Coadministration of Hedera helix L. Extract Enabled Mice to Overcome Insufficient Protection against Influenza A/PR/8 Virus Infection under Suboptimal Treatment with Oseltamivir

Abstract

Several anti-influenza drugs that reduce disease manifestation exist, and although these drugs provide clinical benefits in infected patients, their efficacy is limited by the emergence of drug-resistant influenza viruses. In the current study, we assessed the therapeutic strategy of enhancing the antiviral efficacy of an existing neuraminidase inhibitor, oseltamivir, by coadministering with the leaf extract from Hedera helix L, commonly known as ivy. Ivy extract has anti-inflammatory, antibacterial, antifungal, and antihelminthic properties. In the present study, we investigated its potential antiviral properties against influenza A/PR/8 (PR8) virus in a mouse model with suboptimal oseltamivir that mimics a poor clinical response to antiviral drug treatment. Suboptimal oseltamivir resulted in insufficient protection against PR8 infection. Oral administration of ivy extract with suboptimal oseltamivir increased the antiviral activity of oseltamivir. Ivy extract and its compounds, particularly hedrasaponin F, significantly reduced the cytopathic effect in PR8-infected A549 cells in the presence of oseltamivir. Compared with oseltamivir treatment alone, coadministration of the fraction of ivy extract that contained the highest proportion of hedrasaponin F with oseltamivir decreased pulmonary inflammation in PR8-infected mice. Inflammatory cytokines and chemokines, including tumor necrosis factor-alpha and chemokine (C-C motif) ligand 2, were reduced by treatment with oseltamivir and the fraction of ivy extract. Analysis of inflammatory cell infiltration in the bronchial alveolar of PR8-infected mice revealed that CD11b+Ly6G+ and CD11b+Ly6Cint cells were recruited after virus infection; coadministration of the ivy extract fraction with oseltamivir reduced infiltration of these inflammatory cells. In a model of suboptimal oseltamivir treatment, coadministration of ivy extract fraction that includes hedrasaponin F increased protection against PR8 infection that could be explained by its antiviral and anti-inflammatory activities.

Fig 1. Determination of the effective dose of ivy extract in combination with oseltamivir.

The survival rate (A) and body weight (B) of PR8-infected mice administered oseltamivir (*P<0.05; **P<0.01; ***P<0.001, two-tailed unpaired t-test). C: Survival rate of PR8-infected mice that received ivy extract or vehicle (PBS). D: Survival rate of PR8-infected mice that received oral coadministration of ivy extract and oseltamivir (*P<0.05, log-rank analysis of Mantel-Cox data) E: Body weight of PR8-infected mice that received oral coadministration of ivy extract and oseltamivir (*P<0.05, one-way ANOVA with Tukey’s post hoc test).

Fig 2. Antiviral activity of combined ivy extract and oseltamivir treatment against PR8 virus in vitro and in vivo.

A: CPE reduction assay using SRB assay in A549 cells infected with PR8 virus were treated with oseltamivir for 48 h at the concentrations indicated. (***P<0.0001) B: Antiviral activity of ivy extract (at the concentrations indicated) combined with 25 μg/mL oseltamivir PR8-infected A549 cells. (***P<0.001) C: HSF, HSB, and hederacoside C were used in the presence of 25 μg/mL oseltamivir to identify its anti-PR8 virus activity in A549 cells. After 48 h of incubation, the antiviral activity was investigated by CPE reduction assay using SRB. (***P<0.001), and the antiviral activity was calculated based on the viability of virus infected cells as a percentage of the corresponding untreated control. Data are expressed as the mean ± SD of the percentage values obtained from 3 independent experiments carried out in triplicate. (***P<0.001). D: Survival of mice (n = 5/group) was monitored as depicted in Materials and Methods after treating PR8-infected mice with PBS, oseltamivir, or HSF (*P<0.05, log-rank analysis of Mantel-Cox data). E: Mice were infected with 5 x 10³ pfu/mouse of PR8, orally coadministered with HSF and/or oseltamivir from 2 days after PR8 infection for 5 days. Mice were sacrificed at 6h after final administration, and lung sections were prepared as described in Materials and Methods. Representative H&E stained samples of lung section were shown (left). Pathological grade of each mouse was evaluated (right). CPE, cytopathic effect; HSB, hederasaponin B; HSF, hederasaponin F; SRB, sulforhodamane B; H&E, hematoxylin and eosin. (*P<0.05) using one-way ANOVA with Tukey’s post hoc test.

Fig 3. TIC chromatograms of chromatography fractions.

The HSF content of each fraction analyzed using a chromatogram. HSF, hederasaponin F; TIC, total ion current.

Fig 4. Anti-influenza virus activity of combined treatment with oseltamivir and fraction of ivy extract in mice.

Representative H&E stained samples of lung section from uninfected or PR8-infected mice shown at 200x magnification. A and C: Infected mice were treated with oseltamivir alone or oseltamivir and fraction 4 of ivy extract (n = 4 per group) for 2 (A) or 5 (C) days. B and D: Pathological grade of mice that received oral drug administration for 2 (B) and 5 (D) days. *P<0.05;**p<0.01;***p<0.001; n.s., not significant. one-way ANOVA with Tukey’s post hoc test.

Fig 5. Cytokine production reduced by the combination of oseltamivir and fraction of ivy extract in mice.

A: Body weight of PR8-infected mice administered with oseltamivir alone or coadministratered with oseltamivir and fraction 4 (*P<0.05, one-way ANOVA with Tukey’s post hoc test). B and C: Proinflammatory cytokines and chemokines measured from lung tissue of PR8-infected mice (n = 5 per group) treated with oseltamivir (5 mg/kg) in combination with fraction 4 of ivy extract (30 mg/kg) for 2 (B) and 5 (C) days. *P<0.05;**P<0.01;***P<0.001; n.s., not significant. one-way ANOVA with Tukey’s post hoc test.

Fig 6. CD11b⁺Ly6G⁺ and Ly6G^int population reduced in mice treated with the combination of oseltamivir and fraction of ivy extract.

A: Absolute number of cells in BALF from PR8-infected mice treated with oseltamivir alone or oseltamivir combined with fraction 4 of ivy extract for 5 days. B: Representative plots of CD11b and Ly6G levels assessed by flow cytometry. C and E: Percentages of cell populations in B were shown as a bar graph. D and F: The number of cells calculated were shown, *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with Tukey’s post hoc test), BALF, bronchial alveolar lavage fluid.