Introduction

Molecular Medicine Reports

1791-2997 1791-3004

D.A. Spandidos

10.3892/mmr.2018.9696

mmr-19-01-0563

Articles

(+)-pinoresinol-O-β-D-glucopyranoside from Eucommia ulmoides Oliver and its anti-inflammatory and antiviral effects against influenza A (H1N1) virus infection

Jing

1 * Liang

Xiaoli

1 * Zhou

Beixian

2 * Chen

Xiaowei

1 Xie

Peifang

1 Jiang

Haiming

1 Jiang

Zhihong

3 Yang

Zifeng

1 3 Pan

Xiping

1State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, National Clinical Centre of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510120, P.R. China 2Department of Pharmacy, The People's Hospital of Gaozhou, Gaozhou, Guangdong 525200, P.R. China 3State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau 999078, P.R. China 4Institute of Chinese Integrative Medicine, Guangzhou Medical University, Guangzhou, Guangdong 511436, P.R. China

Correspondence to: Professor Zifeng Yang, State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, National Clinical Centre of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, 151 Yanjiang Xi Road, Guangzhou, Guangdong 510120, P.R. China, E-mail: jeffyah@163.com Professor Xiping Pan, Institute of Chinese Integrative Medicine, Guangzhou Medical University, 195 Dongfeng Xi Road, Guangzhou, Guangdong 511436, P.R. China, E-mail: xppan116@sina.com *

Contributed equally

012019

26112018

19 1 563 572 13042018 01102018

2019

Eucommia ulmoides Oliver (Du-Zhong) is an ancient Chinese herbal remedy used for the treatment of various diseases. To date, the effects of its constituent lignans on influenza viruses remain to be elucidated. In the present study, a lignan glycoside was isolated and purified from Eucommia ulmoides Oliver. Its structures were identified via extensive spectroscopic analysis, and its antiviral and anti-inflammatory activities, specifically against influenza viruses, were determined via a cytopathic effect (CPE) assay, plaque-reduction assays, a progeny virus yield reduction assay, reverse transcription-quantitative polymerase chain reaction analysis and a Luminex assay. Additionally, western blot analysis was performed to investigate the underlying mechanisms of its effects against influenza viruses. The chemical and spectroscopic methods determined the structure of lignan glycoside to be (+)-pinoresinol-O-β-D-glucopyranoside. The CPE assay showed that (+)-pinoresinol-O-β-D-glucopyranoside exerted inhibitory activities with 50% inhibition concentration values of 408.81±5.24 and 176.24±4.41 µg/ml against the influenza A/PR/8/34 (H1N1) and A/Guangzhou/GIRD07/09 (H1N1) strains, respectively. Its antiviral properties were confirmed by plaque reduction and progeny virus yield reduction assays. Additional mechanistic analyses indicated that the anti-H1N1 virus-induced effects of (+)-pinoresinol-O-β-D-glucopyranoside were likely due to inactivation of the nuclear factor-κB, p38 mitogen-activated protein kinase and AKT signaling pathways. Furthermore, (+)-pinoresinol-O-β-D-glucopyranoside exhibited pronounced inhibitory effects on the expression of influenza H1N1 virus-induced pro-inflammatory mediators, including tumor necrosis factor-α, interleukin (IL)-6, IL-8 and monocyte chemoattractant protein 1. The data obtained suggest that (+)-pinoresinol-O-β-D-glucopyranoside may be a candidate drug for treating influenza H1N1 virus infection.

lignin (+)-pinoresinol-O-β-D-glucopyranoside influenza A virus antiviral anti-inflammatory

Introduction

Influenza A virus (IAV; orthomyxoviridae) infection in humans affects the upper and lower respiratory tracts, which often causes acute respiratory diseases ranging from mild to severe. For example, the symptoms of seasonal influenza virus infection can include fever, headache and chills, although patients recover in days (1), whereas lower respiratory tract infections, including 1918 H1N1 or H51N1, can contribute to alveolitis and diffuse alveolar damage leading to mortality (2,3). Clinically available anti-influenza virus medications include neuraminidase (NA) inhibitors, for example. oseltamivir and zanamivir, and M2 ion channel blockers, including amantadine and rimantadine, which have been shown to be ineffective due to viral genome mutations (4). It was previously reported that amino acid substitutions in NA (e.g. NA-R292K and NA-Arg292Lys) of H7N9 confer oseltamivir resistance, raising worldwide concerns on preparedness for an influenza pandemic (5).

Interactions between influenza virus hemagglutinin and cell surface sialic acid receptors are important for infection to establish in target cells (6). During viral replication, structural features, including double stranded RNA or 5′-triphosphate RNA, are sensed by the host immune system, leading to elevated pro-inflammatory mediator production and the recruitment of immune cells to the site of infection (7). It is well recognized that the host immune system orchestrates appropriate pro-inflammatory responses to eliminate invading pathogens and clear infected cells. However, it is also becoming clear that viral factors and host immune responses are involved in the pathogenesis of diseases caused by influenza (8). The PB1-F2 protein of H5N1(HK/97) and 1918 H1N1, with an amino acid change at position 66 (N66S), has been found to increase viral virulence (9). Furthermore, exacerbated cytokine production and the dysregulated recruitment of immune cells, including macrophages, following influenza virus infection contribute to the progression of acute lung injury to acute respiratory distress syndrome (ARDS) (10,11). Therefore, data suggests that the most advantageous strategy for the treatment of influenza diseases combines antiviral compounds with immunomodulators.

The activation of host signaling pathways is essential for viral replication and the expression of pro-inflammatory mediators. Activation of the phosphoinositide-3-kinase (PI3K)/AKT, nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK)1/2 and P38 MAPK signaling cascades triggered by influenza virus infection, is significant in viral entry, replication of the viral genome and the nuclear export of viral ribonucleoproteins (vRNPs), but it also elicits an excessive pro-inflammatory response and collateral lung damage (12–15). Therefore, the development of novel compounds that target certain host signaling pathways may be a promising therapeutic direction for diseases caused by influenza.

The herb Eucommia ulmoides Oliver (Du-Zhong) has been used in various clinical situations (16,17); traditionally, it was used for strengthening muscles and pulmonary function, reducing blood pressure and preventing miscarriages. Numerous active components have been identified from Eucommia ulmoides Oliver, including lignans, polyphenolics, triterpenes and flavonoids (18). Among the active components, the main bioactive components, Eucommia lignans, have protective effects against hypertensive renal injury (19). However, their effects on influenza virus infection remain to be fully elucidated. In the present study, the lignan glycoside (+)-pinoresinol-O-β-D-glucopyranoside was isolated from Eucommia ulmoides Oliver and subjected to various assays to characterize its inhibitory activity, and the underlying mechanisms, against influenza virus infection.

Materials and methods <sec> <title>General experimental procedures

The nuclear magnetic resonance (NMR) spectra were obtained using Bruker AVANCE-400 NMR spectrometers (Bruker Corporation, Billerica, MA, USA). Analytical high-performance liquid chromatography (HPLC) was performed using the Shimadzu LC-10A instrument (Shimadzu Corporation, Kyoto, Japan) equipped with a DAD detector and a reversed-phase C18 column (5-µm, 4.60×250 mm; Shimadzu Corporation). Preparative HPLC was performed on a Shimadzu LC-8A instrument (Shimadzu Corporation) with a UV SPD-20A detector using a reversed-phase C18 column (5 µm, 20×250 mm). Silica gel (200–300 mesh) and silica gel G plates (both from Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) were used for thin layer chromatography analysis.

Plant material

Eucommia ulmoides Oliver was collected from Hubei province (China) and authenticated by Professor Xiping Pan (Guangzhou Medical University, Guangzhou, China).

Extraction and isolation

The air-dried bark (1.5 kg) of Eucommia ulmoides Oliver was refluxed with 95% EtOH (v/v, 3×5 l, 1.5 h each). The combined extracts were concentrated in vacuo to generate a brown residue (120 g), which was dissolved in H₂O (1.5 l) and subjected to column chromatography (CC) over Diaion HP20 macroporous adsorptive resins, prior to elution with MeOH/H₂O (0:100-95:5). The 95% EtOH (v/v) eluate (13.4 g) was subjected to CC on silica gel and eluted with CH₃Cl/MeOH (95:5-0:100) to generate eight fractions (Fr. 1–9). Fr.6 was separated by preparative HPLC and eluted with MeOH/H₂O (2:8-10:0) to obtain one compound (5.6 mg). The purity of the compound was estimated by HPLC to be >95% and identified as (+)-pinoresinol-O-β-D-glucopyranoside by NMR spectroscopy.

Viruses and cell lines

Influenza A/PR/8/34 (H1N1), A/Hongkong/8/68 (H3N2) and A/Hongkong/Y280/97 (H9N2) were obtained from the American Type Culture Collection (Manassas, VA, USA). Influenza A/Guangzhou/GIRD07/09 (H1N1) and B/Lee/1940 (FluB) were isolated from routine clinical specimens. All viral strains used in the present study were propagated in Madin-Darby canine kidney (MDCK) cells. The viral stocks were stored at −80°C and titrated in a 50% tissue culture infectious dose (TCID₅₀) assay prior to use.

The MDCK and human alveolar A549 cells were obtained from the ATCC and maintained at 37°C in a humidified atmosphere of 5% CO₂ in DMEM/F12 (1:1) medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The A549 cells were transfected with 20 ng poly (I:C) from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) using 5 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Cytopathic effect (CPE) inhibition assay

The MDCK cell monolayers (2×10⁴ cells/well) were grown in 96-well plates and inoculated with 100 TCID₅₀ of serial influenza virus strains at 37°C for 2 h. Subsequently, the inoculum was removed and then incubated with 0–1,000 µg/ml of (+)-pinoresinol-O-β-D-glucopyranoside and the positive control oseltamivir carboxylate (TLC PharmaChem., Inc., Canada) at 37°C, respectively. Following 48 h of incubation, the influenza virus-infected cells were stained with 0.5% crystal violet solution and observed under a routine light microscope (DM 3000; Leica Microsystems GmbH, Wetzlar, Germany). The 50% inhibition concentration (IC₅₀) of the virus-induced CPE was calculated as previously described (20).

Plaque-reduction assays

The MDCK cell monolayers (5×10⁵ cells/well) were seeded in 6-well plates and incubated overnight at 37°C to ensure adherence. The cells were then inoculated with 40 PFU/well of influenza virus, including influenza A/PR/8/34 (H1N1) and influenza A/Guangzhou/GIRD07/09 (H1N1), and incubated at 37°C with constant agitation. Following 2 h of incubation, the inoculum was removed and replaced with maintenance DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 1.5% agarose, 1.5 µg/ml TPCK-trypsin and the indicated concentration of (+)-pinoresinol-O-β-D-glucopyranoside. After 3 days, the cells were fixed in 10% formalin and stained with 1% crystal violet.

Progeny virus yield reduction assay

The A549 cells were grown to 90% confluency in 6-well plates and then infected with influenza (MOI=0.1) with or without the indicated concentration of (+)-pinoresinol-O-β-D-glucopyranoside. After 24 h, the supernatants were harvested, and the confluent monolayers of MDCK cells (2×10⁴ cells/well) in the 96-well plate were inoculated with 10-fold dilutions of the supernatants at 37°C for 2 h. Subsequently, the inoculum was removed and replaced with serum-free DMEM containing 1.5 µg/ml TPCK-trypsin. After 48 h, the viral plaques were visualized using trypan blue and observed under a light microscope.

Cell viability assay

The cytotoxic effects induced by (+)-pinoresinol-O-β-D-glucopyranoside in A549 cells were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief, the A549 cells (1×10⁵ cells/well) were seeded into 96-well plates and then incubated with (+)-pinoresinol-O-β-D-glucopyranoside at different concentrations (0–1,000 µg/ml) for 48 h. Subsequently, the cells were washed twice with PBS to remove the drug and incubated with 200 µl MTT solution (5 mg/ml) for an additional 4 h. The formazan crystals generated in each well were dissolved with dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA). The absorbance was determined at 490 nm using a microplate reader (Synergy HT; BioTek Instruments, Inc., Winooski, VT, USA).

Western blot analysis

The following primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) were used for western blot analysis: NF-κB p65 (cat. no. 8242), phosphorylated (phosphor)-NF-κB p65 (Ser⁵³⁶) (cat. no. 3033), p38 MAPK (cat. no. 8690), phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) (cat. no. 4511), AKT (cat. no. 4691), phospho-AKT (Thr³⁰⁸) (cat. no. 13038), ERK1/2 MAPK (cat. no. 4695), phospho-ERK1/2 MAPK (Thr²⁰²/Tyr²⁰⁴) (cat. no. 9101), c-Jun N-terminal kinase (JNK) MAPK (cat. no. 9252), phospho-JNK MAPK (Thr¹⁸³/Tyr¹⁸⁵) (cat. no. 4671), cyclooxygenase-2 (COX-2; cat. no. 12282) and GAPDH (cat. no. 5174). The HRP-conjugated secondary antibody (cat. no. BAB1302) was acquired from Multisciences Biotech Co., Ltd. (Hangzhou, China).

The cells were rinsed twice with ice-cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and protease inhibitors (Sigma-Aldrich; Merck KGaA). The supernatants from each treatment were collected by centrifugation of the lysates at 13,000 × g for 15 min at 4°C, and then evaluated to determine the protein concentration using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equivalent quantities of protein (20 µg/lane) were resolved on a 10% polyacrylamide gel and transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were then blocked with 5% non-fat milk (w/v) in 1X TBS/Tween-20 buffer (0.1%, v/v) for 1 h at room temperature prior to incubation with the primary and secondary antibodies. Then, the membranes were incubated overnight at 4°C with 1:1,000 dilution of primary antibody in 5% BSA (w/v) in TBS/Tween-20 buffer (0.1% v/v). The HRP-conjugated secondary antibody was used to detect the primary antibody at a dilution of 1:500 for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence reaction kit (Amersham; GE Healthcare Life Sciences, Chalfont, UK). The intensity of the phosphorylated bands was quantified using ImageJ software version 1.43 (National Institutes of Health, Bethesda, MD, USA).

RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

RT-qPCR analysis was performed to determine the relative mRNA levels of cytokines and chemokines. Briefly, the influenza A virus-infected cells were treated with the indicated concentrations of (+)-pinoresinol-O-β-D-glucopyranoside. Total cellular RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.); the cDNA was then synthesized from 1 µg total RNA using a PrimeScript™ RT Reagent kit (Takara Bio, Inc., Otsu, Japan), at 37°C for 15 min followed by 5 sec at 85°C to inactivate the reaction. The qPCR analysis was performed using a Premix Ex Taq™ Reagent kit (Takara Bio, Inc.), with initial heating to 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing and elongation at 60°C for 40 sec in an Applied Biosystems 7500 Real-Time PCR system with the primers and probes specified in Table I. GAPDH was used as an internal reference gene. The relative mRNA expression data were calculated using the 2^−ΔΔCq method (21).

Pro-inflammatory mediator measurements

The inhibitory effects of (+)-pinoresinol-O-β-D-glucopyranoside on the influenza virus-induced production of pro-inflammatory mediators were measured via Luminex assays and ELISAs, respectively. Briefly, the A549 cells in 6-well plates were inoculated with A/PR/8/34 (H1N1) for 2 h, followed by treatment with different concentrations of (+)-pinoresinol-O-β-D-glucopyranoside. Following another 24 h of incubation, the culture supernatants were collected to evaluate the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1 (MCP-1) and prostaglandin E2 (PGE2) using a Luminex kit (Bio-Rad Laboratories, Inc.) and ELISA kits (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols.

Statistical analyses

All data were analyzed using SPSS v.18.0 statistical software (SPSS, Inc., Chicago, IL, USA) and are presented as the mean ± standard deviation based on at least three independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Bonferroni's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Structural elucidation of (+)-pinoresinol-O-β-D-glucopyranoside

White amorphous powder, 1H-NMR (MeOD,400 MHz):δ7.15 (1H, d, J=8.4 Hz, H-5′), 6.95 (1H, d, J=1.6 Hz, H-2′), 6.92 (1H, dd, J=8.4, 1.6 Hz, H-6′), 6.82 (1H, br, J=8.4 Hz, H-6), 6.80(1H, d, J=1.6 Hz, H-2), 6.77 (1H, d, J=8.4 Hz, H-5), 4.88 (1H, d, J=6.0 Hz, Glc-1), 4.76 (1H, d, J=4.0 Hz, H-7′), 4.71 (1H, d, J=4.0 Hz, H-7), 3.88 (3H, s, 3′-OMe), 3.87 (3H, s, 3-OMe). 13C-NMR (MeOD, 100 MHz): 148.0 (C-3′), 146.17 (C-3), 144.53 (C-4′), 144.36 (C-4), 134.49 (C-1′), 130.78 (C-1), 117.10 (C-6), 116.84 (C-6), 115.02 (C-5′), 113.12 (C-5), 108.64 (C-2′), 108.0 (C-2), 99.87 (Glc-1), 84.54 (C-7), 84.15 (C-7′), 75.24 (Glc-3), 74.87 (Glc-5), 71.94 (Glc-2), 69.76 (C-9′), 69.72 (C-9), 68.36 (Glc-4), 59.5 (Glc-6), 53.8 (3-OMe), 53.45 (3′-OMe), 52.58 (C-8′), 52.39 (C-8). The data were in accordance with the literature regarding (+)-pinoresinol-O-β-D-glucopyranoside (Fig. 1) (22).

Anti-influenza effects of (+)-pinoresinol-O-β-D-glucopyranoside in vitro

The present study initially evaluated the anti-influenza effects of (+)-pinoresinol-O-β-D-glucopyranoside using a CPE reduction assay. The MDCK cells were inoculated with 100 TCID₅₀ of influenza viruses and then incubated with a concentration series of (+)-pinoresinol-O-β-D-glucopyranoside or oseltamivir carboxylate following removal of the inoculum. (+)-pinoresinol-O-β-D-glucopyranoside was found to reduce the CPE induced by two influenza A/H1N1 viral strains (A/PR/8/34 and A/Guangzhou/GIRD07/09), with IC₅₀ values of 176.24–408.81 µg/ml and SI values of 1.80–4.17 (Table II). However, (+)-pinoresinol-O-β-D-glucopyranoside did not exhibit antiviral effects against influenza virus A/Hongkong/8/68 (H3N2), A/Hongkong/Y280/97 (H9N2) or B/Lee/1940 (FluB) (Table II). The activity against A/PR/8/34 and A/Guangzhou/GIRD07/09 was confirmed using plaque reduction assays and progeny virus yield reduction assays. As shown in Fig. 2A, (+)-pinoresinol-O-β-D-glucopyranoside treatment significantly reduced plaque formation in the A/PR/8/34 and A/Guangzhou/GIRD07/09 (H1N1) virus-infected cells. Furthermore, the progeny virus titers of the two virus strains were significantly decreased by treatment with (+)-pinoresinol-O-β-D-glucopyranoside at the concentration of 250 or 500 µg/ml (Fig. 2B). Together, these results suggested that (+)-pinoresinol-O-β-D-glucopyranoside inhibits influenza A H1N1 viruses.

Effects of (+)-pinoresinol-O-β-D-glucopyranoside on A549 cell viability

To select appropriate concentrations for further experiments, the A549 cells were incubated with increasing concentrations of (+)-pinoresinol-O-β-D-glucopyranoside for 48 h. Following this, cell viability was assessed with an MTT assay to evaluate the potential cytotoxicity of lignan (+)-pinoresinol-O-β-D-glucopyranoside. The results showed that (+)-pinoresinol-O-β-D-glucopyranoside did not affect the viability of A549 cells up to a concentration of 1,000 µg/ml (Fig. 3). Therefore, the pharmacological effects of (+)-pinoresinol-O-β-D-glucopyranoside on viral infection were investigated using a concentration range of 150–450 µg/ml.

Effect of (+)-pinoresinol-O-β-D-glucopyranoside on influenza virus-induced cellular signaling

Studies have revealed that influenza A virus exploits multiple host cell signaling pathways to facilitate self-replication (23,24). It has been suggested that the pharmacological inhibition of cellular signaling may be a potential strategy for controlling viral infection (25). The results of the study indicated that (+)-pinoresinol-O-β-D-glucopyranoside possesses antiviral activity against influenza A H1N1, therefore, whether the anti-H1N1 virus activity was associated with the inhibition of signaling pathways required for influenza virus infection was determined. Treatment with (+)-pinoresinol-O-β-D-glucopyranoside significantly decreased the influenza H1N1-induced activation of multiple cellular signaling pathways, including the NF-κB, p38, MAPK and AKT pathways, but not the JNK or ERK MAPK pathways (Fig. 4A and B). As these pathways may also have been activated by viral products, whether (+)-pinoresinol-O-β-D-glucopyranoside affected synthetic mimics of viral RNA poly (I:C)-mediated pathway activation was investigated. As shown in Fig. 4C and D, it was found that (+)-pinoresinol-O-β-D-glucopyranoside did not affect the poly (I:C)-induced activation of NF-κB, p38 MAPK or AKT signaling. Taken together, these results suggested that (+)-pinoresinol-O-β-D-glucopyranoside inactivates multiple cellular signaling pathways triggered by viral infection, therefore exerting antivirus effects against H1N1.

Effects of (+)-pinoresinol-O-β-D-glucopyranoside on the influenza virus-induced expression of pro-inflammatory mediators

The high or low pathogenic influenza virus-induced hyperinduction of pro-inflammatory mediators was mediated though specific host cellular pathways, which are considered to affect the severity of influenza diseases (26,27). To determine whether (+)-pinoresinol-O-β-D-glucopyranoside can affect the H1N1 influenza virus-induced expression of pro-inflammatory mediators though the inhibition of cellular signaling, the present study assessed the expression of pro-inflammatory mediators at the mRNA and protein levels via RT-qPCR and Luminex assays, respectively. As shown in Fig. 5A and B, treatment with (+)-pinoresinol-O-β-D-glucopyranoside decreased the H1N1 influenza virus-induced upregulation of cytokine and chemokine expression, including that of TNF-α, IL-6, IL-8 and MCP-1, in a concentration-dependent manner. Furthermore, it was found that treatment with (+)-pinoresinol-O-β-D-glucopyranoside suppressed the H1N1 virus-induced production of COX-2 (Fig. 5C and D) and that of the derived PGE2 (Fig. 5E). These results indicated that (+)-pinoresinol-O-β-D-glucopyranoside decreased the H1N1 influenza virus-induced expression of pro-inflammatory mediators via the inhibition of multiple signaling pathways.

Discussion

In previous decades, the increasing incidence of antiviral drug-resistant influenza viruses has highlighted the urgency for novel antiviral drugs. Compounds from Chinese herbal medicines have gained interest in the development of novel antiviral medications as they tend to possess multiple activities and a broad safety window. In the present study, a lignan compound was isolated from Eucommia ulmoides Oliver and its structure was subjected to extensive spectroscopic analysis; it was identified as (+)-pinoresinol-O-β-D-glucopyranoside. Further investigation showed that the antiviral and anti-inflammatory effects of (+)-pinoresinol-O-β-D-glucopyranoside against influenza virus infection likely occur through the inhibition of AKT, NF-κB, and p38 MAPK signaling.

Our previous study reported on the structure of a novel lignan glycoside [(+)-pinoresinol 4-O-[6-O-vanilloyl]-β-D-glucopyranoside)] from the latex of Calotropis gigantean, comprised of (+)-pinoresinol-O-β-D-glucopyranoside moiety and a vanilloyl group, which possessed antiviral activity though the retention of vRNPs in the nucleus (28). Additionally, it was found that (+)-pinoresinol-O-β-D-glucopyranoside did not have any inhibitory effects on influenza A/PR/8/34 (H1N1) virus with an IC₅₀ value >348.6 µM and SI value <1.0 (28). In the present study, the antiviral effect of (+)-pinoresinol-O-β-D-glucopyranoside was re-evaluated, and the compound was found to have antiviral activity against the influenza A/PR/8/34 (H1N1) virus with an IC₅₀ value of 408.81±5.24 µg/ml (785.37±10.07 µM) (Table II), which was higher than previously reported and for that of (+)-pinoresinol 4-O-[6′-O-vanilloyl]-β-D-glucopyranoside. These results suggested that (+)-pinoresinol-O-β-D-glucopyranoside was not potent enough to exert inhibitory effects on the influenza A/PR/8/34 (H1N1) virus at low doses due to the absence of a vanilloyl moiety. The antiviral properties of (+)-pinoresinol-O-β-D-glucopyranoside were confirmed by the result that treatment reduced influenza A/GZ/GIRD07/09 (H1N1) virus-induced CPE in MDCK cells (Table II).

Influenza viruses exploit multiple host cell signaling cascades to facilitate their replication. An increasing number of reports have demonstrated that the suppression of cellular signaling using pharmacological agents can limit the spread of influenza. The inhibition of NF-κB activity by acetylsalicylic acid or Bay 11–7082 inhibited influenza virus propagation via the retention of vRNP in the nucleus, and effectively reduced viral titers in vitro and in vivo (13,29). Furthermore, the synthesis of eight segments of the viral RNA (vRNA) genome was reduced by an NF-κB inhibitor (30). Similarly, the inhibition of PI3K/AKT signaling confirmed its importance in viral processes, including viral uptake, vRNA synthesis and vRNP nuclear export (14,31). Phosphorylation of the early-endosomal protein EEA1 and anti-apoptotic factor B-cell lymphoma 2 by P38 MAPK has been reported to enhance the endocytosis of virus particles and the nucleocytoplasmic export of viral NP proteins, and this was eradicated following treatment with a P38 MAPK-specific inhibitor (15,32). Findings indicated that inhibition of the NF-κB, p38 MAPK, and AKT signaling pathways by specific inhibitors exerted antiviral activity. However, certain compounds from Chinese herbal medicines with NF-κB inhibition activity did not exhibit broad antiviral activity and the detailed mechanism was not revealed (28,33). In concordance, although the present found that the virus-induced NF-κB, p38 MAPK, and AKT signaling pathways were inhibited by (+)-pinoresinol-O-β-D-glucopyranoside, (+)-pinoresinol-O-β-D-glucopyranoside did not exert inhibitory effects on influenza virus A/Hongkong/8/68 (H3N2), A/Hongkong/Y280/97 (H9N2) or B/Lee/1940 (FluB) (Table II). In the present study, the reasons why lignan (+)-pinoresinol-O-β-D-glucopyranoside, with its NF-κB, p38 MAPK, and AKT signaling inhibition properties, did not show broad antiviral activity were not elucidated. The results suggested that the inhibition activity of natural compounds from traditional Chinese medicine on cellular molecules was not potent enough, compared with specific inhibitors. It is anticipated that investigations in the future will elucidate the detailed mechanism. The possible underlying mechanism of (+)-pinoresinol-O-β-D-glucopyranoside against influenza infection may involve inactivation of the NF-κB, P38 MAPK and PI3K/AKT signaling pathways (Fig. 3).

The results of the present study demonstrated that (+)-pinoresinol-O-β-D-glucopyranoside decreased the expression of TNF-α, IL-6, IL-8 and MCP-1 (Fig. 5A and B). During influenza virus infection, the abnormal activation of host signaling pathways leads to an excessive inflammatory response, which is considered to result in lung tissue injury and may progress to ARDS (34). In patients infected with seasonal influenza viruses, the levels of cytokines, including IL-6, TNF-α and interferon (IFN)-γ-inducible protein 10 (IP-10), were elevated on day 1 but had declined rapidly by day 5 (35). By contrast, the persistent elevation of cytokines in patients infected with avian H7N9 or H5N1 viruses resulted in poor outcomes and even mortality (27,36). Dysregulation among pro-inflammatory cytokines has served as a hallmark of influenza disease severity (37). The suppression of NF-κB signaling has been shown to decrease the influenza virus-mediated expression of IL-6, IL-8, MCP-1 and RANTES in vitro and in vivo (13). p50 subunit deficiency in mice attenuated an array of NF-κB-targeted genes induced by influenza A (H5N1) (38). P38-mediated signaling is also involved in the initiation of pro-inflammatory cytokine synthesis. Treatment with a p38 MAPK inhibitor (SB203580) reduced the H5N1 virus-mediated expression of cytokines and chemokines, including TNF-α, IP-10, MCP-1 and RANTES (39). The cytokine levels, including those of IP-10 and MCP-1, in patients with severe influenza A virus infection were positively correlated with the expression of P38 MAPK in CD4⁺ lymphocytes (40). During viral replication, the viral products, including viral RNA sensed by host pattern recognition receptors can also activate cellular signaling and initiate the expression of pro-inflammatory cytokines. In examining whether that the anti-inflammatory effects of (+)-pinoresinol-O-β-D-glucopyranoside is due to its antiviral property or the inhibition of cellular signaling triggered by viral products, the present study found that treatment with (+)-pinoresinol-O-β-D-glucopyranoside did not affect the poly (I:C)-mediated activation of NF-κB, p38 kinase or AKT signaling (Fig. 4B). These results suggested that the anti-inflammatory effects of (+)-pinoresinol-O-β-D-glucopyranoside were a result of its antiviral effects. Therefore, it was hypothesized that the inhibitory effects of (+)-pinoresinol-O-β-D-glucopyranoside on infection-activated NF-κB and p38 kinase led to a decrease in the influenza virus-induced expression of pro-inflammatory cytokines.

Previous studies have reported that NF-κB and p38 kinase signaling are required for the expression of COX-2, which is involved in the pathogenesis of pneumococcal pneumonia and influenza H5N1 viral disease (41–43). From the data presented in the present study, the inhibitory effects on NF-κB and p38 kinase signaling by (+)-pinoresinol-O-β-D-glucopyranoside treatment were correlated with the decreased expression of COX-2 and PGE2 (Fig. 5C-E). Previous studies have demonstrated that COX-2 deficiency or inhibition significantly reduced virus-induced inflammation and changes in body temperature, and protected against life-threatening influenza challenge (44,45). Notably, the delayed combination of antiviral agents with COX-2 inhibitor treatment significantly prolonged the survival of mice infected with H5N1 (46). Additionally, Coulombe et al revealed that PGE2 impaired the type I IFN-mediated antiviral response (47). Therefore, it appears that suppression of the expression of COX-2 and PGE2 by (+)-pinoresinol-O-β-D-glucopyranoside is beneficial to the host during influenza infection.

In conclusion, the present study found that (+)-pinoresinol-O-β-D-glucopyranoside from Eucommia ulmoides Oliver exerts antiviral and anti-inflammatory effects through NF-κB, P38 MAPK and AKT signaling pathway inhibition in influenza virus-infected cells. Therefore, it was hypothesized that the product possesses multiple biological activities and low toxicity, and that it may be a promising anti-influenza candidate drug.

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the Ministry of Science and Technology of China (grant no. 2015DFM30010), the Secondary Development Projects of Guangdong Famous and Excellent Traditional Chinese Patent Medicines (grant no. 20174005), the First Affiliated Hospital of Guangzhou Medical University (grant no. LJ2016) and the International S&T Cooperation Program of Guangdong Province (grant no. 2013B051000085).

Availability of data and materials

The datasets used and analyzed during the present study are available from the corresponding authors on reasonable request.

Author contributions

ZY, XP and ZJ conceived and designed the experiments; JL, XL, BZ, XC, PX and HJ performed the experiments; JL and XL analyzed the data; JL, XL and BZ wrote the manuscript. ZY, XP and ZJ contributed to revisions of the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Hayden

Gwaltney

JMJ

Viral infections. In textbook of respiratory medicine

Murray

Nadel

WB. Saunders Co.

Philadelphia

97710351994

Peiris

Leung

Cheung

Nicholls

Chan

Lai

Lim

Re-emergence of fatal human influenza A subtype H5N1 disease

Lancet3636176192004

10.1016/S0140-6736(04)15595-5

14987888

Kash

Tumpey

Proll

Carter

Perwitasari

Thomas

Basler

Palese

Taubenberger

García-Sastre

Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus

Nature4435785812006

17006449

2615558

Hayden

de Jong

Emerging influenza antiviral resistance threats

J Infect Dis2036102011

10.1093/infdis/jiq012

21148489

3086431

Hai

Schmolke

Leyva-Grado

Thangavel

Margine

Jaffe

Krammer

Solórzano

García-Sastre

Palese

Bouvier

Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility

Nat Commun428542013

10.1038/ncomms3854

24326875

3863970

Gamblin

Skehel

Influenza hemagglutinin and neuraminidase membrane glycoproteins

J Biol Chem28528403284092010

10.1074/jbc.R110.129809

20538598

2937864

Thompson

Locarnini

Toll-like receptors, RIG-I-like RNA helicases and the antiviral innate immune response

Immunol Cell Biol854354452007

10.1038/sj.icb.7100100

17667934

Fukuyama

Kawaoka

The pathogenesis of influenza virus infections: The contributions of virus and host factors

Curr Opin Immunol234814862011

10.1016/j.coi.2011.07.016

21840185

3163725

Conenello

Zamarin

Perrone

Tumpey

Palese

A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence

PLoS Pathog3141414212007

10.1371/journal.ppat.0030141

17922571

Högner

Wolff

Pleschka

Plog

Gruber

Kalinke

Walmrath

Bodner

Gattenlöhner

Lewe-Schlosser

Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia

PLoS Pathog9e10031882013

10.1371/journal.ppat.1003188

23468627

3585175

Hagau

Slavcovici

Gonganau

Oltean

Dirzu

Brezoszki

Maxim

Ciuce

Mlesnite

Gavrus

Clinical aspects and cytokine response in severe H1N1 influenza A virus infection

Crit Care14R2032010

10.1186/cc9324

21062445

3220006

Ludwig

Planz

Influenza viruses and the NF-kappaB signaling pathway-towards a novel concept of antiviral therapy

Biol Chem389130713122008

10.1515/BC.2008.148

18713017

Pinto

Herold

Cakarova

Hoegner

Lohmeyer

Planz

Pleschka

Inhibition of influenza virus-induced NF-kappaB and Raf/MEK/ERK activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo

Antiviral Res9245562011

10.1016/j.antiviral.2011.05.009

21641936

Ehrhardt

Marjuki

Wolff

Nürnberg

Planz

Pleschka

Ludwig

Bivalent role of the phosphatidylinositol-3-kinase (PI3K) during influenza virus infection and host cell defence

Cell Microbiol8133613482006

10.1111/j.1462-5822.2006.00713.x

16882036

Marchant

Singhera

Utokaparch

Hackett

Boyd

Luo

Dorscheid

McManus

Hegele

Toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism

J Virol8411359113732010

10.1128/JVI.00804-10

20702616

2953195

Tang

Liu

Dai

Antioxidant properties of Du-zhong (Eucommia ulmoides Oliv.) extracts and their effects on color stability and lipid oxidation of raw pork patties

J Agric Food Chem58728972962010

10.1021/jf100304t

20499841

Lee

Cho

Kim

Jang

Shin

Park

Lee

Choi

Lee

Du-zhong (Eucommia ulmoides Oliv.) cortex water extract alters heme biosynthesis and erythrocyte antioxidant defense system in lead-administered rats

J Med Food886922005

10.1089/jmf.2005.8.86

15857215

Hussain

Tan

Liu

Oladele

Rahu

Tossou

Yin

Health-promoting properties of eucommia ulmoides: A review

Evid Based Complement Alternat Med201652029082016

10.1155/2016/5202908

27042191

4793136

Yan

Wang

Deng

Jing

Protective effects of Eucommia lignans against hypertensive renal injury by inhibiting expression of aldose reductase

J Ethnopharmacol1394544612012

10.1016/j.jep.2011.11.032

22138658

Reed

Muench

A simple method of estimating fifty percent endpoints

American J Epidemiol274934971938

10.1093/oxfordjournals.aje.a118408

Livak

Schmittgen

Analysis of relative gene expression data using real-time quantitative PCR and the 2^ΔΔCT method

Sugiyama

Kikuchi

Studies on the constituents of osmanthus species. VII. Structures of lignan glycosides from the leaves of osmanthus asiaticus NAKAI

Chem Pharm Bull3964834851991

10.1248/cpb.39.483

Marjuki

Gornitzky

Marathe

Ilyushina

Aldridge

Desai

Webby

Webster

Influenza A virus-induced early activation of ERK and PI3K mediates V-ATPase-dependent intracellular pH change required for fusion

Cell Microbiol135876012011

10.1111/j.1462-5822.2010.01556.x

21129142

Marjuki

Alam

Ehrhardt

Wagner

Planz

Klenk

Ludwig

Pleschka

Membrane accumulation of influenza A virus hemagglutinin triggers nuclear export of the viral genome via protein kinase Calpha-mediated activation of ERK signaling

J Biol Chem28116707167152006

10.1074/jbc.M510233200

16608852

Ludwig

Targeting cell signalling pathways to fight the flu: Towards a paradigm change in anti-influenza therapy

J Antimicrob Chemother64142009

10.1093/jac/dkp161

19420020

Hayden

Fritz

Lobo

Alvord

Strober

Straus

Local and systemic cytokine responses during experimental human influenza A virus infection. Relation to symptom formation and host defense

J Clin Invest1016436491998

10.1172/JCI1355

9449698

508608

de Jong

Simmons

Thanh

Hien

Smith

Chau

Hoang

Chau

Khanh

Dong

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Nat Med12120312072006

10.1038/nm1477

16964257

4333202

Parhira

Yang

Zhu

Chen

Zhou

Wang

Liu

Bai

Jiang

In vitro anti-influenza virus activities of a new lignan glycoside from the latex of Calotropis gigantea

PLoS One9e1045442014

10.1371/journal.pone.0104544

25102000

4125211

Mazur

Wurzer

Ehrhardt

Pleschka

Puthavathana

Silberzahn

Wolff

Planz

Ludwig

Acetylsalicylic acid (ASA) blocks influenza virus propagation via its NF-kappaB-inhibiting activity

Cell Microbiol9168316942007

10.1111/j.1462-5822.2007.00902.x

17324159

Kumar

Xin

Liang

NF-kappaB signaling differentially regulates influenza virus RNA synthesis

J Virol82988098892008

10.1128/JVI.00909-08

18701591

2566266

Shin

Liu

Tikoo

Babiuk

Zhou

Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation

J Gen Virol889429502007

10.1099/vir.0.82483-0

17325368

Nencioni

De Chiara

Sgarbanti

Amatore

Aquilano

Marcocci

Serafino

Torcia

Cozzolino

Ciriolo

Bcl-2 expression and p38MAPK activity in cells infected with influenza A virus: Impact on virally induced apoptosis and viral replication

J Biol Chem28416004160152009

10.1074/jbc.M900146200

19336399

2708894

Guan

Chen

Jiang

Zhang

Wang

Yang

Pan

Pterodontic acid isolated from laggera pterodonta inhibits viral replication and inflammation induced by influenza A virus

Molecules22E17382017

10.3390/molecules22101738

29035328

Bauer

Ewig

Rodloff

Müller

Acute respiratory distress syndrome and pneumonia: A comprehensive review of clinical data

Clin Infect Dis437487562006

10.1086/506430

16912951

Bian

Nie

Zang

Fang

Xiu

Clinical aspects and cytokine response in adults with seasonal influenza infection

Int J Clin Exp Med7559356022014

25664078

4307525

Yang

Mok

Liu

Guan

Pan

Chen

Lin

Clinical, virological and immunological features from patients infected with re-emergent avian-origin human H7N9 influenza disease of varying severity in Guangdong province

PLoS One10e01178462015

10.1371/journal.pone.0117846

25723593

4344233

Ramos

Fernandez-Sesma

Modulating the innate immune response to influenza A virus: Potential therapeutic use of anti-inflammatory drugs

Front Immunol63612015

10.3389/fimmu.2015.00361

26257731

4507467

Droebner

Reiling

Planz

Role of hypercytokinemia in NF-kappaB p50-deficient mice after H5N1 influenza A virus infection

J Virol8211461114662008

10.1128/JVI.01071-08

18768968

2573282

Hui

Lee

Cheung

Poon

Guan

Lau

Peiris

Induction of proinflammatory cytokines in primary human macrophages by influenza A virus (H5N1) is selectively regulated by IFN regulatory factor 3 and p38 MAPK

J Immunol182108810982009

10.4049/jimmunol.182.2.1088

19124752

Lee

Wong

Chan

Lun

Lui

Wong

Hui

Lam

Cockram

Choi

Hypercytokinemia and hyperactivation of phospho-p38 mitogen-activated protein kinase in severe human influenza A virus infection

Clin Infect Dis457237312007

10.1086/520981

17712756

N'Guessan

Hippenstiel

Etouem

Zahlten

Beermann

Lindner

Opitz

Witzenrath

Rosseau

Suttorp

Streptococcus pneumoniae induced p38 MAPK- and NF-kappaB-dependent COX-2 expression in human lung epithelium

Am J Physiol Lung Cell Mol Physiol290L1131L11382006

10.1152/ajplung.00383.2005

16414978

Singer

Baker

McCaffrey

AuCoin

Dechert

Gerthoffer

p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes

Am J Physiol Lung Cell Mol Physiol285L1087L10982003

10.1152/ajplung.00409.2002

12871860

Lee

Cheung

Nicholls

Hui

Leung

Uiprasertkul

Tipoe

Lau

Poon

Hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: A mechanism for the pathogenesis of avian influenza H5N1 infection

J Infect Dis1985255352008

10.1086/590499

18613795

Carey

Bradbury

Seubert

Langenbach

Zeldin

Germolec

Contrasting effects of cyclooxygenase-1 (COX-1) and COX-2 deficiency on the host response to influenza A viral infection

J Immunol175687868842005

10.4049/jimmunol.175.10.6878

16272346

Carey

Bradbury

Rebolloso

Graves

Zeldin

Germolec

Pharmacologic inhibition of COX-1 and COX-2 in influenza A viral infection in mice

PLoS One5e116102010

10.1371/journal.pone.0011610

20657653

2904706

Zheng

Chan

Lin

Zhao

Chan

Zhang

Chen

Wong

Lau

Woo

Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus

Proc Natl Acad Sci USA105809180962008

10.1073/pnas.0711942105

18523003

Coulombe

Jaworska

Verway

Tzelepis

Massoud

Gillard

Wong

Kobinger

Xing

Couture

Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages

Immunity405545682014

10.1016/j.immuni.2014.02.013

24726877

Figure 1.

Chemical structure of (+)-pinoresinol-O-β-D-glucopyranoside.

Figure 2.

Antiviral effects of PG. (A) MDCK cells were infected with 40 PFU/well of influenza virus A/PR/8/34 (H1N1) and A/Guangzhou/GIRD07/09 (H1N1). Following adsorption of the virus for 2 h, the inoculum was removed, and the cells were overlaid with DMEM containing 1.5% agarose with serial dilutions of PG. After 3 days, the cells were stained and images were captured. (B) A549 cells were infected with influenza virus A/PR/8/34 (H1N1) and A/Guangzhou/GIRD07/09 (H1N1) (MOI=0.1). The indicated concentrations of PG were added to the medium. After 24 h, supernatants were collected, and virus titers were determined using a plaque assay. Data are expressed as the mean ± standard deviation. **P<0.01 and ***P<0.001, vs. untreated infected cells. PG, (+)-pinoresinol-O-β-D-glucopyranoside.

Figure 3.

Effect of PG on A549 cell viability. A549 cells were treated with increasing concentrations of PG for 48 h, and cell viability was determined with an MTT assay. The percentage cell viability was calculated as the absorbance of PG-treated cells relative to the untreated cells. Data are expressed as the mean ± standard deviation. PG, (+)-pinoresinol-O-β-D-glucopyranoside.

Figure 4.

PG treatment inhibits IAV-induced activation of cellular signaling pathways. (A) A549 cells seeded in 6-well plates were either mock-infected or infected with influenza virus A/PR8/34 (H1N1) (MOI=0.1), and then cultured in the presence or absence of PG (150–450 µg/ml). Following cell lysis for 24 h, equal quantities of protein lysates were analyzed via western blot analysis using the indicated antibodies. (B) Quantification of indicated phosphorylated proteins. (C) A549 cells were transfected with 20 ng poly (I:C) using Lipofectamine 2000 in the presence or absence of PG (150–450 µg/ml) for 24 h. The cells were lysed and the lysates were subjected to western blot analysis. (D) Quantification of indicated phosphorylated proteins. Quantification was performed using the ImageJ software (normalized to GAPDH protein levels). Data are expressed as the mean ± standard deviation of three separate experiments. *P<0.05, **P<0.01 and ***P<0.001, vs. untreated infected cells. PG, (+)-pinoresinol-O-β-D-glucopyranoside; IAV, influenza A virus; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; P-, phosphorylated.

Figure 5.

PG treatment reduces IAV-induced expression of pro-inflammatory mediators. (A) A549 cells were either mock-infected or infected with IAV/PR8/34 (H1N1) (MOI=0.1) in the presence or absence of PG (150–450 µg/ml), and then lysed with TRIzol reagent for 24 h. Total RNA was isolated and RT-qPCR analyses were performed to measure the gene expression of TNF-α, IL-6, IL-8 and MCP-1. (B) Culture supernatants of H1N1 virus-infected A549 cells treated with different concentrations of PG were harvested for the evaluation of cytokines and chemokines using a Luminex assay. (C) mRNA and (D) protein levels of COX-2 in H1N1 virus-infected A549 cells with treated with different concentrations of PG were analyzed via RT-qPCR and western blot analyses, respectively. (E) PGE2 levels in the culture supernatants from virus-infected A549 cells with/without PG treatment were evaluated using ELISAs. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001, vs. untreated infected cells. PG, (+)-pinoresinol-O-β-D-glucopyranoside; IAV, influenza A virus; Con, control; TNF-α, tumor necrosis factor-α; IL, interleukin; MCP, monocyte chemoattractant protein 1; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Table I.

Primers and probe sequences for reverse transcription-quantitative polymerase chain reaction analysis.

Gene	Primer and probe	Sequence (5′→3′)
TNF-α	Forward	AACATCCAACCTTCCCAAACG
	Reverse	GACCCTAAGCCCCCAATTCTC
	Probe	CCCCCTCCTTCAGACACCCTCAACC
IL-6	Forward	CGGGAACGAAAGAGAAGCTCTA
	Reverse	CGCTTGTGGAGAAGGAGTTCA
	Probe	TCCCCTCCAGGAGCCCAGCT
IL-8	Forward	TTGGCAGCCTTCCTGATTTC
	Reverse	TATGCACTGACATCTAAGTTCTTTAGCA
	Probe	CCTTGGCAAAACTGCACCTTCACACA
MCP-1	Forward	CAAGCAGAAGTGGGTTCAGGAT
	Reverse	AGTGAGTGTTCAAGTCTTCGGAGTT
	Probe	CATGGACCACCTGGACAAGCAAACC
COX-2	Forward	GAATCATTCACCAGGCAAATTG
	Reverse	TTTCTGTACTGCGGGTGGAAC
	Probe	TTCCTACCACCAGCAACCCTGCCA
GAPDH	Forward	GAAGGTGAAGGTCGGAGTC
	Reverse	GAAGATGGTGATGGGATTTC
	Probe	CAAGCTTCCCGTTCTCAGCC

TNF-α, tumor necrosis factor-α; IL, interleukin; MCP, monocyte chemoattractant protein 1; COX-2, cyclooxygenase-2.

Table II.

Anti-influenza virus efficacy of (+)-pinoresinol-O-β-D-glucopyranoside.

	(+)-pinoresinol-O-β-D-glucopyranoside (µg/ml)			Oseltamivir (µg/ml)

Virus type and strain	TC₅₀	IC₅₀	SI^a	TC₅₀	IC₅₀	SI^a
A/PR/8/34 (H1N1)	736.49±34.51	408.81±5.24	1.80±0.11	>1,000	0.041±0.01	>1,000
A/GZ/GIRD07/09 (H1N1)	736.49±34.51	176.24±4.41	4.17±0.30	>1,000	0.022±0.01	>1,000
A/HK/8/68 (H3N2)	736.49±34.51	>737	<1	>1,000	0.098±0.01	>1,000
A/HK/Y280/97 (H9N2)	736.49± 4.51	>737	<1	>1,000	0.756±0.12	>200
B/Lee/1940 (FluB)	736.49±34.51	>737	<1	>1,000	11.51±1.19	>100

SI was calculated as the ratio of TC₅₀ to IC₅₀. SI, selectivity index; TC₅₀, 50% toxic concentration; IC₅₀, 50% inhibition concentration.