Combined Antiviral and Cytoprotective Action of Rosmarinic Acid Against EV-A71 Infection: A Potential Therapeutic Strategy

Abstract

Enterovirus A71 (EV-A71), a major etiological agent of hand-foot-mouth disease, can cause severe neurological complications. However, the mechanisms underlying EV-A71-induced cell damage and potential therapeutic strategies remain inadequately understood. Here, we investigated EV-A71 replication dynamics and associated cytopathic effects in nine distinct cell lines, including epithelial, neuronal, immune, and other cell types. Cell viability, membrane integrity, and energy metabolism were assessed using Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH), and adenosine triphosphate (ATP) assays. The antiviral activity of rosmarinic acid (RA), a natural polyphenol, was evaluated by plaque reduction, qPCR, and Western blot. EV-A71 exhibited cell-type-specific replication and cytotoxicity patterns. RA significantly preserved cell viability, reduced LDH release, maintained ATP levels, and suppressed IL-6 expression. Mechanistically, RA inhibited viral replication by downregulating VP1 expression and viral RNA levels. Molecular docking indicated strong binding of RA to the hydrophobic pocket of VP1, potentially disrupting virus-host interactions. Collectively, these findings highlight RA's combined antiviral and cytoprotective potential, supporting its candidacy as a therapeutic agent against EV-A71 infection.

Keywords: Enterovirus A71; cell damage; inflammatory response; rosmarinic acid; viral replication.

Figure 1

Rate of multiplication of EV-A71 infection in multiple host cell lines. Intracellular EV-A71 mRNA levels were quantified in (A) RD, (B) HeLa, (C) Vero, (D) HUVEC, (E) Hep-2, (F) BHK, (G) NSC34, (H) bEnd.3, and (I) RAW264.7 cells at 1, 12, 24, 36, and 48 h post-infection (hpi). Cells were seeded in 24-well plates and infected with EV-A71 at a multiplicity of infection (MOI) of 1. Total RNA was extracted at each time point, and viral RNA levels were measured by quantitative real-time PCR (qPCR). Relative viral RNA abundance was calculated using the 2^−ΔΔCt method, normalized to GAPDH expression and compared to MOCK (uninfected control) cells (n = 4).

Figure 2

Effects of EV-A71 infection on host cell viability and cytotoxicity across multiple MOI gradients. (A) RD, (B) HeLa, (C) RAW264.7, (D) Vero, (E) HUVEC, (F) bEnd.3, and (G) NSC34 cells were infected with EV-A71 at increasing multiplicities of infection (MOI 0, 0.5, 1, 2, 5, and 10). The MOI = 0 group received only virus dilution buffer and served as the uninfected control. After 48 h, cell viability and damage were assessed using the CCK-8 assay, intracellular ATP quantification, and LDH release assay. Panels A–G present the respective results for each cell line across different MOI conditions. Data are expressed as mean ± SD of three independent experiments. Statistical significance was evaluated using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Rosmarinic acid improves the viability of infected cells. (A) Chemical structure of rosmarinic acid (RA). (B) Cytotoxicity curves for RA and ribavirin (RBV) in HeLa cells after 48 h treatment. Compounds were tested in twofold serial dilutions to determine the half-maximal cytotoxic concentration (CC₅₀). Data are presented as mean ± SD (n = 6). (C–E) Experimental overview: HeLa cells were infected with EV-A71 (MOI = 1) and treated simultaneously with graded concentrations of RA (RA-H: 62.5 µM; RA-M: 31.3 µM; RA-L: 15.6 µM) or RBV (15.6 µM). MOCK refers to the uninfected, untreated group. Cell viability was assessed via CCK-8, intracellular ATP levels, and LDH release after 48 h. ** p < 0.01, *** p < 0.001.

Figure 4

Time-dependent effects of RA on mRNA expression of inflammatory cytokines in EV-A71-infected cells. qPCR analysis of IL-6, IL-1β, and TNF-α mRNA expression at 12, 24, 36, and 48 h post-infection (hpi). Cells were infected with EV-A71 at a multiplicity of infection (MOI) of 0.5 and treated with high-dose RA (RA-H: 62.5 µM) or ribavirin (RBV: 15.6 µM). mRNA levels were normalized to GAPDH and calibrated against the MOCK group (uninfected and untreated), which was set to 1. Data are presented as bar graphs (mean ± SD, n = 6) for each cytokine and time point. Statistical significance was assessed by one-way ANOVA; *** p < 0.001.

Figure 5

Effects of rosmarinic acid on EV-A71 viral RNA, protein expression, and plaque formation. (A) qPCR analysis of EV-A71 viral RNA levels in HeLa cells 48 h post-infection following treatment with RA-H (62.5 µM), RA-M (31.3 µM), RA-L (15.6 µM), or RBV (15.6 µM). MOCK: uninfected, untreated control. EV-A71: infected, untreated group. Data are expressed as mean ± SD (n = 6). (B) Time-course analysis of EV-A71 mRNA expression at 12, 24, 36, and 48 hpi in HeLa cells treated with RA-H or RBV. EV-A71 group: black circles; RA-H group: blue squares; RBV group: red triangles (n = 3). (C) Viral titers in the infected HeLa cells treated with RA-H or RBV were measured by TCID₅₀ assay in RD cells at 48 hpi. Results are shown as mean ± SD (n = 3). (D) Plaque reduction assay performed in Vero cells. Cells were infected with EV-A71 and overlaid with agar containing RA (62.5, 31.3, or 15.6 µM) or RBV (15.6 µM). Both RA and RBV treatments led to reduced plaque formation. (E) Western blot analysis of VP1 protein levels in HeLa cells 48 h post-infection and treatment. GAPDH served as the loading control. All experiments were conducted at MOI = 0.5. Statistical significance was determined by one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. EV-A71 group.

Figure 6

Binding mode of RA with key residues in the binding pocket of EV-A71 VP1 protein. The left panel shows the overall 3D structure of the VP1 protein (green) with the ligand-binding region highlighted. The right panel provides a magnified view of the binding pocket, illustrating the interaction between rosmarinic acid (RA, cyan) and key residues TYR-201, ASN-228, MET-225, and GLY-223 (magenta). Hydrogen bonds are shown as dashed lines, and their bond lengths (in Å) are indicated. RA forms three hydrogen bonds with VP1, and the interaction with MET-225 is hydrophobic in nature, with a distance of 3.8 Å, exceeding the conventional hydrogen bond threshold. Therefore, this interaction is classified as hydrophobic rather than a hydrogen bond.