Isochlorogenic acid C prevents enterovirus 71 infection via modulating redox homeostasis of glutathione

www.nature.com/scientificreports OPEN Received: 15 June 2017 Accepted: 13 November 2017 Published: xx xx xxxx Isochlorogenic acid C prevents enterovirus 71 infection via modulating redox homeostasis of glutathione Zeyu Cao, Yue Ding, Liang Cao, Gang Ding, Zhenzhong Wang & Wei Xiao Enterovirus 71 (EV71) is a key pathogen of hand, foot and mouth disease (HFMD) in children under 6 years of age. The antiviral potency of antioxidant isochlorogenic acid C (ICAC) extracted from foods was evaluated in cellular and animal models. First, the cytotoxicity of ICAC on Vero cells was investigated. The viral plaques, cytopathic effects and yield induced by EV71 infection were obviously reduced by ICAC, which was consistent with the investigation of VP1 transcripts and protein expression. Moreover, the mortality, weight loss and limb paralysis of mice caused by EV71 challenge were remarkably relieved by ICAC injection, which was achieved through decreases in the viral load and cytokine secretion in the mouse brain. Further biochemical assays showed that ICAC modulated several antioxidant enzymes involved in reduced and oxidized glutathione (GSH and GSSG) homeostasis, including glutathione reductase (GR), glutathione peroxidase (GPX), and glucose-6phosphate dehydrogenase (G6PD), resulting in restoration of the GSH/GSSG ratio and reactive oxygen species (ROS) level. Finally, the antiviral effects of ICAC were dose-dependently disrupted by BSO, a biosynthesis inhibitor of GSH. This study indicated that ICAC acted as an antioxidant and prevented EV71 infection by modulating the redox homeostasis of glutathione. Human enterovirus 71 (EV71), which belongs to the genus Enterovirus, has been confirmed as a critical pathogen of hand, foot and mouth disease1. EV71 infection can cause severe symptoms, such as pulmonary oedema and brainstem and cerebellar encephalitis, which can lead to respiratory failure and death2–5. Cytokines in tissues, such as tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1), can be induced by an excess viral load, resulting in tissue damage and chronic inflammation6, which play key roles in the pathogenicity and severity of EV71 infection7–11. In 2015, a vaccine against EV71 was approved as a new tool to control hand, foot and mouth disease (HFMD) outbreaks12. However, no specific chemical drug targeting EV71 has been approved. Thus, antiviral compounds must be screened for new drug development, and the mechanisms should be carefully discussed. Viral infection can lead to oxidative stress13. A previous study showed that the redox status modulated host cell susceptibility to viruses14. For example, a shift in the intracellular redox milieu towards the oxidizing end enhanced viral replication and CPE15. Interestingly, the viral infection could be reversed by supplementing with glutathione (GSH), which indicated roles for GSH in antiviral defence16. GSH plays a central role in reactive oxygen species (ROS) detoxification17. Briefly, glutathione peroxidase (GPX) catalyses the degradation of ROS coupled with the conversion of GSH to its oxidized form (GSSG). Glutathione reductase (GR) is an essential factor responsible for reducing GSSG back to GSH in the presence of NADPH, which is provided by glucose-6-phsophate dehydrogenase (G6PD)17. Upon viral infection, an imbalance in GSH redox homeostasis in host cells is observed15 due to the accumulation of ROS18, which can be reversed by exogenous supplementation of antioxidants15,16. L-buthionine sulfoximine (BSO) is a specific and selective inhibitor of γ- glutamylcysteine synthase and consequently of GSH synthesis19. Previous report20 showed BSO significantly reduced GSH level. Thus, BSO is usually used to deplete GSH in cells. State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Jiangsu Kanion Pharmaceutical Co., Ltd, Lianyungang, 222001, China. Correspondence and requests for materials should be addressed to W.X. (email: ) SCieNtifiC Reports | 7: 16278 | DOI:10.1038/s41598-017-16446-7 1 www.nature.com/scientificreports/ Figure 1. The molecular structure of ICAC and its effects on Vero cell viability. (A) The molecular structure of ICAC. (B) The compound was diluted to various concentrations as indicated in triplicate. The cytotoxicity of ICAC was determined by MTS assay after incubation with the drug for 48 h. The cell viability in DMEM without ICAC (control) was set as 100% (n = 6). All results were expressed as the means ± SEs. Asterisks indicate that the data significantly differ from the control group at the P < 0.05 level according to one-way analysis of variance. Isochlorogenic acid C (ICAC), which is a di-O-caffeoyl derivative of chlorogenic acid (CHA), is a well-known antioxidant21–23 from herbal plants21 that has revealed more potent effects than other isomers24,25. Previous reports showed that ICAC and its isomers exhibited a broad-spectrum antiviral potency against respiratory syncytial virus (RSV)26, human immunodeficiency virus (HIV)27–29, and coxsackievirus30. However, the potential effects of ICAC against EV71 are unknown and should be investigated. Only CHA (the parent nucleus of ICAC) has been reported to inhibit EV71 replication in vitro31. However, ICAC extracted from Flos Lonicerae was indicated to reverse acetaminophen-induced liver injury via modulating the GSH content32. Thus, the potential regulation of GSH metabolism should be discussed with a focus on ICAC-derived inhibition of EV71 infection. In this study, the antiviral efficacy of ICAC against EV71 was confirmed in both cellular and animal models. The compound was shown to reduce the mortality of mice upon EV71 challenge by decreasing the viral load and cytokine secretion. Further biochemical assays suggested that ICAC restored the GSH/GSSG ratio by regulating the enzymes responsible for GSH metabolism, resulting in a decreased ROS level. Taken together, the data in this study indicated that ICAC acted as an antioxidant and prevented EV71 infection via modulating GSH redox homeostasis. Results Cytotoxicity of ICAC. The molecular structure of ICAC, which is also called 4,5-O-dicaffeoylquinic acid, is displayed in Fig. 1A. To investigate the toxicity of ICAC on Vero cells, the cell viability was determined after ICAC supplementation for 48 h. The compound exhibited minor cytotoxicity at concentrations up to 250 µM (Fig. 1B, P > 0.05). However, the cell viability decreased gradually and clearly when the concentration was increased to 375 µM or more in comparison with the viability of the blank control (Fig. 1B, P < 0.05). According to the results, the median toxic concentration (TC50) of ICAC for the cells was approximately 429 µM. Antiviral effects of ICAC against EV71 in vitro. The antiviral potency of ICAC was estimated in a cellular model. As expected, the plaques and CPE caused by EV71 infection were significantly reduced by ICAC in a dose-dependent manner (Fig. 2 (...truncated)