Astragaloside IV inhibits inflammation caused by influenza virus via reactive oxygen species/NOD-like receptor thermal protein domain associated protein 3/Caspase-1 signaling pathway

Abstract

Background: Astragaloside IV (AS-IV) is the most active monomer in the traditional Chinese herbal medicine Radix Astragali, which has a wide range of antiviral, anti-inflammatory, and antifibrosis pharmacological effects, and shows protective effects in acute lung injury.

Methods: This study utilized the immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, and hematoxylin and eosin staining methods to investigate the mechanism of AS-IV in reducing viral pneumonia caused by influenza A virus in A549 cells and BALB/c mice.

Results: The results showed that AS-IV suppressed reactive oxygen species production in influenza virus-infected A549 cells in a dose-dependent manner, and subsequently inhibited the activation of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 inflammasome and Caspase-1, decreased interleukin (IL) -1β and IL-18 secretion. In BALB/c mice infected with Poly (I:C), oral administration of AS-IV can significantly reduce Poly (I:C)-induced acute pneumonia and lung pathological injury.

Conclusions: AS-IV alleviates the inflammatory response induced by influenza virus in vitro and lung flammation and structural damage caused by poly (I:C) in vivo.

Keywords: Caspase‐1; NOD‐like receptor thermal protein domain associated protein 3; astragaloside IV; inflammation; influenza virus; reactive oxygen species.

Figure 1

The cytotoxicity effects of astragaloside IV (AS‐IV) on the A549 cells. (A) Chemical structure of AS‐IV. (B) Cell Counting Kit‐8 (CCK‐8) reagent was used to detect A549 cell viability. Cell viability was defined as the percentage of the 0‐μM group (n = 3). (C) The growth of A549 cells was observed under the microscope (100x magnifying power). ns, no significant difference, ** p < 0.01, when compared to the 0 group.

Figure 2

Protective effect of astragaloside IV (AS‐IV) on A549 cells infected with influenza virus. Cell Counting Kit‐8 (CCK‐8) reagent was used to detect A549 cell viability. Cell viability was defined as the percentage of the NC group (n = 3). NC, negative control group; IAV, influenza A virus. ^## p < 0.01, when compared to the NC group; * p < 0,05, ** p < 0.01, when compared to the IAV group.

Figure 3

Effects of astragaloside IV (AS‐IV) on influenza virus in A549 cells. (A) A549 cells infected with influenza A virus (IAV) were treated with 50‐, 75‐, and 100‐μM AS‐IV, and the infection rate of IAV in A549 cells in each group was observed after 16 h (100x magnifying power). (B) and (C) Western blot detected NP protein expression in A549 cells treated with AS‐IV after influenza virus infection (n = 3). (D) 100‐μM AS‐IV treated A549 cells infected with IAV, and immunofluorescence was performed at different time points (2, 4, 6, 8, 10, and 12 h) to observe the effect of AS‐IV on nuclear membrane shuttle transport of IAV NP protein in A549 cells (200x magnifying power). ns, no significant difference, compared to the IAV group. NC, negative control group. NP, nucleoprotein.

Figure 4

Antioxidative effect of astragaloside IV (AS‐IV). (A)–(D) Effects of AS‐IV on total antioxidant capacity, superoxide dismutase, glutathione peroxidase, catalase, and malondialdehyde in A549 cells infected with influenza virus (n = 3). (F) Effects of AS‐IV on reactive oxygen species in A549 cells infected with influenza virus. GPX, glutathione peroxidase; NC, negative control group; IAV, influenza A virus. ^## p < .01, when compared to the NC group; ns, no significant difference, * p < 0.05, ** p < 0.01, when compared to the IAV group.

Figure 5

Effect of astragaloside IV (AS‐IV) therapy on the NOD‐like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome pathway in A549 cells infected with influenza virus. (A)–(D) Real‐time polymerase chain reaction analysis was used to analyze the mRNA levels of NLRP3, Caspase‐1, IL‐1β, and IL‐18 in different groups. (E)–(H) The protein expression levels of NLRP3 inflammasome, Pro‐Caspase‐1, and Cleaved Caspase‐1 in each group were detected by western blot. (I)–(J) Enzyme‐linked immunosorbent assay was used to detect the expression levels of inflammatory cytokines IL‐18 and IL‐1β in each group. n = 3, NC, negative control group; IAV, influenza A virus. ^# p < 0.05, ^## p < 0.01, when compared to the NC group; * p < 0.05, ** p < 0.01, when compared to the IAV group. IL, interleukin; mRNA, messenger RiboNucleic acid; NOD, nucleotide‐binding oligomerization domain.

Figure 6

Effects of astragaloside IV (AS‐IV) treatment on poly (I:C)‐induced lung inflammatory lesions in mice. (A) Survival curve of each group of mice. (B) Lung index for mice killed on Day 6. (C) Pathological scores of lung tissue in mice of each group. (D) and (E) Enzyme‐linked immunosorbent assay detects levels of IL‐18 and IL‐1β in mice lung epithelial cells. (F) and (G) Quantitative reverse transcription‐polymerase chain reaction detects levels of IL‐18 and IL‐1β in mice lung epithelial cells. (H) Hematoxylin and eosin staining of lung tissue, the red arrow points to inflammatory cells (200x magnifying power). The number of samples in each group is n = 6. NC, negative control group. ^## p < 0.01, when compared to the NC group; * p < 0.05, ** p < 0.01, when compared to the poly (I: C) group. IL, interleukin.