Evaluation and mechanism study of Pien Tze Huang against EV-A71 infection

Figures

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  FIGURE 1. PZH inhibits EV-A71 replication. Vero and RD cells were mock-infected or infected with EV-A71 (H, MOI = 0.01) for 1 h. Then, cells were subjected to PZH or RBV treatment for 24 h. The cells were collected for WB assay using specific antibodies (A), In-cell WB assay using indicated antibodies (B), qRT-PCR assay (C) (n = 3) and viral titers detection by the end-point dilution assay (TCID50) in Vero cells (D) (n = 3). Statistical analysis was conducted using one-way ANOVA with Holm-Sidak multiple comparisons test compared to the control group and significance was indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

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  FIGURE 2. PZH protects mice from EV-A71 infection. (A–C) 11-Day ICR mice were intraperitoneally inoculated with EV-A71-H-MA (20000 TCID50) for 1 h. Following the infection, the infected mice received intragastric administration of PZH (170, 56.7 and 18.9 mg/kg) and intraperitoneal injection of RBV (50 mg/kg) daily for 7 days. The survival of the mice was monitored daily for 14 days (A, B) and the clinical scores were recorded for the same duration (C). **p < 0.01 by A Log-Rank (Mantel-Cox) assay (A,B) and ***p < 0.001 by Ridit assay (C). (D–F) The mice were dissected at day 5 post-infection. Muscle tissues were subjected to viral titer assays [(D), n = 5]. Immunohistochemistry analyses were performed on muscle tissue sections using EV-A71 VP1 antibody [(E), n = 5)]. Paraffin-embedded sections of muscle tissues were prepared and examined using H&E stain [(F), n = 5]. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Holm-Sidak multiple comparisons test or Ridit assay, compared to the vehicle group. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA with Holm-Sidak multiple comparisons test (D,E) or Ridit assay (F), compared with vehicle group.

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  FIGURE 3. PZH downregulates cytokines in mice serum. 11-Day ICR mice were intraperitoneally inoculated with EV-A71-H-MA (20000 TCID50) for 1 h. Following the infection, the infected mice received intragastric administration of PZH (170, 56.7 and 18.9 mg/kg) and intraperitoneal injection of RBV (50 mg/kg). Serum from mice was collected at 5 dpi for testing. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Holm-Sidak multiple comparisons test, compared with vehicle group.

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  FIGURE 4. Gene ontology annotation and KEGG pathway analysis of EV-A71-infected RD cells after PZH treatment. (A) GO analysis of upregulated proteins. (B) GO analysis of downregulated proteins. (C) KEGG analysis of upregulated proteins. (D) KEGG analysis of downregulated proteins.

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  FIGURE 5. Validation of differently expression proteins. RD cells were mock-infected or infected with EV-A71 (H, MOI = 0.01) for 1 h. Then, cells were subjected to treatment with either PZH or RBV for 24 h. The cells were harvested and the expression of VP1 (A), p-AKT (B), p-mTOR (C), p-PI3K (D), p-p65 (E), p-JNK (F) was examined by Western blot. *p < 0.05, **p < 0.01 by t-test.