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Introduction

Enteroviruses are a group of small single-strand, positive-sense RNA viruses in the Enterovirus genus of the Picornaviridae family (1-3). Infection with certain enteroviruses, including enterovirus 71 (EV71) and coxsackievirus B, lead to severe diseases including aseptic meningitis, brainstem encephalitis, myocarditis and pancreatitis (4,5). Enterovirus infections are particularly common among children <5 years old, and it is one of the major causative pathogens of hand, foot and mouth disease (HFMD), which affects millions of children in the Asia-Pacific region (6,7). In 2015, there were up to 2 million patients with HFMD, with 129 mortalities reported in China. The clinical presentation of HFMD is characterized by buccal ulcerative lesions with pain, and skin rashes on hands and feet without pain (8). A small number of children may exhibit central nervous system and respiratory system damage, causing aseptic meningitis, encephalitis, acute flaccid paralysis, neurogenic pulmonary edema and myocarditis. Critically ill children progress rapidly, and are at risk of mortality (9). In recent years, there have been a number of treatments for EV infection, including vaccines, antivirals and interferon (10,11). Unfortunately, the efficacy of these treatments is not satisfactory.

The primary active ingredient of Polygonum cuspidatum is polydatin and resveratrol (RES) (12). Polydatin has exhibited antitussive, hepatoprotective and anti-shock effects, and the ability to inhibit platelet aggregation, among other effects (13). RES is the most valuable and promising ingredient in P. cuspidatum, and it has antibacterial, anti-inflammatory, anti-allergic, anti-thrombotic, anti-oxidant, anti-cancer and anti-mutation effects (14-16). In addition, RES also has antiviral effects against certain viruses, including influenza virus, hepatitis C virus, respiratory syncytial virus, Epstein-Barr virus, African swine fever virus, enterovirus and duck enteritis virus (17-18).

However, the solubility of RES is poor in aqueous solution, and it is easily degraded (12,14). Therefore, RES has a low bioavailability, which results in a limited curative effect. Nanoparticle (NP) technology may improve the solubility and therapeutic effects of hydrophobic drugs (19-21). In the present study, in order to effectively inhibit the replication of EV71 virus, RES-loaded nanoparticles (RES-NPs) were used, and the protective mechanism of RES-NPs against rhabdosarcoma (RD) cells infected with EV71 was investigated.

Materials and methods

Materials

RD cells were purchased from Nanjing Kangxi Biotechnology Co., Ltd. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and collagenase type II were purchased from Gibco; Thermo Fisher Scientific, Inc. The polyvinylidene fluoride membranes and enhanced chemiluminescence (ECL) western blot detection system were purchased from Pierce; Thermo Fisher Scientific, Inc. The malondial-dehyde (MDA) assay and bicinchoninic acid (BCA) kits was purchased from Nanjing Jiancheng Bioengineering Institute. The superoxide dismutase (SOD) assay kit was purchased from Dojindo Molecular Technologies, Inc. The reactive oxygen species (ROS) assay kit was obtained from Applygen Technologies, Inc. Rabbit polyclonal antibodies against GAPDH, protein kinase R-like endoplasmic reticulum-resident kinase (PERK), phosphorylated-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phosphorylated-eIF2α (p-eIF2α), activating transcription factor 4 (ATF4), glucose regulated protein 78 (GRP78), transcription factor CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) and light chain 3A/B (LC3-I/II) were purchased from Cell Signaling Technology, Inc. Major capsid protein VP1 (VP1), LC3B (LC3-II), horseradish peroxidase-linked rabbit anti-mouse IgG and Cy3-labeled goat anti-rabbit IgG were purchased from Abcam. MTT, RES, apocynin, 4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA) reagents were purchased from Sigma-Aldrich; Merck KGaA.

Synthesis of RES-NPs

The synthesis of copolymer monomethoxy poly (ethylene glycol)-b-poly (D,L-lactide) (PEG-PDLLA) was performed as described previously (22). In detail, 4 ml RES (1 mg) and PEG-PDLLA (9 mg) solution in tetrahydrofuran (THF) was slowly added into 10 ml distilled water at room temperature and stirred; the mixture was dialyzed to remove THF. RES-NPs were obtained for subsequent use.

Characterization of RES-NPs

The morphology of RES-NPs was observed using transmission electron microscopy (TEM) as follows: Following dropping of the sample liquid on a carbon coated copper grid, it was dyed with 1% uranyl acetate. The size distribution of NPs was determined by dynamic light scattering (DLS) with a vertically polarized He-Ne laser (DAWN EOS; Wyatt Technology Corporation). Zeta potential measurement was performed using Zetasizer Nano-ZS (Malvern Instruments, Ltd.).

In vitro release

A total of 2 ml PBS (pH 7.4, 1.0 wt % Tween-80) solution and RES-NPs were added to the dialysis bag (Molecular weight cutoff=3.5 kDa), and the dialysis bag was placed in 8 ml release medium and oscillated at 37°C. At intervals of 2 h, 1 ml buffer solution was removed from the dialysis bag for UV visualization, and 1 ml fresh buffer solution was added to the dialysis bag. The calibration curve of RES was obtained based on the absorbance of RES at 306 nm. Finally, the cumulative release weight and relative percentage of RES were obtained.

Cytotoxicity test

Cell suspensions were seeded in 96-well plates (1×105 cells per well, 100 µl per well). The culture solution containing RES (200 µg/ml) or RES-NPs (RES concentration, 200 µg/ml) was added to each well, and the culture plate was placed in an incubator (37°C and 5% CO2) for 48 h. Following incubation, the supernatant was discarded and 20 µl MTT solution (5 mg/ml in PBS) was added to each well. The culture plate was placed in the incubator (37°C and 5% CO2) and incubated for 4 h prior to removal of the supernatant. Dimethyl sulfoxide (150 µl) was added to each of the 96-well plates, and the absorbance at 570 nm was measured by a microplate reader (Bio-Rad Laboratories, Inc.). Finally, the 50% cytotoxic concentration (CC50) was calculated.

Virus isolation and propagation

A total of 4 ml EV71 medium (MOI=2) was prepared as a virus inoculum. Following culture for 24 h, RD cells were washed once with PBS, diluted to a density of 1×106 cells, and incubated with the prepared EV71 medium for 1.5 h at 37°C. To remove unbound EV71, the cells were washed with fresh medium. Cell supernatants were collected at 0, 1, 3, 6, 12, 16, 18, 20 and 24 h after infection, virus titers were determined by cytopathic effect (CPE), and the median tissue culture infectious dose (TCID50) per ml was calculated. The results were observed and recorded daily for 5-7 days; CPE was observed under a light microscope (magnification, ×100) and categorized according to the following criteria: i) −: no cytopathic changes; ii) ±: 1-25% of the cells had pathological changes; iii) ++: 26-50% of the cells had pathological changes; iv) +++: 51-75% of the cells had pathological changes; and v) +++: 76-100% of the cells had pathological changes. The number of positive holes in each row was counted and the virus TCID50 was calculated using the Reed-Muench method (23) or Karber method (24). If there was no CPE in the negative control group and the cells grew well, the lowest dilution was 100% positive and the highest dilution was 100% negative, the test would be effective.

Antiviral activity assay

Following culture of the RD cells in 96-well plates for 24 h, 30 µl EV71 (MOI=2) medium and diluted RES-NPs solution were added and incubated for 1.5 h at 37°C. In order to remove the virus that was not absorbed, the medium was aspirated. The cells were washed with PBS and RES (200 µg/ml) or RES-NPs (RES concentration, 200 µg/ml of each group (doses were ≤ toxic dose) were added. The CPE of each group was observed using a light microscope (magnification, ×100) and subjected to MTT measurement as aforementioned, and finally the half-maximal inhibitory dose value (IC50) was calculated.

Time course analysis of RES-NPs on EV71 replication

RD cells (3×105 cells/well) were inoculated in a 96-well plate for 24 h, and the control, RES (200 µg/ml) or RES-NPs (RES concentration=200 µg/ml) groups were infected with EV71 (MOI=2) for 1.5 h. Cell supernatants were then collected at different assay times (0, 1, 3, 6, 12, 16, 18, 20 and 24 h) post-infection, and EV71 titers were determined by calculating the TCID50 values.

Measurement of ROS levels

Dihydroethidium [DHE; Weigras Biotechnology (Beijing) Co., Ltd], a ROS-level indicative fluorescence probe (λex=535 nm, λem=610 nm), was used to detect intracellular superoxide anions. Briefly, RD cells were seeded

in 6-well plates (2×104 cells/well) and cultured with complete medium for 24 h. Cells were then divided into different groups and exposed to culture medium (control) or EV71 or EV71 + RES-NPs (RES concentration=200 µg/ml), respectively, for 24 h at 37°C. Subsequently, cells were washed with cold PBS and additionally incubated with fresh medium containing 10 µM DHE at 37°C in the dark for 20 min to stain the nuclei. The harvested cells were resuspended in PBS at a density of 2×107 cells/ml, transferred to a light-shielded 96-well plate (100 µl cell suspension per well), followed by determination of DHE intensity using a fluorescence microplate reader (Bio-Rad Laboratories, Inc.). Fluorescence images of the cells were captured using an electronic camera (Olympus Corporation).

Determination of major biochemical parameters

MDA and SOD levels were measured using MDA and SOD activity kits, respectively. Briefly, RD cells that were cultured with complete medium for 24 h were divided into different groups, and these groups were treated with culture medium (control) or EV71 or EV71 + RES-NPs (RES concentration=200 µg/ml) for 24 h at 37°C. The harvested cells were lysed and the supernatant was collected by centrifugation at 12,000 × g for 10 min at 4°C. Protein content in the supernatant was detected using a BCA kit. In addition, 100 µl supernatant was placed into a centrifuge tube, and the MDA testing solution (200 µl) was added. Following mixing, the mixture was boiled for 15 min and cooled to room temperature, followed by centrifugation at 1,000 × g for 10 min at 4°C. A total of 200 µl prepared supernatant was added to 96-well plate, and the absorbance was measured at 532 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

SOD was detected based on its ability to inhibit super-oxide-mediated reduction. A SOD assay kit (cat. no. S311; Dojindo Molecular Technologies, Inc.) was used to detect its activity. The harvested cells were homogenized, and the obtained homogenate was centrifuged at 12,000 × g for 10 min at 4°C. The protein content in supernatant was determined using the BCA kit. A mixture of 20 µl supernatant and 160 µl nitro-blue tetrazolium chloride/enzymatic working solution was added to a centrifuge tube, and mixed at 4°C for 5 min. Then, 20 µl reaction-initiating working solution was added and incubated at 37°C for 30 min. The resultant mixture was detected for its absorbance at 560 nm using the aforementioned microplate reader.

Western blot analysis

The cells in each group were cultured in a 37°C, 5% CO2 incubator for 24 h and the supernatant was discarded. The cells were lysed with 1X RIPA lysis buffer (Cell Signaling Technology, Inc.). The cells were then collected in a centrifuge tube and centrifuged at 12,000 × g for 15 min at 4°C. The lysates were clarified, and the supernatants fractions were isolated. Protein concentrations in cells were determined using a bicinchoninic acid protein assay. In total, 30-50 µg of protein was loaded and separated by SDS-PAGE on 8-12% gels and then transferred to a PVDF membrane. After blocking the membrane with 5% non-fat milk for 1 h at room temperature, the following primary antibodies were used for western blotting: VP1 (cat. no. ab53977; 1:10,000), PERK (cat. no. 3192; 1:1,000), p-PERK (cat. no. 3179; 1:1,000), eIF2α (cat. no. 9722; 1:1,000), p-eIF2α (cat. no. 3398; 1:1,000), ATF4 (cat. no. 11815; 1:1,000), GRP78 (cat. no. 3183; 1:1000), CHOP (cat. no. 2895; 1:1,000), LC3-I/II (cat. no. 4108; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000). Primary antibodies were incubated with membranes overnight at 4°C. The membrane was incubated with horseradish peroxidase-linked rabbit anti-mouse IgG (cat. no. ab6728; 1:2,000) for 2 h at room temperature. Finally, the blots were visualized using a chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.). GeneTools software 4.1 (Syngene) was used to quantify the immunoblots.

Immunofluorescence analysis

The cells in each group were cultured in a 37°C, 5% CO2 incubator for 24 h, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and blocked with 1% bovine serum albumin (cat. no. B2064; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. After washing the cells with PBS, the cells were incubated with primary antibodies against LC3-II (cat. no. ab48394; 1:400) for 24 h at 4°C. After washing with PBS, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (cat. no. ab150077; 1:200) for 60 min at room temperature. DAPI (5 mg/ml) was used to stain all cell nuclei at room temperature for 10 min. A fluorescence microscope (magnification, ×400; Olympus Corporation) was used to observe the morphology of the cells and the intensity of the fluorescent signals.

Detection of inflammatory cytokines

RD cells infected with EV71 (MOI=2) were centrifuged at 1,500 × g for 10 min at 4°C, and the supernatant was collected at 0, 3, 6, 12 and 24 h after infection. The concentration of interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) in the supernatant was measured by Milliplex magnetic beads (EMD Millipore) and the FLEXMAP 3D platform (Luminex Corporation). The resulting data was analyzed by MILLIPLEX Analyst v5.1 (VigeneTech, Inc.).

Statistical analysis

All experiments were performed a minimum of 3 times, and the data were analyzed using GraphPad Prism v5 software (GraphPad Software, Inc.). All data are presented as the mean ± standard deviation, and were analyzed using one-way analysis of variance followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Preparation of RES-NPs

The THF solution containing RES and PEG-PDLLA was dropped into water, and ideal RES-NPs were obtained. Fig. 1A illustrates the Gaussian distribution of RES-NP size. The particle size and zeta potential of the obtained RES-NPs were 144.7 nm (polydispersity index=0.31) and 0.47±0.012 mV, respectively. TEM data indicated that RES-NPs were spherical nanoparticles with diameters of ~145 nm (Fig. 1B). All these data indicate that RES-NPs had small sizes and nearly neutral surface charge, suggesting that they have potential for practical applications.

Figure 1 Size distribution and TEM image of RES-NPs. (A) Size distribution of RES-NPs determined by dynamic light scattering. (B) Representative TEM image of RES-NPs. Scale bar, 100 nm. TEM, transmission electron microscope; RES-NPs, resveratrol-loaded nanoparticles.

Release profile of RES-NPs

In order to calculate the cumulative release rate of RES, the released RES were collected at different time points. As demonstrated in Fig. 2, the release of RES was rapid during the first 4 h, with a cumulative release rate of up to 40%, but after 4 h, RES release became slow, due to the strong hydrophobicity of RES (25).

Figure 2 Cumulative release profiles of resveratrol from resveratrol-loaded nanoparticles in pH 7.4 PBS for 24 h.

Effect of RES-NPs on viability of RD cells

To understand the toxicity of RES-NPs and RES on RD cells, different concentrations of RES-NPs and RES were added to RD cells and incubated for 48 h to detect cell viability. As indicated in Fig. 3, RES-NPs and RES were not significantly toxic to RD cells at 200 and 40 µg/ml, respectively. However, when the concentration of RES-NPs and RES was >200 and 40 µg/ml, respectively, the toxicity increased markedly. The CC50 of RES-NPs and RES were 618.54 and 82.77 µg/ml, respectively.

Figure 3 Cytotoxic effects of RES-NPs or RES on rhabdosarcoma cells. The cytotoxicity of RES-NPs or RES on rhabdosarcoma cells was determined by quantifying the cell viability by an MTT assay. The absorbance was measured using a microplate reader at 570 nm. Data from three repeat experiments are presented as the mean ± standard deviation. *P<0.05 vs. 0 RES-NPs µg/ml group (white bar); #P<0.05 and ##P<0.01 vs. 0 µg/ml RES group (gray bar). RES, resveratrol; RES-NPs, resveratrol-loaded nanoparticles.

Antiviral effect of RES-NPs

To understand the association between RES-NPs treatment time and antiviral efficacy, 2 assays were performed. The first assay involved the addition of RES-NPs and EV71 to RD cells concomitantly, and the virus titer was detected after 48 h. The second assay involved the addition of RES-NPs following EV71 infection of RD cells for 1.5 h, and the virus titer was detected after 48 h of RES-NPs treatment. As indicated in Fig. 4A, RES-NPs significantly inhibited the replication of EV71 in a dose-dependent manner in both groups, and there was no significant difference between the two groups.

Figure 4 Antiviral effects of RES-NPs on EV71 replication in RD cells. (A) Various doses of RES-NPs were added with EV71 concomitantly, or following EV71 absorption for 1.5 h. (B) RES (200 µg/ml) or RES-NPs (RES concentration=200 µg/ml) were added following EV71 absorption for 1.5 h. The culture supernatants were collected at 0, 1, 3, 6, 12, 16, 18, 20 and 24 h. Data from three repeat experiments are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. control group. (C) Inhibition of RES or RES-NPs on the expression of VP1 protein. At 0, 3, 6, 12 and 24 h, RD cell lysates were subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes to determine the expression level of VP1. Data from three repeat experiments are presented as the mean ± standard deviation. VP1 levels were normalized with GAPDH. *P<0.05 vs. EV71. RES, resveratrol; RES-NPs, resveratrol-loaded nanoparticles; EV71, enterovirus 71; RD, rhabdosarcoma; VP1, major capsid protein VP1; CPE, cytopathic effect.

To additionally understand the time-dependent antiviral effect of RES or RES-NPs, EV71 was added to RD cells and incubated for 1.5 h, then added RES (200 µg/ml) or RES-NPs (RES concentration=200 µg/ml) and incubated for different times. As demonstrated in Fig. 4B, RES or RES-NPs significantly inhibited EV71 replication during incubation for 6-24 h. On the basis of this result, the effect of RES or RES-NPs on VP1 synthesis was additionally analyzed. The results indicated that VP1 was significantly decreased in the presence of RES (200 µg/ml) or RES-NPs (RES concentration=200 µg/ml) at 6, 12 and 24 h, suggesting that RES or RES-NPs may effectively inhibit viral replication in EV71-infected RD cells (Fig. 4C). These results suggested that there was no significant difference in the antiviral efficacy of RES-NPs (200 µg/ml) and RES (200 µg/ml) within 24 h of stimulation. The reason was that, although the cumulative release of RES released by RES-NPs was only ~40% at 24 h of stimulation, the sustained release of NPs maintained a high level of local RES cellular concentration and prolonged the effective drug action time. Therefore, the antiviral effect was able to reach the levels observed for the small molecule RES.

Effect of RES-NPs on EV71-induced ROS level, MDA content and SOD activity in RD cells

EV71-induced ROS levels were first detected by using DHE as a fluorescent probe, and the results are presented in Fig. 5. The images denote that the RD cells treated with EV71 alone exhibited brighter red fluorescence signals compared with the control group, suggesting that EV71 infection may lead to an increase in intracellular ROS levels. Conversely, the red fluorescence corresponding to the cells treated with the combination of EV71 and RES-NPs exhibited largely decreased brightness when compared with those cells treated by EV71 only, demonstrating that RES-NPs may significantly prevent intracellular ROS production. DHE fluorescence intensity was quantified, and the data suggested that RES-NPs may completely inhibit the EV71-induced increase in ROS levels (Fig. 5B).

Figure 5 Fluorescence images and major biochemical parameters of RD cells treated with EV71 alone and the combination of EV71 and RES-NPs for 24 h. (A) Representative images of ROS-positive cells (RES concentration, 200 µg/ml; scale bar, 50 µm). (B) Fluorescence intensity of dihydroethidium signals. (C) MDA levels in RD cells. (D) SOD activity in RD cells. Data of three repeat experiments are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. control; #P<0.05 and ##P<0.01 vs. EV71. RD, rhabdosarcoma; EV71, enterovirus 71; RES-NPs, resveratrol-loaded nanoparticles; ROS, reactive oxygen species; RES, resveratrol; MDA, malondialdehyde; SOD, super oxide dismutase.

The data presented in Fig. 5C and D demonstrate that the EV71 infection resulted in a marked increase in the level of MDA, while leading to a significant decrease in SOD activity; these changes are markedly associated with the rise of intracellular oxidative stress levels. In contrast to these observations, RES-NPs (RES concentration=200 µg/ml) markedly inhibited the increase of MDA levels and regulated the SOD activity, ensuring that these indexes remained at normal levels.

Effect of RES-NPs on EV71-induced endoplasmic reticulum stress (ERS) upregulation in RD cells

The endoplasmic reticulum (ER) is an organelle responsible for the synthesis and folding of secreted proteins and transmembrane proteins (26). When the ER is in a resting state, GRP78 binds to baroreceptors and maintains the ER homeostasis (26). Viral infection may lead to the accumulation of misfolded proteins in the ER, which triggers ERS (27). ERS may contribute to autophagy via activation of GRP78, PERK, eIF2α, ATF4 and CHOP. In the present study, EV71 infection upregulated the expression of p-PERK, p-eIF2α and ATF4 in RD cells at 6, 12 and 24 h (Fig. 6A), but a significant decrease in their expression in the presence of RES-NPs (RES concentration=200 µg/ml) was observed (Fig. 6B). Concomitantly, RES-NPs failed to inhibit the expression of p-PERK, p-eIF2α and ATF4 when pretreated with apocynin, an oxidative stress inhibitor (Fig. 6C). In addition, EV71 infection upregulated the expression of GRP78 and CHOP in RD cells at 6, 12 and 24 h (Fig. 6D), but a significant decrease in their expression in the presence of RES-NPs (RES concentration=200 µg/ml) was observed (Fig. 6E). Furthermore, RES-NPs failed to inhibit the expression of GRP78 and CHOP when pretreated with apocynin (Fig. 6F). These data indicated that the RES-NPs inhibited the EV71-induced activation of the oxidative stress-mediated ERS signal pathway.

Figure 6 RES-NPs alters the expression levels of the endoplasmic reticulum stress-associated proteins p-PERK, PERK, p-eIF2α, eIF2α, ATF4, GRP78 and CHOP in EV71-infected RD cells. The levels of p-PERK, PERK, p-eIF2α, eIF2α and ATF4 were measured in RD cells pretreated with (A) culture medium, (B) RES-NPs (RES concentration, 200 µg/ml) alone or (C) RES-NPs (RES concentration, 200 µg/ml) + apocynin (300 µM) for 0, 3, 6, 12 and 24 h using a western blot assay following protein extraction. p-PERK and p-eIF2α levels were normalized to PERK and eIF2α, and then normalized to GAPDH. ATF4 levels were normalized to GAPDH. The levels of GRP78 and CHOP were measured in RD cells pretreated with (D) culture medium for 0, 3, 6, 12 and 24 h using a western blotting assay following protein extraction. Data from three repeat experiments are presented as the mean ± standard deviation. *P<0.05 vs. 0 h p-PERK/PERK group; #P<0.05 vs. 0 h p-eIF2α/eIF2α group; $P<0.05 vs. 0 h ATF4 group. The levels of GRP78 and CHOP were measured in RD cells pretreated with (E) RES-NPs (RES concentration, 200 µg/ml) alone or (F) RES-NPs (RES concentration, 200 µg/ml) + apocynin (300 µM) for 0, 3, 6, 12 and 24 h using a western blotting assay following protein extraction. Data from three repeat experiments are presented as the mean ± standard deviation. *P<0.05 vs. 0 h p-PERK/PERK group; #P<0.05 vs. 0 h p-eIF2α/eIF2α group; $P<0.05 vs. 0 h ATF4 group. RES, resveratrol; RES-NPs, resveratrol-loaded nanoparticles; PERK, PKR-like endoplasmic reticulum-resident kinase; p-PERK, phosphorylated PERK; eIF2α, eukaryotic initiation factor 2α; p-eIF2α, phosphorylated eIF2α; ATF4, activating transcription factor 4; GRP78, glucose regulated protein 78, CHOP, transcription factor CCAAT/enhancer binding protein-homologous protein; EV71, enterovirus 71; RD, rhabdosarcoma.

Effect of RES-NPs on EV71-induced autophagy upregulation in RD cells

Autophagy is a type of programmed cell death through which the body degrades old proteins and senescent organelles, to achieve their own metabolic needs and organelle renewal (28-30). Under normal conditions, the level of autophagy is low. However, in the case of pathogen infection, autophagy may be rapidly upregulated (31). In the present study, EV71 infection increased the ratio of LC3-II/cytosolic LC3 (LC3-I) in RD cells at 6, 12 and 24 h (Fig. 7A), but they revealed a significant reduction in the presence of RES-NPs (RES concentration=200 µg/ml) (Fig. 7B). Furthermore, RES-NPs failed to inhibit the ratio of LC3-II/LC3-I when pretreated with the 4-PBA (eIF2α-mediated ERS pathway inhibitor) (Fig. 7C). Concomitantly, red fluorescent-labeled LC3-II antibody was used to detect the level of autophagy and DAPI was used to localize the cells. Fig. 7D demonstrated that the RD cells infected with EV71 alone exhibited brighter red fluorescence compared with the control group, signifying that the EV71 infection may lead to an increase in intracellular LC3-II expression. Conversely, the red fluorescence signals corresponding to the cells treated with the combination of EV71 and RES-NPs were markedly decreased when compared with those cells treated with EV71 only, demonstrating that the use of RES-NPs may significantly prevent intracellular LC3-II expression. These red fluorescence intensity data were quantified, and suggested that RES-NPs may almost completely inhibit the EV71-induced increase in LC3-II expression (Fig. 7E). These data indicated that the RES-NPs inhibited the EV71-induced activation of the ERS-mediated autophagy signaling pathway.

Figure 7 RES-NPs alters the expression levels of autophagy-associated protein LC3-I in EV71-infected RD cells. The RD cells were pretreated with (A) culture medium, (B) RES-NPs (RES concentration, 200 µg/ml) alone or (C) RES-NPs (RES concentration, 200 µg/ml) + 4-PBA (5 mM) for 0, 3, 6, 12 and 24 h. The expression levels of LC3-I and LC3-II was measured using a western blotting assay following protein extraction. LC3-I and LC3-II levels were normalized to GAPDH, and the LC3-II/LC3-I ratio was quantitatively analyzed. Data from three repeat experiments are represented as the mean ± standard deviation. *P<0.05 vs. 0 h group. (D) Fluorescence images of RD cells treated with EV71 alone and the combination of EV71 and RES-NPs (RES concentration, 200 µg/ml) for 24 h. Scale bar, 50 µm. (E) Fluorescence intensity of LC3-II expression was quantified. Data from three repeat experiments are presented as the mean ± standard deviation. **P<0.01 vs. control; ##P<0.01 vs. EV71. RES, resveratrol; RES-NPs, resveratrol-loaded nanoparticles; EV71, enterovirus 71; RD, rhabdosarcoma; PBA, 4-Phenylbutyric acid; LC3, microtubule-associated proteins 1A/1B-light chain 3.

Effect of RES-NPs on EV71-induced inflammatory cytokines secretion and VP1 synthesis in RD cells

Previous data have indicated that the occurrence and development of HFMD are associated with excessive inflammation and the immune response in the body (32). Cytokines are a type of low-molecular-weight protein with a broad range of biological activities, induced by immunogens and other stimuli (32). They are able to regulate cell growth and differentiation by regulating their corresponding receptors, and regulate immune responses (32). It has been identified that inflammatory cytokines including IL-6, IL-8 and TNF-α were over-secreted in serum and body fluid of children with HFMD (33). In the present study, compared with the control group, EV71-infected cells exhibited increased secretion levels of IL-6, IL-8 and TNF-α at 6, 12 and 24 h (Fig. 8A-C). The treatment of RES-NPs (RES concentration=200 µg/ml) suppressed the secretion of IL-6, IL-8 and TNF-α in EV71-infected RD cells, and no difference between the control and RES-NPs groups was observed (Fig. 8A-C). These data suggested that RES-NPs may effectively inhibit the secretion of inflammatory cytokines and mitigate the damage of RD cells. Concomitantly, RES-NPs failed to inhibit the secretion of inflammatory cytokines when pretreated with apocynin, the oxidative stress inhibitor, 4-PBA, the ERS inhibitor, or 3-MA, the autophagy inhibitor. Fig. 8D indicated that RES-NPs may effectively inhibit the synthesis of VP1 and mitigate the virus replication in EV71-infected RD cells. In addition, RES-NPs failed to inhibit the synthesis of VP1 when pretreated with the apocynin, 4-PBA or 3-MA (Fig. 8D). These results suggested that oxidative stress/ERS/autophagy signal transduction may be involved in the anti-inflammatory effect and antiviral effect of RES-NPs on EV71-infected RD cells.

Figure 8 Inhibition of RES-NPs on the several chemokine releases and expression of VP1 protein in EV71-infected RD cells at 24 h. Promotion of (A) IL-6, (B) IL-8 and (C) TNF-α release in RD cells with EV71 infection. The culture supernatants were harvested at 0, 3, 6, 12 and 24 h after infection to measure the release of cytokines using the FLEXMAP 3D platform. (D) VP1 levels were measured using a western blotting assay following protein extraction. VP1 levels were normalized to GAPDH. Quantitative analysis of the expression of VP1. Data from three repeat experiments are presented as the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. EV71; $P<0.05 vs. EV71 + RES-NPs. IL-6, interleukin-6; IL-8, interleukin-8; TNF-α, tumor necrosis factor-α; RD, rhabdosarcoma; EV71, enterovirus 71; RES, resveratrol; RES-NPs, resveratrol-loaded nanoparticles; Control group, uninfected RD cells; RES-NPs group, uninfected RD cells treated with RES-NPs; EV71 group, EV71 infected RD cells; RES-NPs + EV71 group, RD cells pretreated with RES-NPs (RES concentration, 200 µg/ml) for 1.5 h prior to EV71 infection; VP1, major capsid protein VP1.

Discussion

EV71 infection may lead to HFMD, aseptic meningitis, encephalitis and poliomyelitis-like paralysis and other diseases (34). At present, the effectiveness of antiviral drugs, vaccines and interferon treatments in their abilities to completely eradicate the virus is limited (10,11). It has been observed in clinical practice that certain traditional Chinese medicines may be effective in the prevention and treatment of EV71 (35). RES exhibits multiple pharmacological properties, including anti-inflammatory, antibacterial, anti-tumor and antifungal effects, lipid metabolism regulation and cardiovascular protection (14). In addition, RES usually interferes with the signal transduction pathway in the cell to exert its extensive anti-viral effects (36,37). As RES is a polyphenolic compound, it is very polar, with low solubility in water and high solubility in strong polar solvents including chloroform, methanol, ethanol and acetone, resulting in low bioavailability. It is unstable to light and therefore inconvenient for clinical use. NPs use block copolymers to entrap hydrophobic drugs in hydrophobic cores. NP systems may increase the solubility and biocompatibility of hydrophobic drugs. In the present study, an efficient antiviral effect was obtained by using NPs formulations of RES in EV71 infected-RD cells. However, the antiviral effects and mechanism of RES-NPs on EV71 have not been widely studied. The present study confirmed that there was no cytotoxicity of RES-NPs on RD cells at the dose of 200 µg/ml. Concomitantly, RES-NPs were able to significantly inhibit CPE caused by EV71, and the inhibitory activity of RES-NPs on EV71 replication was observed to be dose-dependent. When RES-NPs (RES concentration=200 µg/ml) were added simultaneously with or after EV71 absorption for 1.5 h, the viral titers in cell supernatant decreased, suggesting that RES-NPs may inhibit EV71 replication in RD cells. These data suggested that the inhibitory activity of RES-NPs on EV71 replication was time-dependent, but independent of viral attachment and penetration.

VP1 is an enterovirus-encoded capsid protein that is exposed on the surface of viral particles and is the major antigenic determinant of EV71 (38). It is also a specific indicator of EV71 replication (38). The data from the present study indicated that the synthesis of VP1 in RD cells was significantly increased at 6-24 h after EV71 infection, whereas RES-NPs were able to effectively prevent the synthesis of VP1 in RD cells infected with EV71. These results suggested that RES-NPs may exert strong antiviral effects in EV71-infected RD cells, and the mechanism requires additional exploration.

Rhinovirus and respiratory syncytial virus induce oxidative stress in cells (39,40). It has been previously identified that EV71 infection may lead to increased ROS generation (41). MDA is the preferred indicator to evaluate the ROS content in vivo (42). SOD is an important antioxidant enzyme that maintains the free radical homeostasis in the human body (43). The present study demonstrated that RES-NPs may significantly decrease the production of ROS and MDA in EV71-infected RD cells. Concomitantly, RES-NPs may significantly increase the production of SOD in EV71-infected RD cells.

Cells may affect the homeostasis and function of the ER in stress conditions, including bacterial or viral infections (44). A previous research demonstrated that EV71 infection might cause upregulation and aberrant redistribution of GRP78 to the cytosol, thereby facilitating virus replication (45). Under pathological states including viral infection, ER-localized chaperones, including GRP78, are activated, and then the unfolded protein response system is initiated, including the PERK/eIF2α/ATF4 pathway (44). CHOP, also known as growth arrest and DNA damage-inducing gene 153, is a pro-apoptotic transcription factor (46) belonging to the C/EBP transcription factor family that heterodimerizes with other C/EBPs and may be upregulated by the PERK/eIF2α/ATF4 pathway (47). In the present study, the western blot analysis data revealed that exposure to EV71 in RD cells increased the expression of p-PERK, p-eIF2α, ATF4, GRP78 and CHOP, indicating that the PERK/eIF2α/ATF4 pathway participated in EV71-induced virus replication. Following pretreatment with RES-NPs, the increased expression of p-PERK, p-eIF2α, ATF4, GRP78 and CHOP was significantly inhibited in the EV71-infected RD cells. Notably, RES-NPs failed to inhibit the expression of p-PERK, p-eIF2α, ATF4, GRP78 and CHOP when pretreated with apocynin. These results indicated that RES-NPs may effectively protect the RD cells against EV71 infection, through inhibiting the activation of the oxidative stress-mediated ERS pathway and extenuated damage of the RD cells.

The importance of autophagy in viral infections has become increasingly apparent and has become an important topic of study (48). Cell autophagy is closely associated with the innate immune system of the body. It may serve roles in the identification and presentation of viral antigens, activation of natural immune responses and clearance of the virus. Conversely, the virus may also use the regulation of autophagy to escape the anti-virus response of the body, and to maintain their own survival and reproduction (49). There have been a number of studies on the induction of cell autophagy by viral infection. For example, membrane cofactor protein is used as a receptor for cell surface-mediated invasion of various pathogens (50). The successive waves of autophagy result from distinct molecular pathways and appear to be associated with anti- and/or pro-measles virus consequences (51). Previous studies have also identified that viral infection may induce autophagy through the activation of the ERS signaling pathway (52,53). When autophagy occurs, LC3-I in the cytoplasm is recruited to the autophagosome to form LC3-II (49). Therefore, the LC3-II/LC3-I ratio may be used as an indicator to reflect the level of autophagy in cells (49). In the present study, the western blot analysis and immunofluorescence data revealed that exposure to EV71 in RD cells increased the LC3-II/LC3-I ratio and LC3-II expression, indicating that the autophagy pathway participated in EV71 induced virus replication. Following pretreatment with RES-NPs, the increased LC3-II/LC3-I ratio and LC3-II expression was significantly inhibited in EV71-infected RD cells. Notably, RES-NPs failed to inhibit the LC3-II/LC3-I ratio when pretreated with 4-PBA, the ERS inhibitor. These data indicated that RES-NPs may effectively protect the host cells against EV71 infection through inhibition of the activation of ERS-mediated autophagy pathway and extenuated damage to the RD cells.

Cytokines serve an important role in the inflammatory response in HFMD, are involved in the immune and inflammatory reactions and affect the intensity and duration of the reaction (33). The investigation of cytokines has identified that there are >200 types of human cytokines, including IL-6, IL-8 and TNF-α (54,55). The results from the present study suggested that the secretion of IL-6, IL-8 and TNF-α significantly increased in EV71 infected RD cells. Concomitantly, the western blot analysis results suggested that the expression of VP1 was significantly increased in EV71-infected RD cells. Following pretreatment with RES-NPs, VP1 expression levels and the secretion of IL-6, IL-8 and TNF-α were observed to be decreased in EV71-infected RD cells. These data indicated that RES-NPs may effectively inhibit the inflammatory reaction and virus replication in EV71-infected RD cells. Notably, 3 inhibitors targeting oxidative stress, ERS or autophagy inhibited the anti-inflammatory and antiviral effects of RES-NPs on EV71-infected RD cells, suggesting that the RES-NPs-induced anti-inflammatory and antiviral effects were closely associated with the oxidative stress/ERS/autophagy pathway.

In conclusion, the present study demonstrated that RES-NPs inhibited viral replication and the inflammatory reaction of EV71-infected RD cells by inhibiting the synthesis of VP1 protein and the secretion of cytokines, and that RES-NPs served their role through inhibiting the oxida-tive stress-mediated ERS/autophagy pathway. These results indicate that RES-NPs may be a feasible alternative strategy for the treatment of EV71 infection. The underlying mechanism requires additional in vitro and in vivo studies, in order to examine the oxidative stress-mediated ERS/autophagy pathway.

Acknowledgments

The authors would like to thank Mr. Ming Liu of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences for offering technical guidance for the synthesis of nanoparticles.

Funding

The present study was supported by a grant from the Jilin Provincial Science and Technology Department in China (grant no. 20130206040SF).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

ND and FW conceived and designed the study. XL, WB, BW, GX and FW performed the experiments. ND, XL, WB, BW and GX wrote the paper. FW reviewed and edited the manuscript. All authors read and approved the final manuscript.

Ethics 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