Inhibitory effects and mechanisms of proanthocyanidins against enterovirus 71 infection

Figures

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  Fig. 1. Inhibitory effects of PC on EV71 infection in vitro. (A) Schematic diagram of the chemical structure of PC. (B) The cytotoxicity of PC was determined by MTT assay. Values are means ± S.D. (n = 5). (C) The virus yields in EV71 (MOI = 0.1) infected cells at 16 h p.i. were determined by plaque assay. Values are means ± S.D. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 vs. virus control group. (D) EV71 (MOI = 0.5, 1.0, or 2.0) infected Vero cells were treated with PC for 30 h, then the antiviral activity was determined by CPE inhibition assay. (E) Approximately 50–100 PFU/well of EV71 was pretreated with PC (0.0625–1 μM) for 60 min at 37 °C before infection. Then the virus-PC mixture was transferred to Vero cells, incubated at 37 °C for 1 h and subjected to plaque reduction assay. (F) Plaque numbers from plaque reduction assays performed on EV71 infected Vero cells (E). The percent inhibition was determined relative to the virus control. Values are means ± S.D. (n = 5). *P < 0.05, ***P < 0.001 vs. virus control.

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  Fig. 2. Influence of different treatment conditions of PC on EV71 infection. (A) Schematic diagram of the experiment processes in time of addition assay. (B) Time of addition assay: EV71 (MOI = 0.2) infected Vero cells were treated with PC (5 μM) under four different conditions: pre-treatment of viruses, pre-treatment of cells, during virus adsorption, or post adsorption. At 24 h p.i., virus yields were determined by plaque assay. Values are means ± S.D. (n = 3). *P < 0.05, **P < 0.01 vs. virus control group. (C) Vero cells were infected with EV71 (MOI = 0.2) under four treatment conditions of PC (10 μM), and the expression of VP1 was detected by immunofluorescence assay. Scale bar represents 50 μm. (D and E) EV71 (MOI = 5.0) infected RD cells were treated with or without PC (20 μM) during adsorption (D) or entry process (E), then after washing, the immunofluorescence assay of VP1 was performed. Scale bar represents 20 μm. (F-H) The average fluorescence intensity of VP1 during adsorption (F) or entry process (G) was measured by Image J (NIH) version 1.33 u (USA) to calculate the average intensity per cell of different images (n = 10). The average intensity of VP1 on cell membrane during…

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  Fig. 3. PC may interfere with EV71 infection through targeting VP1 protein. (A) The cell lysates of pCDNA3.1-SCARB2-Myc transfected RD cells were incubated with anti-myc magnetic beads overnight at 4 °C before washing the beads three times with PBST. Then the PC (100, 500 μM)-EV71 mixture was added to SCARB2-myc coupled magnetic beads and incubated at 4 °C for 2 h. After washed thrice with PBST, the VP1 and SCARB2-myc proteins on the beads was detected by western blot. (B) The EV71 VP1 proteins were immobilized onto the surface of a CM5 sensor chip. DMSO was first used to flow through the chip surface to reset in order to remove the influence of DMSO solvent. PC (50, 25, 12.5, 6.25, 3.125 μM) was then flowed over the biosensor chip surface. The sensorgram for all binding interactions were recorded in real time and the changes in mass due to the binding response were recorded as resonance units (RU). (C-E) The 2D and 3D binding modes of PC with the three binding pockets (Glu92-Tyr106, Gly212-Met225, Ala280-Ala293) of VP1 (PDB ID: 3VBH) for SCARB2 binding were shown, respectively. PC is colored in red, the surrounding residues are colored in purple. The backbone of the receptor is depicted…

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  Fig. 4. Transcriptome analysis identifies the differential expressed genes in PC treated EV71-infected cells. (A) The EV71 (MOI = 0.5) infected or non-infected RD cells were treated with or without PC (10 μM) for 10 h after virus adsorption. Then the purified RNAs isolated from four experimental groups (Mock, PC, EV71, EV71+PC) were subjected to transcriptome analysis. Volcano plots indicate up-regulated (red) and down-regulated (green) mRNA transcripts in the Mock vs EV71 group, EV71 vs EV71+PC group or Mock vs PC group. (B) Venn analysis indicates about 170 differentially expressed genes (DEGs) are up-regulated in the Mock vs EV71 group and also down-regulated in the EV71 vs EV71+PC group. (C) The KEGG annotation analysis was performed to display the 170 DEGs related biological processes in EV71 infected cells (Mock vs EV71). (D) Some DEGs with up-regulation after EV71 infection (log2FC(EV71/Mock)>1) in RD cells (P < 0.01) were shown. The relative gene levels of FOS, JUN, CXCL8, GADD45β, TXNIP and MALAT1 were calculated by SAM analysis. The relative RNA levels for non-infected control cells (Mock) were assigned values of 1.0. Values are means ± SD (n = 3). ##P < 0.01 vs. mock control…

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  Fig. 5. Inhibition effects of PC on EV71 induced inflammatory responses. (A-F) EV71 (MOI = 1.0) infected Vero cells, and the cell lysates were collected at different time points post infection (1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h p.i.). Then the phosphorylation and total levels of SAPK/JNK (A), p38MAPK (C) and ERK (E) was evaluated by western blot. Quantification of immunoblot for the ratio of p-SAPK/JNK (B), p-p38MAPK (D) or p-ERK (F) to GAPDH was also shown. (G-I) EV71 (MOI = 1.0) infected Vero cells were treated with or without PC (1.25, 2.5, 5, 10 μM) for 10 h. Then the levels of phosphorylated and total SAPK/JNK (G), p38MAPK (H) and ERK (I) were detected by western blotting. Blots were also probed for α-tubulin or GAPDH proteins as loading controls. Quantification of immunoblot for the ratio of SAPK/JNK (G), p38MAPK (H) and ERK (I) to α-tubulin or GAPDH, respectively. The ratio for non-infected cells (Mock) was assigned values of 1.0. Values are means ± S.D. (n = 3). ###P < 0.001 vs. normal control group (Mock); *P < 0.05, **P < 0.01, ***P < 0.001 vs. virus control group (EV71).

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  Fig. 6. PC also possessed anti-EV71 activities in vivo. (A) The EV71 infected neonatal mice model was established by intraperitoneal injection of EV71 into three-day-old ICR mice and drug treatment was given by intramuscular injection at 12 h, 24 h and 48 h p.i. Created with BioRender.com. (B) Body weights. The average body weights in each group were monitored daily for 8 days and expressed as a percentage of the initial value. (C) Survival rate. EV71 infected mice were received intramuscular treatment of PC (5 or 10 mg/kg), Guanidine Hydrochloride (10 mg/kg), or PBS at 12, 24 and 48 h after EV71 infection. Results are expressed as percentage of survival, evaluated daily for 8 days. (D) The clinical scores indicating the clinical manifestations of infected mice in each group were monitored daily for 6 days. (E) Three different clinical manifestations: single limb weakness, double hind limb weakness and death.

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  Fig. 7. A proposed model underlying the anti-EV71 mechanisms of PC. PC can block EV71 adsorption and entry via targeting VP1 protein to interfere with the binding of VP1 to its receptor SCARB2. PC can also inhibit cellular MAPK pathways to attenuate virus infection and inflammatory responses. Created with BioRender.com.