Antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza A (H1N1) virus infection

Abstract

Aim: To investigate the effects of arbidol hydrochloride (ARB), a widely used antiviral agent, on the inflammation induced by influenza virus.

Methods: MDCK cells were infected with seasonal influenza A/FM/1/47 (H1N1) or pandemic influenza A/Hubei/71/2009 (H1N1). In vitro cytotoxicity and antiviral activity of ARB was determined using MTT assay. BALB/c mice were infected with A/FM/1/47 (H1N1). Four hours later the mice were administered ARB (45, 90, and 180 mg·kg(-1)·d(-1)) or the neuraminidase inhibitor oseltamivir (22.5 mg·kg(-1)·d(-1)) via oral gavage once a day for 5 d. Body-weight, median survival time, viral titer, and lung index of the mice were measured. The levels of inflammatory cytokines were examined using real-time RT-PCR and ELISA.

Results: Both H1N1 stains were equally sensitive to ARB as tested in vitro. In the infected mice, ARB (90 and 180 mg·kg(-1)·d(-1)) significantly decreased the mortality, alleviated virus-induced lung lesions and viral titers. Furthermore, ARB suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10 in the bronchoalveolar lavage fluids and lung tissues. However, ARB did not significantly affect the levels of IFN-α and IFN-γ, but reduced the level of IFN-β1 in lung tissues at 5 dpi. In peritoneal macrophages challenged with A/FM/1/47 (H1N1) or poly I:C, ARB (20 μmol/L) suppressed the levels of IL-1β, IL-6, IL-12, and TNF-α, and elevated the level of IL-10. Oseltamivir produced comparable alleviation of virus-induced lung lesions with more reduction in the viral titers, but less effective modulation of the inflammatory cytokines.

Conclusion: ARB efficiently inhibits both H1N1 stains and diminishes both viral replication and acute inflammation through modulating the expression of inflammatory cytokines.

Figure 1

ARB alleviated IFV A (H1N1)-induced clinical signs in mice. BALB/c mice (n=12 mice/group) were infected with the Influenza A/FM/1/47 (H1N1) virus (10 LD₅₀ per mouse, in). After 4 h, mice were treated with 0.5% methylcellulose solution (normal control, NC; viral control, VC), ARB (180, 90, or 45 mg·kg⁻¹·d⁻¹), or OSE (oseltamivir, 22.5 mg·kg⁻¹·d⁻¹) qd for 5 d, respectively. Body weight (A) and lethality (B) were collected daily for 15 d. Viral titers of lungs (C) at 5 dpi were determined by plaque assay. ^cP<0.01.

Figure 2

ARB reduced IFV A (H1N1)-induced lung lesion in mice. BALB/c mice (n=12 mice/group) were infected with Influenza A/FM/1/47 (H1N1) virus (10 LD₅₀ per mouse, in). After 4 h, mice were treated with 0.5% methylcellulose solution (normal control, NC; viral control, VC), ARB (180, 90, or 45 mg·kg⁻¹·d⁻¹) or OSE (O, 22.5 mg·kg⁻¹·d⁻¹) qd for 5 d. All the mice were sacrificed at the 5th dpi. (A) Lung index of each group. The index was determined as lung weight/final body weight (LW/BW). (B) Pathological examination for each group. (C) Cytokine profile in BALF of mice. ^bP<0.05, ^cP<0.01. UD=under detectable level.

Figure 3

ARB decreased the transcription of cytokines in murine lung following the IFV A (H1N1) Infection. BALB/c mice (n=12 mice/group) were infected with influenza A/FM/1/47 (H1N1) virus (10 LD₅₀ per mouse, in) or PBS. After 4 h, mice were treated with 0.5% methylcellulose solution (normal control, NC; viral control, VC) or ARB (180, 90, or 45 mg·kg⁻¹·d⁻¹) qd for 5 d. Mock infected group was treated with 90 mg·kg⁻¹·d⁻¹ of ARB. The mice were scheduled for sacrifice at 1, 3, and 5 dpi. Lungs of the mice were collected and homogenated. Real-time PCR analysis was used to determine the mRNA expression level of cytokines normalized to cellular GAPDH. ^bP<0.05, ^cP<0.01.

Figure 4

ARB suppressed the acute inflammation in peritoneal macrophage following the IFV A (H1N1) Infection or poly I:C. Murine peritoneal macrophage were isolated and cultured as the protocol of Zhang et al. Within 24 h the cells were stimulated with 2 MOI of influenza A/FM/1/47 (H1N1) for 1 h or 20 μg/mL poly I:C for 30 min. The cells were then treated with ARB (20, 10, or 5 μmol/L) or free serum medium (normal control, NC; viral control, VC). Mock infected group (M) was treated with 20 μmol/L of ARB. Cells were collected at 2, 4, or 6 h post activation. Transcription for cytokine response was used to determine the mRNA expression level of cytokines normalized to cellular GAPDH by RT-PCR analysis. Supernatants from the groups of ARB (20 μmol/L), mock, positive control and normal control at the point of 6 h were collected for ELISA assay. (A and C: Influenza challenged; B and D: poly I:C challenged). ^bP<0.05, ^cP<0.01. UD=under detectable level.

Figure 5

ARB modulates the production of cytokine in the serum of poly I:C stimulated mice. Mice (n=4 mice/group) were treated with 0.5% methylcellulose solution (normal control, NC; viral control, VC) or ARB via gavage once a day for 2 d. Two hours after the last administration, mice were given an intraperitoneal injection of PBS or poly I:C (pIC, 100 μg/mouse). Mock infected group (M) was treated with 90 mg·kg⁻¹·d⁻¹ of ARB. After another 4 h, mice were sacrificed, serum was isolated and cytokines were tested by ELISA. ^bP<0.05. UD=under detectable level.