Korean Chestnut Honey Suppresses HSV-1 Infection by Regulating the ROS-NLRP3 Inflammasome Pathway

Abstract

Herpes simplex virus 1 (HSV-1) is double-stranded DNA virus that belongs to the Orthoherpesviridae family. It causes serious neurological diseases of the central nervous system, such as encephalitis. The current U.S. Food and Drug Administration (FDA)-approved drugs for preventing HSV-1 infection include acyclovir (ACV) and valacyclovir; however, their long-term use causes severe side effects and often results in the emergence of drug-resistant strains. Therefore, it is important to discover new antiviral agents that are safe and effective against HSV-1 infection. Korean chestnut honey (KCH) has various pharmacological activities, such as antioxidant, antibacterial, and anti-inflammation effects; however, antiviral effects against HSV-1 have not yet been reported. Therefore, we determined the antiviral activity and mechanism of action of KCH after HSV-1 infection on the cellular level. KCH inhibited the HSV-1 infection of host cells through binding and virucidal steps. KCH decreased the production of reactive oxygen species (ROS) and calcium (Ca²⁺) following HSV-1 infection and suppressed the production of inflammatory cytokines by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) activity. Furthermore, we found that KCH inhibited the expression of the nod-like receptor protein 3 (NLRP3) inflammasome during HSV-1 infection. Taken together, the antiviral effects of KCH occur through multiple targets, including the inhibition of viral replication and the ROS-mediated NLRP3 inflammasome pathway. Our findings suggest that KCH has potential for the treatment of HSV-1 infection and related diseases.

Keywords: HSV-1; KCH; NF-кB; NLRP3 inflammasome; ROS; mitochondria.

Figure 1

Korean chestnut honey.

Figure 2

KCH inhibits HSV-1 and HSV-2 replication in Vero cells. The cells were infected with HSV-1 or HSV-2 for 2 h and treated with KCH (1.25, 2.5, and 5 mg/mL) or ACV (30 μmol/L) for 48 h. ACV was used as a positive control. After incubation, the virus was quantitated using a plaque assay for (A) HSV-1 and (B) HSV-2. Cytotoxicity induced by (C) HSV-1 and (D) HSV-2 was measured by the crystal violet method. Bar graph (mean ± SEM) statistical data were measured using a one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001 compared with the untreated group. ### p < 0.001; ## p < 0.01 and # p < 0.05 compared with the HSV-1-infected group.

Figure 3

KCH suppresses the infection of Vero cells by HSV-1. The cells were infected with GFP encoding HSV-1 for 2 h and treated with KCH (1.25, 2.5 and 5 mg/mL) or ACV (30 μmol/L) for 48 h. (A) GFP was detected using fluorescence microscopy and flow cytometry. Scale bar = 100 μm. (B) Western blot analysis of the HSV viral proteins, gB and ICP8, and (C) quantitative analysis of the protein bands using ImageJ software. Three independent experiments were performed. Bar graph (mean ± SEM) statistical data were determined using a one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001 compared with the untreated group. ### p < 0.001; ## p < 0.01 and # p < 0.05 compared with the HSV-1-infected group.

Figure 4

Effect of KCH on the time-of-addition assay. (A) Following the entry assay. (B) Following the binding assay. (C) Following the virucide assay. Bar graph (mean ± SEM) statistical data were determined using a one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001 compared with the untreated group. ### p < 0.001; ## p < 0.01 and # p < 0.05 compared with the HSV-1-infected group.

Figure 5

KCH reduced infection of HSV-1-induced mitochondrial stress in Vero cells. Intracellular ROS and Ca²⁺ were measured using (A) DCF-DA and (B) Fluo-4AM. Mitochondrial ROS and Ca²⁺ were determined using the (C) MitoSoX and (D) Rhod-2 staining methods. (E) Mitochondrial membrane potential (MMP, Δψm) was measured using DioC6(3). All fluorescence values were detected by flow cytometry. (F) CAT and (G) SOD activities were measured using an antioxidant enzyme assay kit. (H) The expressions of MFN2 and VDAC were detected via Western blot analysis. Bar graph (mean ± SEM) statistical data were determined using a one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001 compared with the untreated group. ### p < 0.001; ## p < 0.01 and # p < 0.05 compared with the HSV-1-infected group.

Figure 6

KCH decreased infection and HSV-1- induced phosphorylation of NF-κB and inflammatory cytokines. Vero cells were infected with HSV-1 for 2 h and treated with KCH (1.25, 2.5, and 5 mg/mL) or ACV (30 μmol/L) for 48 h. After the reaction, (A) staining for p-NF-κB determined via the IF method, and also (B) cytosol and nuclear isolation via the nuclear isolation kit for the expression of p-NF-κB were performed. Scale bar = 100 μm (C) levels of TNF-α, IL-6, and IL-1β were measured using an ELISA kit. Bar graph (mean ± SEM) statistical data were calculated using one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001 compared with the untreated group. ### p < 0.001 and ## p < 0.01 compared with the HSV-1-infected group.

Figure 7

KCH suppresses infection of HSV-1-induced expression of NLRP3 inflammasome. (A) Expression of the NLRP3 inflammasome, including ACS, caspase-1, and IL-1β, was analyzed via Western blot. (B) Quantification of protein bands using the ImageJ software. Bar graph (mean ± SEM) statistical data were determined using one-way analysis of variance with Tukey’s post hoc test. *** p < 0.001; ** p < 0.01 and * p < 0.05 compared with the untreated group. ## p < 0.01 and # p < 0.05 compared with the HSV-1-infected group.

Figure 8

Antiviral mechanism of KCH upon HSV-1 infection in Vero cells. KCH attenuates viral infection by suppressing ROS-mediated NLRP3 inflammasome after HSV-1 infection.