Urolithin A inhibits enterovirus 71 replication and promotes autophagy and apoptosis of infected cells in vitro

Hand, foot, and mouth disease (HFMD) is a serious threat to the health of infants, and it can be caused by enterovirus 71 (EV71). The clinical symptoms are mostly self-limiting, but some infections develop into aseptic meningitis with poor prognosis and even death. In this study, urolithin A (UroA), an intestinal metabolite of ellagic acid, significantly inhibited the replication of EV71 in cells. Further evaluation showed that UroA was better than ribavirin in terms of its 50% cytopathic concentration (CC50), 50% inhibitory concentration (IC50), and selectivity index. Moreover, UroA inhibited the proliferation of EV71 by promoting autophagy and apoptosis of infected cells. Therefore, UroA is a candidate drug for the treatment of EV71 infection.


Introduction
Hand, foot, and mouth disease (HFMD) is a common infectious disease that occurs worldwide, most frequently in children under 5 years old [28]. It is mostly self-limiting, with symptoms such as fever, rash on the hands and feet, and oral sores [25,30]. However, in some children, the disease progresses rapidly and can lead to severe pulmonary edema, neurological and systemic complications, and even death [27,35]. Enterovirus 71 (EV71), a single-stranded positivesense RNA virus belonging to the genus Enterovirus of the family Picornaviridae, is one of the major pathogens causing severe and fatal cases of HFMD [29]. In 1997, an outbreak of HFMD occurred in the Asia-Pacific region, and in China, approximately 2 million cases, including more than 100 deaths, have been reported every year since 2008. Therefore, HFMD poses a serious threat to public health. At present, there are no effective drugs for the prevention and treatment of HFMD [26,27].
Ellagic acid, a natural polyphenolic compound, is widely distributed in pomegranates, strawberries, grapes, walnuts, chestnuts, and other fruits and nuts [5]. It has various biological activities, such as anti-oxidation, anti-inflammation, and anti-tumor activity, and it is able to regulate gut microbes [2,19,24]. However, it is quickly eliminated from the body after administration [18,21]. Studies have shown that ellagic acid is metabolized by gut microbes into the more easily absorbed urolithin, which is the main active form for its function in vivo [1,7,19,32].
Urolithins are a class of dibenzo-6-ketone derivatives containing different phenolic hydroxyl groups, and they are formed by the loss of a lactone ring of ellagic acid and successive removal of hydroxyl groups [11,23]. In the present study, the antiviral activity of urolithins A, B, and C (UroA, UroB, and UroC) was analyzed. The results showed that UroA, one of the main bioactive forms of ellagic acid in vivo, has very low toxicity and strong antiviral activity. Therefore, UroA is an excellent candidate for clinical use, especially as an antiviral agent for infants and young children.

Cells and viruses
African green monkey kidney cells (Vero cells), rhabdomyosarcoma cells (RD cells), human neuroblastoma SK-N-SH cells (SK-N-SH cells), and Madin-Darby canine kidney cells (MDCK cells) were purchased from the American Type Culture Collection (ATCC) and cultured in modified Eagle's medium (BasalMedia, China) supplemented with 10% inactivated fetal bovine serum (FBS; Gibco) and 1% penicillinstreptomycin (Solarbio, China). Human lung adenocarcinoma A549 cells (A549 cells) were purchased from the ATCC and cultured in F-12 medium (BasalMedia, China) supplemented with 10% inactivated FBS and 1% penicillinstreptomycin. EV71 strain BrCr (VR-1775) was purchased from the ATCC and passaged in Vero cells. Influenza A virus strain A/Puerto Rico/8/34(H1N1) was purchased from the ATCC and passaged in MDCK cells.

Compounds and antibodies
UroA, UroB, UroC, and ribavirin were purchased from MedChemExpress (MEC; Shanghai, China). The purity was at least 98%, and all of the compounds were dissolved in DMSO (Solarbio, China).

CPE inhibition assay for anti-EV71 activity
RD cells (3 × 10 4 cells/well) were placed in 96-well culture plates and incubated for 24 h. The medium was then removed, and the cells were infected with EV71 (MOI = 0.1) in serumfree medium for 1 h at 37 ℃. Then, the unbound viruses were removed, various concentrations of the compound were added, and the cells were incubated for another 48 h. The cells were then stained with 0.1% crystal violet at room temperature for 20 min, washed with phosphate-buffered saline (PBS), and dried. The OD value of each well was measured at 570 nm, and the 50% inhibitory concentration (IC 50 ) was calculated using GraphPad Prim 5.0 software. The selectivity index (SI) was calculated as the ratio CC 50 /IC 50 .

TCID 50 assay
The virus was serially diluted tenfold and added to Vero cells in 96-well plates, and the cells were cultured at 37 ℃ for 48 h. Median tissue culture infectious dose (TCID 50 ) values were determined based on CPE. Further calculations were conducted using the Reed-Muench method.

qRT-PCR assay
Total cellular RNA was isolated using a TransZol Up Kit, reverse transcribed using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix, and analyzed using PerfectStartTM Green qPCR SuperMix, following the instructions. All reagents were purchased from Transgen Biotech, Beijing, China. The sequences of the primers used for qPCR analysis are as follows: sense primer 5′-GCA GCC CAA AAG AAC TTC AC-3′ and antisense primer 5′-ATT TCA GCA GCT TGG AGTGC-3′ targeting a conserved region of the EV71-VP1 gene; sense primer 5′-GAC AAC TTT GGT ATC GTG GAA-3′ and antisense primer 5′-CCA GGA AAT GAG CTT GAC A-3′ targeting a conserved region of the human GAPDH gene. All primers were synthesized by Sangon Biotech, Shanghai, China.

Western blot assay
Total cellular proteins were extracted using 1× RIPA buffer (CST) and denatured by adding SDS loading buffer and boiling for 6 min at 100 ℃. Twenty µg of protein was resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were blocked with 2% bovine serum albumin (Sigma Aldrich, Germany) and then incubated overnight with the appropriate primary antibody at 4 ℃. The membranes were washed with 1× TBST and incubated with a 1:5000 dilution of anti-rabbit or anti-mouse horseradish-peroxidase-conjugated antibody for 1.5 h. Following incubation, the membranes were washed extensively with 1× TBST. Immunoreactive bands detected using ECL reagent (Advansta, USA) were analyzed using Image Lab (Bio-Rad, USA).

Screening of antiviral drugs
The cytotoxicity of the three urolithins was determined by CCK-8 assay in RD cells. The results showed that the maximum nontoxic concentration of UroA, UroB, and UroC was 50, 25, and 25 µM, respectively (Fig. 1A).
RD cells were infected with EV71 and maintained with UroA, UroB, and UroC at nontoxic concentrations (25 µM) in cell culture medium to detect the antiviral effects of the three urolithins. The total protein of the cells was extracted at 24 h postinfection. Expression of the EV71-VP1 protein was detected by Western blot assay. The results showed that UroA had strong antiviral effect (Fig. 1B).

UroA inhibits EV71 replication
The antiviral effects of UroA on intracellular viral RNA levels, intracellular viral VP1 protein levels, and the viral titer in the cell culture medium were further evaluated. The results showed that UroA effectively inhibited EV71 RNA replication ( Fig

UroA is an effective anti-EV71 compound
The antiviral effects of UroA were compared with those of ribavirin for further evaluation. First, a CCK-8 assay was used to evaluate the viability of RD cells incubated with a series of concentrations of ribavirin and UroA for 48 h. The CC 50 was calculated for both compounds. The results indicated that UroA is less cytotoxic than ribavirin (Fig. 3A). The anti-EV71 activity of ribavirin and UroA at different non-cytotoxic concentrations was then assessed at 48 hpi using a CPE inhibition assay (Fig. 3B and Table 1). The SI was calculated as the CC 50 /IC 50 ratio. The SI of UroA was much higher than that of ribavirin ( Table 1). The results suggest that UroA is superior to ribavirin.
Three types of antiviral effects of UroA were evaluated: prevention, treatment, and direct viricide (Fig. 4). The results showed that UroA exhibited significant anti-EV71 effects under treatment, but neither prevention nor direct viricide.

UroA promotes autophagy and apoptosis of infected cells
Autophagy and apoptosis are self-defense mechanisms of host cells during EV71 [3,6,31,35]. A Western blot assay showed that the level of LC3-II, a marker for autophagy, increased in the presence of 25 µM UroA during EV71 infection for 12 h. The level of p62, which functions as a bridge between LC3B and ubiquitinated substrates to be degraded [26], decreased for 12 h (Fig. 5A). cleaved-caspase-3, which is associated with apoptosis, also increased in this process (Fig. 5B). These results showed that autophagy and apoptosis were activated for 12 h after EV71 infection with UroA treatment.

UroA also exhibits an antiviral effect in SK-N-SH cells
EV71 infection can affect the nervous system of children [12]. We therefore examined the effects of UroA on the proliferation of EV71 in SK-N-SH cells, which are human neuroblastoma cells. The nontoxic concentration of UroA was determined by CCK-8 assay (Fig. 6A), and the anti-EV71 effect was assessed by Western blot assay (Fig. 6B). The results showed that UroA could significantly inhibit the proliferation of EV71 in SK-N-SH cells. The antiviral effects of UroA and ribavirin were also compared in SK-N-SH cells (Fig. 6C). The results showed that UroA had a stronger antiviral effect in these cells than ribavirin.

UroA has no effect on the proliferation of influenza virus
The effect of UroA on the proliferation of influenza virus was examined. A549 cells infected with influenza A virus strain A/Puerto Rico/8/34(H1N1) were treated with a nontoxic concentration of UroA for 24 h, and intracellular viral NP protein was detected by Western blot assay ( Fig. 7A and B). The result indicated that UroA does not inhibit influenza virus replication.

Discussion
EV71 belongs to the genus Enterovirus of the family Picornaviridae, and it is one of the main pathogens causing HFMD in infants. The development of antiviral drugs is a slow process, especially those intended for use in infants and young children, and safe and effective drugs against EV71 infection are urgently needed [26,27].
Ellagic acid, a natural polyphenolic compound that is widely distributed in fruits and nuts, has various biological functions. However, its low absorption efficiency restricts its application as a drug [18]. Urolithins are metabolites of ellagic acid that are also biologically active but can reach appropriate concentrations in the blood after absorption through the intestinal tract [8,23]. In the present study, UroA, UroB, and UroC were screened for their antiviral effects. The results showed that UroA had a significant antiviral effect (Fig. 1B), which was confirmed by examining viral protein expression, the levels of viral RNA, and virus titers ( Fig. 2A-C). In addition, the antiviral effect of UroA was found to be dose-dependent (Fig. 2D). The cytotoxicity and anti-EV71 activities of UroA was also investigated in terms of CC 50 , IC 50 , and SI. The results showed that UroA has better safety and stronger antiviral activity than ribavirin. Thus, it has potential as a candidate drug for treating EV71 infection ( Table 1). The functions of UroA in the prevention and treatment of infection and its direct viricidal effect on EV71 were also tested. The results showed UroA to be effective in the treatment of EV71 infection, but it had little preventative effect and no direct viricidal effect of EV71 (Fig. 4).  Table 1 The CC 50 , IC 50 , and SI of UroA and ribavirin Data shown are the mean ± SD for three independent experiments. a CC 50 , cytotoxic concentration required to reduce the viability of uninfected RD cells by 50% determined by CCK-8 assay b IC 50 , inhibitory concentration required to reduce the cytopathic effect (CPE) caused by EV71 MOI = 0.1) by 50% determined by CPE inhibition assay c SI, selectivity index, the CC 50 /IC 50 ratio Viral infection induces different cellular stress responses in infected cells, including autophagy and apoptosis, which are self-defense mechanisms of host cells that are activated during infection [17]. Autophagy and apoptosis induced by viral infection can have both positive and negative effects of the proliferation on the virus [3,31]. In viral infection, autophagy initially triggers an innate immune response that induces interferon production and clears aggressive viruses [20]. However, the autophagy induced by EV71 provides support for viral replication in vitro and in vivo [13]. In  the present study, the effects of UroA on the autophagy and apoptosis of EV71-infected cells were examined. The results revealed that LC3-II levels increased in the presence of UroA. In addition, the level of p62, a selective autophagy receptor [4], decreased at 12 h and then returned to normal at 24 h. (Fig. 5A). EV71 induces cell apoptosis by activating caspase-3 [14]. However, virus-infected cells are eliminated through apoptosis to prevent the generation of progeny virions and inhibit the proliferation of the virus [9,22]. As shown in Fig. 5B, after 12 h, the level of cleaved-caspase-3 was higher and that of the EV71-VP1 protein was significantly lower in treated group than in infected control cells.
After the breakdown of infected cells during treatment with UroA, the surviving cells continued to grow. Accordingly, the change in expression of cleaved-caspase-3 protein was the opposite at 24 h. The results showed that UroA promoted cell autophagy and apoptosis for 12 h after EV71 infection (Figs. 5A and B), which might facilitate viral clearance, as suggested previously [10]. This may also be the reason why there was significantly more autophagy and apoptosis of UroA treated cells than of infected control cells at 12 h after EV71 infection.
Some clinical case reports have shown that EV71 infection causes nerve damage, and the replication of EV71 in   [12,15,16,33]. In the present study, the effects of UroA on the proliferation of EV71 in SK-N-SH cells were examined. The results showed that UroA could significantly inhibit the proliferation of EV71 (Fig. 6B), and the antiviral effects of UroA were superior to those of ribavirin in SK-N-SH cells ( Fig. 6A and C).
We also tested the effect of UroA on influenza virus infection. A Western blot assay showed that UroA did not inhibit influenza virus replication (Fig. 7A and B), suggesting that UroA does not have a broad-spectrum antiviral effect. However, the effect of UroA on the proliferation of other viruses still needs to be tested.
In conclusion, UroA exerted more-potent antiviral activity against EV71 in vitro and exhibited less cytotoxicity than ribavirin. The anti-EV71 mechanism of UroA may be related to the promotion of the autophagy and apoptosis of infected cells. Therefore, UroA could be used as a potential drug candidate and is worthy of further study.