DMO-CAP inhibits influenza virus replication by activating heme oxygenase-1-mediated IFN response

Abstract

Background: As a leading cause of respiratory disease, influenza A virus (IAV) infection remains a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapeutic drugs, development of new drugs is urgently required. Flavonoids extracted from Artemisia rupestris L. have an inhibitory effect on virus infections. Despite this fact, the antiviral properties of 6-demethoxy-4'-O-methylcapillarisin (DMO-CAP), one of such flavonoids, against the influenza virus have not been reported. Thus, the aim of this study is to investigate the anti-IAV virus efficacy and antiviral mechanism of DMO-CAP.

Methods: The inhibitory activity of DMO-CAP against IAV was detected in vitro using viral titers by Western blot analysis, qRT-PCR, and immunofluorescence assays. The mechanism of DMO-CAP against influenza virus was analyzed by Western blot analysis, qRT-PCR, and luciferase assay.

Results: DMO-CAP exhibits broad spectrum of antiviral activities against IAV in vitro. Mechanistically, DMO-CAP treatment induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), JNK MAPK, and ERK MAPK, which led to the activation of Nrf2/heme oxygenase-1 (HO-1) pathway. Then, the up-regulation of HO-1 expression activated the IFN response and induced the expression of IFN-stimulated genes, thereby leading to efficient anti-IAV effects.

Conclusions: DMO-CAP inhibited IAV replication by activating HO-1-mediated IFN response. DMO-CAP may be a potential agent or supplement against IAV infection.

Conflict of interest statement

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Competing interests

The authors have declared that they have no competing interests.

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Figures

Fig. 1

The structure and cytotoxicity of compound DMO-CAP. a The structure of DMO-CAP. b The viabilities of DMO-CAP on MDCK and RAW264.7 cells was measured by CCK assay

Fig. 2

Antiviral activity of DMO-CAP against IAV. a Antiviral activity of DMO-CAP in RAW264.7 cells against A/Fort Monmouth/1/1947 (H1N1) and IAV A/Wuhan/359/1995 (H3N2) was tested by viral titers assay. b and c DMO-CAP reduced the expression of M2 RNA and protein in MDCK cells by one-step qRT-PCR assay and Western blot assay. d DMO-CAP reduced the expression of M2 protein by immunofluorescence. e DMO-CAP reduced the expression of M2 protein of influenza resistant strains by Western blot assay. Mock: normal cells without treatment; H1N1: cells were infected with IAV A/FM1/1947 at 0.01MOI; H3N2: cells were infected with IAV A/Wuhan/359/1995 (H3N2) at 0.01MOI. Con: cells were infected pathognomonic viral strains and treated with equal amounts of DMSO or DMO-CAP. The experiments were performed in triplicate and the data represents mean ± SD. ***P < 0.001, **P < 0.01, *P < 0.05 versus Con

Fig. 3

DMO-CAP inhibited IAV replication through up-regulating HO-1 expression. a HEK293T-17 cells were infected with IAV A/FM1/1947 (0.2 MOI) and then treated with DMO-CAP (50 μM)、CoPP (2 μM) together with DMO-CAP (50 μM) and CoPP (2 μM) for 24 h. b HEK293T-17 cells were transfected with HO-1 siRNA or SCR siRNA for 24 h and then infected with IAV A/FM1/1947 (0.2 MOI) in the absence or presence of DMO-CAP for 24 h. The expression of M2 and HO-1 proteins were analyzed by Western blot

Fig. 4

DMO-CAP activates the interferon response by stimulating the Nrf2/ARE pathway to up-regulate the expression of HO-1. a Nrf2 DNA binding activity was analyzed in HEK293T-17 cells co-transfected with pGL4.37 [luc2P/ARE/Hygro]/pAP-1-Luc/pNF-κB-Luc and pRL-SV40 vector, the results were presented as Nrf2 DNA binding activity relative to its basal levels in mock 293 T. **P < 0.01 versus Mock. b DMO-CAP promoted Nrf2 nuclear transcription. RAW264.7 cells were infected with IAV A/FM1/1947 (0.2 MOI) for 2 h and treated with 50 μM DMO-CAP for another 3 h. The total amount of cellular, cytoplasmic and nuclear Nrf2 protein were analyzed by Western blot. c DMO-CAP activated the phosphorylation of p38 MAPK, JNK MAPK and ERK MAPK. RAW264.7 cells were infected with IAV A/FM1/1947 (0.2 MOI) and treated with 50μΜ DMO-CAP for 15 min and then phospho-p38、phospho-JNK and phospho-ERK proteins were valued by Western blot. d RAW264.7 cells were infected with IAV A/FM1/1947 (0.2 MOI) for 2 h, followed by treating with or without DMO-CAP (50μΜ or 25μΜ). The mRNA level of IFN-α and IFN-β were detected by qRT-PCR assay. **P < 0.01 versus Mock. e RAW264.7 cells were infected with IAV A/FM1/1947 (0.2 MOI) for 2 h and the protein levels of ISGs were measured by Western blot after treatment with DMO-CAP for 24 h

Fig. 5

Schematic showing that DMO-CAP inhibits influenza virus replication. DMO-CAP activated the MAPK pathways, thereby leading to Nrf2 expression and subsequent activation of HO-1 gene expression, as well as the up-regulation HO-1 activities host cellular type I IFN response with induction of ISGs expression, which finally leads to inhibition of influenza virus replication