Total astragalosides decrease apoptosis and pyroptosis by inhibiting enterovirus 71 replication in gastric epithelial cells

Abstract

Enterovirus 71 (EV71) is one of the primary pathogens involved in severe hand, foot and mouth disease in children. EV71 infection causes various types of programmed cell death. However, there are currently no clinically approved specific antiviral drugs for control of EV71 infection. Astragalus membranaceus (AM), a Traditional Chinese medicine, has been used in antiviral therapy in China. The aim of the present study was to determine whether total astragalosides (ASTs), bioactive components of AM, protect against EV. DAPI nuclear staining was used to observe morphological changes of the nucleus and the protective effect of ASTs, which revealed that the nucleus shrank following EV71 infection, while ASTs reversed it. Cell Counting Kit-8 assay found that human normal gastric epithelial cell (GES-1 cell) viability decreased following EV71 infection, while lactate dehydrogenase (LDH) assay showed that EV71 infection induced GES-1 cell damage. Western blotting was used to measure the expression levels of apoptosis and pyroptosis marker protein to determine whether EV71 infection induced apoptosis and pyroptosis in GES-1 cells. Reverse transcription-quantitative PCR was used to determine the anti-EV71 effect of ASTs. The results showed that ASTs protected GES-1 cells from EV71-induced cell apoptosis and pyroptosis. Furthermore, the present data demonstrated that the protective effect of ASTs was exerted by suppressing EV71 replication and release. These findings suggested that ASTs may represent a potential antiviral agent for the treatment of EV71 infection.

Keywords: apoptosis; enterovirus 71; pyroptosis; replication; total astragaloside.

Figure 1

EV71 infection causes direct damage to cells. (A) GES-1 cells were infected with EV71 at MOIs of 0, 1, 3 and 5. Cell morphology was observed under an inverted microscope (magnification, x100). (B) GES-1 cell activity was evaluated using a Cell Counting Kit-8 assay. (C) GES-1 cell damage was evaluated by LDH release. Data are presented as the mean ± SD (n=3). ^**P<0.01 vs. 0. EV71, enterovirus 71; LDH, lactate dehydrogenase; MOI, multiplicity of infection.

Figure 2

EV71 induces apoptosis and pyroptosis. GES-1 cells were harvested and total protein was extracted to determine the expression of (A) apoptosis-associated proteins PARP, c-PARP, Caspase-3 and c-caspase-3 and (B) pyroptosis-associated proteins GSDMD and pro-IL-1β using western blotting. The expression of β-actin served as an internal control. Data are presented as the mean ± SD (n=3). ^**P<0.01 vs. 0. EV71, enterovirus 71; PARP, poly (ADP-Ribose) polymerase; GSDMD, gasdermin D protein; MOI, multiplicity of infection; c-, cleaved.

Figure 3

ASTs alleviate EV71 infection-induced cell damage. (A) To determine the protective effect of ASTs, GES-1 cells were treated with ASTs prior to EV71 infection for 2 h. Cell morphology was observed using a light microscope (magnification, x100). (B) Cell Counting Kit-8 assay was performed to detect the viability of treated cells. (C) LDH release was measured to assess cell damage. Data are presented as the mean ± SD (n=3). ^*P<0.05 and ^**P<0.01 vs. control; ^##P<0.01 vs. EV71. ASTs, total astragalosides; EV71, enterovirus 71; LDH, lactate dehydrogenase.

Figure 4

ASTs inhibit GES-1 cell apoptosis. (A) Cells were placed on the culture plates and treated with 10 µg/ml ASTs. After 2 h, cells were infected with EV71 at an MOI of 5 in AST medium. DAPI was used to stain the nucleus (magnification, x200). (B and C) Western blotting was used to measure expression levels of apoptosis-associated proteins PARP, c-PARP, caspase-3 and c-caspase-3. Data are presented as the mean ± SD (n=3). ^**P<0.01 vs. control. ^##P<0.01 vs. EV71. EV71, enterovirus 71; ASTs, total astragalosides; PARP, poly (ADP-ribose) polymerase; MOI, multiplicity of infection; c-, cleaved.

Figure 5

ASTs inhibit pyroptosis in GES-1 cells. (A) To determine the effect of ASTs on pyroptosis, GES-1 cells were seeded in culture plates and infected with EV71 at a MOI of 5 in AST medium. The cells were collected and total protein was measured using western blotting. Anti-NLRP3, anti-GSDMD, anti-pro-caspase-1, anti-c-caspase-1 and anti-IL-1β antibodies were used to analyze the levels of pyroptosis in cells. (B) Relative protein expression levels are also presented. Data are presented as the mean ± SD (n=3). ^*P<0.05 and ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01 vs. EV71. ASTs, total astragalosides; EV71, enterovirus 71; NLRP3, NLR family, pyrin domain containing 3; GSDMD, gasdermin D protein; MOI, multiplicity of infection; c-, cleaved.

Figure 6

ASTs inhibit EV71 replication. (A) To determine whether ASTs inhibit replication of EV71, GES-1 cells were incubated with EV71 and ASTs. Western blotting was used to assess the expression levels of VP1 and host β-actin. (B) Reverse transcription-quantitative PCR was used to detect levels of VP1 mRNA. (C) Supernatant from treated cells was collected, and a median tissue culture infectious dose assay was performed to detect the virus titer. Data are presented as the mean ± SD (n=3). ^#P<0.05 and ^##P<0.01 vs. EV71. ASTs, total astragalosides; EV71, enterovirus 71; VP1, virus structural protein; MOI, multiplicity of infection.