1: rutin; 2: quercitrin; 3: quercetin. B) PCR amplification electrophoresis results of PCV2. The results of PCV2 detection in PK-15 cells. Lane C: PK-15 cells without PCV2 infection; Lanes 1-5: times at 4, 8, 12, 24 and 48 hr after PK-15 cells were infected with PCV2; Lane 6: positive control; Lane M: DNA Marker 2000. C) The results of PCV2 detection in 3D4/2 cells by PCR and Lane Marker: DNA Marker 2000; Lane C: 3D4/2 cells without PCV2 infection; Lanes 1-4: times at 4, 8, 12 and 24 hr after 3D4/2 cells were infected with PCV2; Lane 5: positive control

Fig. 2

Cell viability and ROS generation. A) Cell viability in LPS treated 3D4/2 cells. The cells were incubated with a series of concentrations of LPS at 0.10, 1.00, 5.00 and 10.00 μg mL^-1 for 4, 8, 12 and 24 hr. B) Cell viability in FEA treated 3D4/2 cells. The cells were treated with a series concentration of FEA at 25.00, 50.00, 100, 200, 400 and 800 μg mL^-1) for 24 hr. C) Effects of FEA on ROS generation in PCV2 infected 3D4/2 cells. The 3D4/2 cells were incubated with PCV2 for 2 hr, subsequently, treated with FEA at 25.00, 50.00 and 100 μg mL^-1 for 8 hr. ROS production from 3D4/2 cells was measured by DCFH-DA probe. The values are presented as mean ± standard deviation

Fig. 3

The effects of FEA on secretion of inflammatory cytokines. 3D4/2 cells were incubation with PCV2 for 2 hr, subsequently, treated with FEA (25.00, 50.00 and 100 μg mL^-1) for 8 hr. The secretion of IL-1β, TNF-α and IL-8 in the supernatant of cultured cells was determined using commercial ELISA kits (A, B and C). The mRNA expression of IL-1β, TNF-α and IL-8 were analyzed using qRT-PCR (D, E and F). Values are presented as mean ± standard deviation

Fig 4

Effects of FEA on PI3K/Akt pathways and NF-κB pathways. The 3D4/2 cells were incubated with PCV2 for 2 hr, subsequently, treated with FEA at 25.00, 50.00 and 100 μg mL^-1 for 8 hr. The translocation of p65 to nuclear and IκBα protein expression were analyzed by western blotting analysis. The protein expression of PI3K, Akt and p-Akt were also analyzed by western blotting. A) p65 mRNA expression; B) Akt mRNA expression; C-D) Protein bands; E) the ratio of p-p65/p65. F) The ratio of p-Akt/Akt. G-H) IκBα/β-actin, PI3K/β-actin. The values are presented as mean ± standard deviation