The Chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function

Abstract

Background: Lianhuaqingwen Capsule (LH-C) is a traditional Chinese medicine (TCM) formula used to treat respiratory tract infectious diseases in Chinese. The aim of this study was to determine the antiviral activity of LH-C and its immunomodulatory effects on viral infection.

Method: The in vitro cytotoxicity and antiviral activity of LH-C was determined by MTT and Plaque reduction assays. Time course study under single-cycle virus growth conditions were used to determine which stage of viral replication was blocked. The effect of LH-C on the nuclear export of the viral nucleoprotein was examined using an indirect immunofluorescence assay. The regulation to different signaling transduction events and cytokine/chemokine expression of LH-C was evaluated using Western blotting and real-time RT-PCR. After virus inoculation, BALB/c mice were administered with LH-C of different concentrations for 5 days. Body-weight, viral titers and lung pathology of the mice were measured, the level of inflammatory cytokines were also examined using real-time RT-PCR.

Results: LH-C inhibited the proliferation of influenza viruses of various strain in vitro, with the 50% inhibitory concentration (IC₅₀) ranging from 0.35 to 2 mg/mL. LH-C blocked the early stages (0-2 h) of virus infection, it also suppressed virus-induced NF-kB activation and alleviated virus-induced gene expression of IL-6, IL-8, TNF-a, IP-10, and MCP-1 in a dose-dependent manner. LH-C treatment efficiently impaired the nuclear export of the viral RNP. A decrease of the viral titers in the lungs of mice were observed in groups administered with LH-C. The level of inflammatory cytokines were also decreased in the early stages of infection.

Conclusions: LH-C, as a TCM prescription, exerts broad-spectrum effects on a series of influenza viruses, including the newly emerged H7N9, and particularly regulates the immune response of virus infection. Thus, LH-C might be a promising option for treating influenza virus infection.

Keywords: Antiviral; Immuno-regulation; Influenza virus; Lianhuaqingwen capsule.

Fig. 1

Plaque reduction assay of LH-C against influenza viruses. MDCK cells were infected with influenza viruses, including A/GIRD02/09 (H1N1), A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) (H274Y mut) at 0.01 MOI for 2 h at 34 °C. After viral adsorption, cell monolayer was covered with overlay medium containing LH-C in different concerntration. After incubation for 72 h, the cells were fixed and stained with crystal violet

Fig. 2

MDCK cells in 48-well plates were prepared and subsequently infected with virus A/PR/8/34 (H1N1) (MOI =0.01) for 2 h, after that LH-C (2 mg/mL) was added to the cells at −2, 0, 2, 4, 6 and 8 h after infection. The supernatants were collected and infectious titers were determined by MTT assay (a) and real time PCR assay (b). The data represent the means ± SD of 3 biological samples. The data are presented as the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Indirect immunofluorescence microscopy. A549 cells were infected with A/PR/8/34 (H1N1) (MOI = 0.1) and treated with LH-C (2 mg/mL) or vehicle. After 6 and 8 h post infection, the cells were fixed for 30 min at 4 °C. Cell nuclei were stained with DAPI (blue) and viewed using a fluorescence microscope (Magnification 400×)

Fig. 4

Effect of LH-C on influenza virus-induced signaling expression in A549 cells. A549 cells infected with A/PR/8/34(H1N1) (MOI = 0.1) were treated with 1.5–3 mg/ml LH-C for 24 h. Whole-cell lysates were prepared at the indicated time points, and an immunoblot analysis of the activity of NF-κB and MAPKs ERK1/2 was performed using phospho-specific and total expression antibodies

Fig. 5

Effect of LH-C on influenza virus-induced cytokine/chemokine mRNA expression in A549 cells. A549 cells infected with A/PR/8/34(H1N1) (MOI = 0.1) were treated with 1.5–3 mg/ml LH-C for 24 h prior to extracting RAN. The cytokine/chemokine mRNA level of IL-6, IL-8, IP-10, TNF-α, and MCP-1 was analyzed using qRT-PCR. The data are presented as the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Administration of LH-C efficiently reduced influenza A virus replication in vivo. Three BALB/c mice per group were intranasally infected with 2 MLD₅₀ of A/PR/8/34 (H1N1) virus. The mice were orally administered LH-C (650 or 1300 mg/day). a Mice were monitored for changes in body weight daily. b Influenza virus titers were detected in mice lungs at 2, 4, 6 and 8 days post inoculation. The data are presented as the means ± SEM of 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Inflammatory cytokines and chemokines were detected at the indicated days post infection. The cytokine/chemokine mRNA level was analyzed using qRT-PCR. The data are presented as the means ± SD.*P < 0.5, **P < 0.01, ***P < 0.001

Fig. 8

lung histology was examined at 4 and 6 days post inoculation. Sections of the lung tissues were visualized using hematoxylin and eosin (H&E) staining. (Magnification: 100×)