Hand, foot, and mouth disease (HFMD) stands as a prevalent childhood infectious ailment primarily attributed to more than 20 enteroviruses. The prognosis for HFMD is generally optimistic, characterized by self-limiting symptoms that typically abate within a week (Zhang et al., 2022). However, certain neurological complications such as encephalomyelitis, brainstem encephalitis, and aseptic meningitis (Gonzalez et al.,2019) can rapidly lead to neurogenic pulmonary edema (Wang et al.,2019) and, in severe cases, even death. Enterovirus type 71 (EV71)is the most common viral culprit behind these severe complications (Yang et al., 2022). Currently, no clinically effective drugs exist, and symptomatic treatment remains the primary approach (Liu et al., 2015). HFMD has experienced multiple outbreaks worldwide, posing significant public health concerns and imposing substantial life safety risks and economic burdens on many countries (Solomon et al., 2010).

Chinese medicine considers virus infections as the invasion of cold and malevolent energy into the body. When the body’s defense is weak, this energy can easily invade. However, if the body’s defense is strong, it can resist such invasion (Li et al., 2020). Traditional Chinese medicine, with a history spanning five thousand years, has been routinely used to treat pandemics and endemic diseases, forming a comprehensive theoretical system for the prevention and treatment of deadly epidemics, referred to as “plagues” in ancient China (Qi and Tang, 2021). Traditional Chinese medicine is widely employed for antiviral purposes (Wu t al. 2021) by restoring the body’s overall balance to counteract the harmful effects of viral infections (Chen and Ye, 2022). Numerous traditional Chinese medicines have been proven to possess antiviral properties (Guan et al., 2020; Kang et al., 2021; Lee et al., 2021; Cui et al., 2022) capable of directly acting on viruses and stimulating the immune system to induce interferon production, thereby indirectly inactivating viruses (Huang et al., 2022).

Mulberry leaves (ML), derived from the Moraceae plant, are known for their antibacterial and antiviral effects (Chen et al., 2021). However, their potential anti-EV71 virus effect remains unexplored. This study aimed to investigate the potential of mulberry leaves in EV71 virus infection by predicting key target genes and signaling pathways involved in ML-mediated antiviral mechanisms through network pharmacology, bioinformatics, in vitro experiments, and medical statistics.

HFMD is a highly contagious viral illness primarily caused by enteroviruses, notably coxsackievirus A16 (CV-A16) and enterovirus 71 (Aswathyraj et al., 2016; Xing et al., 2014). Among them, EV71 is associated with the most severe symptoms and highest mortality rate (Yang et al., 2009; Zhang et al., 2010; Li et al., 2017). Unfortunately, no specific drugs are currently available for the treatment of these viruses (Ng et al., 2015). Extensive research efforts have been dedicated to developing anti-EV71 drugs over the past 50 years (Wang et al., 2023). Traditional Chinese medicine has revealed promising monomers, such as glycyrrhizic acid, baicalin, indigo root, Quercetin, kaempferol, and apigenin, which possess anti-EV71 effects (Tsai et al., 2011; Yang et al., 2012; Wang et al., 2013; Zhang et al., 2014; Li et al., 2015; Wang et al., 2016; Yao et al., 2018; Dai et al., 2019). Some of these components are already utilized in clinical practice. While Western medicines exhibit satisfactory inhibitory effects on EV71 replication, their mechanisms often target a single aspect and carry a risk of carcinogenesis (Friedman et al., 2009; Brambilla et al., 2010). In contrast, traditional Chinese medicine embraces a holistic approach, exerting antiviral effects through overall balance. Chinese medicine is distinguished by its multifaceted components and targets, affordability, low toxicity, and minimal cancer risk (Csikós et al., 2021; Zhu et al., 2022). Its antiviral properties are achieved through antioxidant, immune regulatory effects (Colunga Biancatelli et al., 2020), inhibition of viral-induced inflammatory responses (Lim et al., 2018; Saeedi-Boroujeni and Mahmoudian-Sani, 2021), and suppression of virus replication by inhibiting oxidative stress (Chen et al., 2022).

ML, a traditional Chinese medicine, has significant medicinal value and has shown efficacy in treating metabolic disorders such as diabetes, dyslipidemia, obesity, atherosclerosis, and hypertension (Zhang et al., 2022; Cheng et al., 2022). Furthermore, ML has been identified as a potential treatment option for viral diseases (Pronin et al., 2021). Our research was primarily dedicated to unraveling the antiviral attributes of mulberry leaves (ML), identifying multiple active constituents with potent antiviral effects. An extensive body of research has effectively showcased the antiviral prowess of compounds such as Quercetin, kaempferol, and beta-carotene (Sheehan et al., 2012; Ding et al., 2018; Shen et al., 2021; Chen et al., 2022; Shokry et al., 2023). Furthermore, stigmasterol has demonstrated its multifaceted potential, encompassing antioxidant, antiviral, antifungal, antibacterial, and anticancer properties, achieved through immune regulation and anti-inflammatory mechanisms (Petrera et al., 2014). Iristectorigenin A possesses antioxidant and anti-inflammatory effects (Al-Qudah et al., 2015; Lim et al., 2017). Moracin D and Moracin E exhibit antioxidant effects and hold substantial medicinal value in antiviral and anticancer research (Yoon et al., 2021; Mohan Kumar et al., 2022). These active constituents of ML exert antiviral effects through diverse mechanisms, highlighting the potential of ML in countering the EV71 virus. Furthermore, the collective antiviral impacts of these active constituents might demonstrate synergistic properties.

By examining 29 common targets shared by ML and EV71, we predicted the potential function of these genes in ML anti-E71 virus through network pharmacology studies. A number of these genes are involved in oxidative stress, inflammation, vascular permeability, and immune function. For example, AKT1 activation promotes cell proliferation and suppresses cell apoptosis, making it a significant participant in EV71’s immune-inflammation mechanism (Shi et al., 2013). IL-6, IL-1B, and TNF-α exhibit immunomodulatory and pro-inflammatory effects (Luo et al., 2019; Yi et al., 2020). MAPK1 is crucial in the inflammatory response (Xu et al., 2023). Inhibiting CASP3 activity reduces EV71 virus protein expression and replication and can trigger pyroptosis as an alternative to apoptosis, thereby hindering EV71 infection (Song et al., 2018). HMOX1 contributes to the host’s resistance to the virus through the oxidative stress defense system (Zhang et al., 2022). In summary, these target genes of ML employ diverse mechanisms to inhibit EV71 virus infection and exert their influence at various stages of disease development.

To elucidate the mechanism of ML against the EV71 virus, we conducted GO enrichment analysis on the key targets of Quercetin, the primary anti-EV71 component of ML, and performed KEGG enrichment analysis to predict associated signaling pathways responsible for the antiviral effect. The KEGG database encompasses diverse biological domains, furnishing extensive data and insights spanning genomics, proteomics, metabolomics, and more. This encompassing repository facilitates the identification of promising targets associated with drugs and ailments, along with pertinent signaling pathways and biological functions (Lu, et al., 2020). Rigorous procedures have been implemented to ascertain the dependability of our KEGG enrichment analysis outcomes. In this process, data sources are carefully reviewed, statistical significance is evaluated, cross-validation is carried out thoroughly, and the biological relevance of the data is carefully interpreted. The findings suggest a potential relationship between Quercetin and the NF-κB signaling pathway. This pathway, comprising canonical and non-canonical pathways, plays a vital role in various biological processes, including the regulation of B and T-cell immunity (Lu et al., 2021). Many viruses activate or evade antiviral immune responses through this pathway (Struzik and Szulc-Dąbrowska, 2019; Khatiwada et al., 2017). EV71 triggers NLRP3 inflammasome activation via the NF-κB pathway, and inhibiting this pathway aids the host’s defense against EV71 infection in the central nervous system (Gong et al., 2022). Severe EV71 infection is associated with significantly elevated TNF levels (Duan et al., 2014; Sun et al., 2018). The TNF-α-mediated NF-κB pathway is essential for inflammatory responses, and the 2C protein of EV71 promotes NF-κB activation via TNF-α. The activation of NF-κB can be triggered by the TNF-α-related factor 2, the MEK kinase 1, the IKKα, or the IKKβ (Zheng et al., 2011).

To validate the antiviral effect and mechanism of ML, we conducted in vitro experiments to verify the antiviral activity of Quercetin, the primary active compound derived from ML. Immunofluorescence analysis was performed to assess the expression of essential proteins in the NF-κB signaling pathway. The results demonstrated that Quercetin exhibited significant inhibition against the EV71 virus. Pretreatment of the virus with Quercetin weakened its toxicity and directly killed it, leading to its antiviral effect in cell experiments. Immunofluorescence analysis revealed reduced protein levels of p-NF-κB p65, TNF-α, and IL-1β in the quercetin-treated group compared to the EV71 group. The NF-κB signaling pathway is crucial in the inflammatory response (Oeckinghaus et al., 2011). TNF-α activates the non-canonical NF-κB pathway, leading to an inflammatory response (Yu et al., 2020). Downstream kinase IKKα is activated by TNF-α, promoting NF-κB phosphorylation (Sun, 2011). IL-1β induces NF-κB inhibitor phosphorylation, translating NF-κB to the nucleus and transcription of cytokine and chemokine genes (Cheng et al., 2019). The mammalian NF-κB family comprises five distinct proteins: p50, p52, p65 (also recognized as RelA), RelB, and c-Rel. These NF-κB family constituents engage in diverse combinations of homo- and heterodimeric associations with each other, culminating in the formation of biologically active protein complexes. Among these, the p65-p50 heterodimer is the prevailing form within cellular contexts (Chen, et al., 1998). Notably, the p65 protein garners substantial attention among the five NF-κB family members due to its extensive scrutiny. This heightened focus is partly attributable to its role as an activating component within the p65-p50 heterodimeric complex (Lecoq, et al., 2017). Upon infection with a pathogen, the activation of the predominant p65-p50 heterodimer of NF-κB occurs, leading to the translocation of p65 and p50 into the nucleus (Medina et al., 2002). Additionally, the phosphorylation of IκBα, mediated by IκB induced by TNF-α, results in its ubiquitination. This process ultimately leads to the nuclear translocation of NF-κB and the regulation of target gene transcription (Papa et al., 2009). The nuclear translocation of NF-κB p65 and NF-κB p50 has been observed in the mouse liver following infection with the Dengue virus (DENV) (Sreekanth et al., 2020). This study investigated p65 and phosphor-p65 in the cytoplasm and nucleus of whole-cell lysates. Separate studies on these fractions would provide more insight into Quercetin’s role in regulating nuclear translocation of p65. The inhibitory effect of Quercetin on the NF-κB signaling pathway and its ability to decrease the synthesis of pro-inflammatory cytokines, specifically TNF-α and IL-1β, as observed in the study conducted by (Bezzi et al., 2001), aligns with the findings of the current investigation, suggesting that this mechanism may contribute significantly to Quercetin’s antiviral activity against EV71.

Moreover, our investigation into the conduction of the MAPK signaling pathway unveiled that Quercetin’s impact on this pathway parallels that of the NF-κB signaling pathway. Notably, the inhibitory effect of Quercetin on both pathways follows a dose-dependent pattern. The MAPK signaling pathway employs at least three activation routes to transmit extracellular signals to the nucleus. These include the classical MAPK pathway, MAPK/ERK, the JNK/MAPK signaling pathway, and the p38/MAPK signaling pathway. The MAPK/ERK signaling pathway is activated by signals from cell surface receptors like receptor tyrosine kinases (RTKs) or G protein-coupled receptors (GPCRs) (Delire and Stärkel, 2015). The process of ERK activation entails phosphorylation by activated RAF, subsequently activating MEK, resulting in the direct phosphorylation of ERK (Liu et al., 2018). It has been documented that garlic extract inhibits reticuloendotheliosis virus (REV) replication by suppressing ERK expression (Wang et al., 2017). DNA viruses, including Herpes Simplex Virus 1 (HSV-1), exploit the MAPK/ERK pathway for intercellular dissemination (DuShane and Maginnis, 2019; Watanabe et al., 2021). JNK, also recognized as stress-activated protein kinase (SAPK), represents a subfamily within the canonical MAPK signal transduction cascade (Zeke, et al., 2016; Pua, et al., 2022). JNK proteins promptly respond to various cellular stimuli, including inflammatory cytokines, growth factors, ultraviolet radiation, bacterial and viral infections, heat shock, and osmotic and genotoxic stress (Kusumaningrum, et al., 2018). The promotion of Duck Plague Virus (DPV) proliferation has been documented through the inhibition of the immune interferon (IFN) signaling pathway and inflammatory pathways by JNK, as reported by Wu, et al. (2022). Furthermore, the activation of JNK plays a crucial role in Varicella-Zoster Virus (VZV) protein expression and replication, as highlighted by Kurapati et al. (2017). p38 mitogen-activated protein kinases form a class of evolutionarily conserved serine/threonine kinases. They function as intermediaries, connecting extracellular signals to intracellular processes that regulate a multitude of cellular functions (Falcicchia, et al., 2020; Romero-Becerra, et al., 2020). Various extracellular stimuli can phosphorylate p38 through the classical MAPK kinase (MAP3K)–MAP kinase kinase (MKK) pathway. Phosphorylated p38, in turn, activates an array of transcription factors, protein kinases, cytoplasmic and nuclear proteins, and substrates downstream of this phosphorylation encompass the regulation of inflammatory responses, cell differentiation, apoptosis, and more (Yao, et al., 2020; García-Hernández, et al., 2021; O'Neil, et al., 2018). Indeed, recent studies have demonstrated that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce the activation of p38/MAPK, resulting in an upregulation of proinflammatory cytokines and an enhanced replication of the virus (Bouhaddou et al., 2020). It has been observed that inhibiting the p38/MAPK signaling pathway can effectively mitigate the Influenza virus (IV) replication and the excessive production of proinflammatory mediators (Yang et al., 2022). Additionally, the infection of Newcastle Disease Virus (NDV) has been shown to induce the activation of p38/MAPK/Mnk1 signaling, facilitating the efficient synthesis of viral proteins (Zhan et al., 2020). Previous research has indicated that inhibiting the MAPK signaling pathway has significant implications in the context of viral infections (Sun et al., 2023). The infection caused by EV71 is closely linked to the signaling pathways of JNK and p38 MAPK, which in turn activate the MAPK pathway, increasing virus production and releasing proinflammatory cytokines (Peng et al., 2014). EV71 triggers the activation of the ERK MAPK pathway through the induction of c-Src-mediated epidermal growth factor receptor (EGFR) activation (Wong et al., 2005; Tung et al., 2011). Inflammation occurs as a result of the activation of the MAPK pathway downstream of the NF-κB signaling pathway (Roth Flach et al., 2015; Ramalingam et al., 2020). In contrast, an overabundance of phosphorylation in downstream proteins of MAPK has the potential to induce the release and nuclear translocation of NF-κB, thereby intensifying the inflammatory response (Schulze-Osthoff et al., 1997; Papa et al., 2009; Sreekanth et al., 2020; Liu et al., 2022). Our research findings underscore that Quercetin orchestrates the modulation of the inflammatory response through these three MAPK pathways. This influence encompasses the phosphorylation of p65 by inhibiting the MAPK signaling pathway. Quercetin further curtails the inflammatory response by inhibiting the NF-κB signaling pathway, thus presenting a multifaceted mechanism for mitigating inflammation.

In conclusion, this study employed network pharmacology and in vitro experiments to investigate the mechanism of action of ML against EV71 virus infection. The findings reveal that ML exerts a substantial pharmacological effect against EV71 virus infection through a multi-component, multi-target, and multi-pathway approach. Quercetin, as the primary active component of ML, plays a pivotal role in inhibiting EV71 virus infection by targeting the NF-κB signaling pathway. These results provide valuable insights into the therapeutic mechanism of ML in the treatment of EV71 virus infection.