Myricetin possesses the potency against SARS-CoV-2 infection through blocking viral-entry facilitators and suppressing inflammation in rats and mice

Abstract

Background

Myricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood.

Purpose

The aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo.

Methods

The inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models.

Results

The results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC₅₀ 55.18 μM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice.

Conclusion

Our findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

Graphical abstract

Introduction

Coronavirus disease 2019 (COVID-19) is a pandemic pulmonary infection caused by the etiological agent SARS-CoV-2 since 2019 (Dong et al., 2020). Millions of people have suffered from fever, fatigue, and dyspnea or even lost their lives due to infection with this virus (Wang et al., 2020). SARS-CoV-2 variants continue to emerge, with the World Health Organization (WHO) designating several variants of concern including the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529), which were classified based on serotype and genotype (Cameroni et al., 2022). These variants exhibited increased transmissibility and/or virulence, and reduced sensitivity to antibody neuralization (Souza et al., 2021). Thus, the identification of novel anti-COVID-19 agents remains in high demand.

SARS-CoV-2 enters host cells through the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. The S protein “priming process” involves cleavage of the S protein by transmembrane protease serine protease 2 (TMPRSS2) and TMPRSS4, which are located on the host cell surfaces, to generate the S1 and S2 subunits (Hoffmann et al., 2020). During the entry process, S1 binds to ACE2, and S2 facilitates subsequent fusion membranes (Jackson et al., 2022). Therefore, the S protein is an effective target for antiviral drugs against SARS-CoV-2.

Several antiviral drugs have been used to treat COVID-19, among which remdesivir was the first to be approved by the Food and Drug Administration in the United States of America (Bravo et al., 2021). Recently, molnupiravir has been used in England to treat mild-to-severe COVID-19 in adult patients via targeting of the viral RNA-dependent RNA polymerase (RdRp) (Kabinger et al., 2021), and the anti-inflammatory drugs dexamethasone, lopinavir, chloroquine or hydroxychloroquine have also been reported to be effective in some COVID-19 patients (Shahsavarinia et al., 2021). However, clinical sample sizes are too small to judge the efficacy and safety of these drugs for COVID-19 treatment (Cao et al., 2020), and side effects such as worsening or respiratory failure, multiple-organ-dysfunction, diarrhea, renal impairment, hypotension, and kidney injury occurred often. To mitigate the pandemic, new drugs against SARS-CoV-2 infection are urgently needed.

Previously, myricetin ((3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) showed to be an inhibitor of SARS-CoV-2 Mpro and 3CL-pro enzymatic activity (Kuzikov et al., 2021; Xiao et al., 2021). However, its antiviral mechanism and protective value on SARS-CoV-2 infection have not yet been comprehensively understood.

Materials and methods

Source and purity of myricetin

The myricetin was obtained from Macklin Inc. (Shanghai, China) with a minimum purity of 97%.

Cell viability assay

Vero-E6 or Huh-7 cells were seeded in a 96-well plate at a density of 20,000 cells/well and incubated at 37 °C in 5% CO₂ for 24 h. After that, different concentrations of myricetin (Macklin Inc., Shanghai, China) ranging from 15.63 to 1000 μM were added into individual wells, and the cells were further incubated for 72 h. Control wells were treated with medium alone. Cell viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazolium bromide (MTT) assay. Briefly, 10 μl of MTT solution (Cat # A600799-0001; Sangon Biotech, Shanghai, China) was added to each well and incubated for an additional 4 h. The medium was then removed, and 150 μl of methyl sulfoxide (DMSO) was added to each well. The absorbance (A) of each well was measured at 490 nm using a microplate reader. The cell viability of myricetin in Vero-E6 or Huh-7 cells was calculated as 100% × (A _myricetin – A _control)/A _control. The concentration of myricetin that reduced cell viability by 50% in Vero-E6 or Huh-7 cells was assessed by 50% cytotoxic concentration (CC₅₀). Meanwhile, the cytotoxicity of myricetin was also assessed in HEK293, THP1, or A549 cells using the MTT assay with concentrations ranging from 3.13 to 400 μM and stimulation for 24 h.

SARS-CoV-2 infection assay

Vero-E6 or Huh-7 cells were incubated at 37 °C in 5% CO₂ for 24 h in a 96-well plate at a density of 20,000 cells/well. After that, clinical isolated SARS-CoV-2 virus (GenBank accession no. MT123290.1) at 50 median tissue culture infective dose (TCID₅₀) of SARS-CoV-2 was used to infect the cells for 1 h, with plates rocked every 15 min. Subsequently, 2-fold dilutions of myricetin ranging from 1.95 to 31.25 μM were added to individual wells. Myricetin was maintained with the virus inoculum during the 72 h incubation period. Cells were stained with MTT for 1 h. The inhibitory effects of viral replication were determined by the 50% effective concentrations (EC₅₀) of myricetin that inhibit viral replication by 50% in Vero-E6 or Huh-7 cells. The CC₅₀ was tested by the concentration of myricetin that reduced cell viability by 50%. The selectivity index (SI) was calculated by dividing CC₅₀ by EC₅₀ (Indrayanto et al., 2021). A high SI indicated that the compounds were able to inhibit viral replication without having a cytotoxic effect on the host cells.

Molecular docking of myricetin to SARS-CoV-2 and ACE2

The protein crystal structures of ACE2 and SARS-CoV-2, including receptor binding domain (RBD) (RBD^WT, a virus that differs from the Wuhan-Hu-1 strain), RBD^N501Y, RBD^N439K, RBD^Y453F, and S-D614G were obtained from the PDB (Table 1 ) for virtual screening, and nonstandard residues were removed. The myricetin coordinates were downloaded from PubChem and docked with the S proteins and ACE2 in Auto Dock (Scripps).

Table 1.

The affinity of target proteins and myricetin via molecular docking.

No.	Protein	PDB	Affinity (kcal/mol)
1	ACE2	2AJF	-7.74
2	Spike	2AJF	- 5.42
3	S-D614G	6XS6	-7.19
4	RBDN501Y	7MJG	-5.78
5	RBDN439K	7L0N	-6.85
6	RBDY453F	7EKH	-6.31

Bilayer interferometry (BLI) assay

To analyze the role of myricetin in the intermolecular interaction between WT and mutant SARS-CoV-2 RBD and the ACE2 protein, a FortéBio Octet® RED96 BLI system (Octet RED96, FortéBio, Fremont, CA, USA) was applied. Fc-tagged RBD^WT protein was immobilized onto Super Streptavidin (SSA) biosensors (FortéBio), and each His-tagged mutant and hACE2 were immobilized onto Ni-NTA biosensors (FortéBio). Experiments were run at 25 °C, and 1000 rpm. 6 diluted myricetin solutions including 3.13, 6.25, 12.5, 25, 50 and 100 μM were dissolved in 1 × PBS, and the binding kinetics including the KD (equilibrium dissociation constant), Kon (association rate constant), Kdis (dissociation rate constant) and R² values were reported. KD is calculated by dividing the kdis value by the kon value (Orthwein et al., 2021).

Immunocytochemistry (ICC) and confocal microscopy

The plasmid pLV [Exp]-Puro-EF1A>hACE2[NM_021804.3]/EGFP fused to the C-terminus (Vector Builder, Guangzhou, China) was transfected into HEK293 cells; to overexpress human ACE2 cells expressing this plasmid was defined as HEK293-hACE2-EGFP cells. RBD^WT or mutant proteins were mixed with 0, 100, 200, or 300 μM myricetin and incubated for 40 min, and Fc-tagged (Invitrogen, Carlsbad, CA, USA) or His-Tagged antibody (Cell Signaling Technology, Boston, MA, USA) added and incubated for another 120 min. The nucleic acid stain DAPI (Thermo Fisher Scientific, Waltham, MA, USA) was used to label the cell nuclei 5 min before images were acquired with the Carl Zeiss LSM780 system (Zeiss, Oberkochen, Germany) as previously described (Chu et al., 2020).

Pseudo typed lentiviral particle assay

HEK293 cells that express human ACE2 via transfection with pLV[Exp]–mCherry/NeoEF1A>hACE2[nm_02184.3] (Vector Builder, Guangzhou, China) were called HEK293-hACE2-mCherry cells. SARS-CoV-2 pseudo viruses (Vector Builder) including those expressing RBD^WT, RBD^N501Y, RBD^N439K, RBD^Y453F, and S-D614G, were mixed with 0, 100, 200, and 300 μM myricetin and incubated for 30 min, after which the pseudo viruses were incubated with HEK293-hACE2-mCherry cells for another 72 h. The colocalization of SARS-CoV-2 and ACE2 was similar to that observed in our ICC assay. Furthermore, to verify the inhibitory effect of myricetin on pseudo virus infection, flow cytometry (FCM) was also performed.

Carrageenan-induced paw edema in rats

Each group of 9 male Sprague-Dawley (SD) rats was orally administered with either 50, 100, or 200 mg/kg of myricetin or 1 mg/kg of dexamethasone (DEX). The rats in the myricetin and DEX groups were pretreated for 7 day, while those in the healthy control and model groups received a solvent as a control. On the 7th days, after 1 h of administration, the sub-plantar tissues of the right hind paw, except for those in the healthy control group, were subcutaneously injected with 5 mg/kg of carrageenan. The volume of the rats' right paws was measured at 0, 1, 2, 3, 4, and 5 h after injection.

Delayed type hypersensitivity (DTH) mouse model

BALB/c male mice in each group were given 15 mg/kg of diclofenac preparation and myricetin at doses of 50, 100, and 200 mg/kg were given orally every day from day 1 to day 9. Similarly, the mice in the healthy control and model groups were given normal saline at the same time. Ear tumefaction was induced with 1-fluoro-2,4-dinitrobenzene (DNFB) on days 0, 1 and 6 to provoke the sensitization phase of DTH. The healthy control group was treated with acetone/olive oil as reference (Ochiai et al., 2021). On day 9 and after 72 h the last DTH treatment, the degree to which the ear edema had been inhibited was determined in the auricle edema mouse model.

LPS-induced acute lung injury (ALI) mouse model

C57BL/6 mice were orally administered with myricetin at doses of 200, 300, and 400 mg/kg, while mice in the diclofenac group were orally pretreated for a week. The healthy control and model mice received solvent. To establish a mouse ALI model, LPS was administered via intratracheal instillation, 1 hour after dosing on the 6th day. 24 h later, the orbital blood and lung tissues were harvested, and the serum levels of cytokines were measured. Hematoxylin and eosin (H&E) staining was performed, and additional experiments with the lung tissues were carried out.

Real-time quantitative PCR

Total RNA was extracted from mouse lung tissues and THP1 macrophages with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was generated with Easy Script TM Reverse Transcriptase (Roche Molecular Systems, Inc, Basel, Swiss), and an ABI Real-Time Quantitative Thermal Cycler (Bio-Rad, Hercules, CA, USA) used to detect mRNA expression. The primer sequences for IL-6, NFκB p65, RIPK1, RIPK3 and other genes are provided in Supplementary Table 1 and 2.

Western blotting

Protein was extracted from lysates of THP1 macrophages. Protein concentrations were measured with a BCA Protein Assay Kit (Thermo Fisher Scientific), and the protein samples were then mixed with 4 × LDS sample buffer (Thermo Fisher Scientific). The samples were then loaded onto 4–10% gradient Bis-Tris acrylamide gels separated, and transferred onto nitrocellulose membranes (Pall, New York, USA). The membranes were incubated with anti-NFκBp65, anti-phosphorylated-NFκBp65 (p-NFκBp65), anti-RIPK1, anti-phosphorylated-RIPK1 (p-RIPK1), and anti-GAPDH (1:1000 dilution, Cell Signaling Technology) primary antibodies overnight at 4 °C before a series of tests, and HRP-conjugated secondary antibodies (1:5000 dilution, Cell Signaling Technology) were then incubated with the membranes for another 2 h at room temperature. Finally, electrochemiluminescence (ECL) reagents (Bio-Rad) were used to develop the signals of the bands.

Statistical analysis

Statistical analysis was performed, and figures were drawn with GraphPad Prism version 8 (GraphPad, San Diego, CA, USA). The student t-test was applied for two-group comparisons, and one-way ANOVA was applied for multiple-group comparisons. A p values less than 0.05 was used to indicate statistically significant.

Results

Myricetin inhibits SARS-CoV-2 replication in the infected cells

The chemical structure of myricetin was shown in Fig. 1 A. The cytotoxicity of myricetin was evaluated in Vero E6, Huh-7, and A549, THP1, and HEK293 cells by MTT assay. Myricetin showed cytotoxicity on Vero E6 or Huh-7 cells with a CC₅₀ of 247.54 μM and 81.23 μM, respectively (Fig. 1B). Moreover, myricetin also showed no marked cytotoxicity in A549, THP1, and HEK293 cells with a CC₅₀ higher than 400 μM (Suppl. Fig. 1A).

Fig. 1.

Cytotoxic effect and antiviral activity of myricetin. (A) The chemical structure of myricetin; (B) The cytotoxicity of myricetin in Huh-7 cells and Vero E6 cells after 72 h of incubation; (C) The inhibitory effects of myricetin on SARS-CoV-2 replication in Vero E6 cells; (D) The inhibitory effects of myricetin on HCoV-229E replication. Data are expressed as the mean ± SD, (n=3).

To investigate the anti-SARS-CoV-2 effect of myricetin, the Vero E6 or Huh-7 cells were infected with the SARS-CoV-2 virus at a multiplicity of infection (MOI) of 0.01 and incubated with myricetin ranging from 1.95 to 31.25 μM for 72 h. Myricetin was shown to inhibit the replication of SARS-CoV-2 with an EC₅₀ of 55.18 μM in Vero E6 cells (Fig. 1C) and suppress the replication of HCoV-229E virus with an EC₅₀ of 53.51 μM based on CPE assay and 61.52 μM based on MTT assay in Huh-7 cells. In addition, myricetin displayed a SI equal to 4.02. Meanwhile, we observed that remdesivir showed cytotoxicity on Huh-7 cells with a CC₅₀ of 108.1 μM (Suppl. Fig. 1 B). Remdesivir inhibited the SARS-CoV-2 proliferation in VeroE6 cells (Suppl. Fig. 1C) and with an EC₅₀ of 0.05617 μM in CPE assay and 0.05834 μM in MTT assay in HCoV-229E treated Huh-7 cells (Suppl. Fig. 1 D).

Myricetin binds to multiple SARS-CoV-2 viral-entry facilitators with good affinity

To verify whether myricetin is active against the SARS-CoV-2 RBD^WT and mutant proteins, we employed molecular docking. The results showed that myricetin might bind to SARS-CoV-2 RBD^WT and the mutant proteins (Fig. 2 A, Table. 1). A BLI assay was applied for further validation. Biotinylated RBD^WT protein was immobilized onto SSA biosensors, and His-tagged S-D614G, RBD^N501Y, RBD^N439K and RBD^Y453F proteins were immobilized onto Ni-NTA biosensors (Fig. 2B). Immobilized SSA or Ni-NTA biosensors were sequentially immersed in myricetin solutions with a concentration range between 100 to 3.13 μM. Myricetin was detected to bind to RBD^WT and variants protein of S-D614G, RBD^N501Y, RBD^N439K, RBD^Y453F with a KD value of 2.67 μM, 2.64 μM, 2.67 μM, 2.61 μM and 2.92 μM, respectively (Fig. 2C).

Fig. 2.

Blocking effects of myricetin via viral-entry facilitators. (A) Molecular docking of the SARS-CoV-2 spike protein binding domain and myricetin. (B) BLI assay of myricetin and SARS-CoV-2 spike protein with the RBD^WT, S-D614G, RBD^N501Y, RBD^N439K and RBD^Y453F proteins. KD is the dissociation constant and is calculated by dividing the Koff (dissociation rate constant) value by the Kon (association rate constant) value. RBD is the receptor binding domain; ACE2 is the angiotensin-converting enzyme 2; WT is wild type; S-D614G is the spike aspartic acid (D) changed into glycine (G) at 614 positions; N501Y is the asparagine broken into tyrosine at position 501 on RBD domain; N439K is a mutation from asparagine (N) to lysine (K) at the 439th amino acid on RBD domain; Y453F is the tyrosine that broken into phenylalanine substitution (Y453F) on RBD domain. The experimental data for different myricetin dilution series are shown in different colors.

Myricetin inhibits the interaction between ACE2 and SARS-CoV-2 or its variants

SARS-CoV-2 enters into the host cells via the S-protein and engages ACE2 as the host cell-entry receptor (Hoffmann et al., 2020). We firstly used immunocytochemistry to detect the expression of human ACE2 on HEK293 cells with fluorescence. By applying ELISA, we found that myricetin significantly inhibited the interactions between the RBD^WT and ACE2 protein (Suppl. Fig. 2). In addition, we visualized these interactions in HEK293-hACE2-EGFP cells and found that the binding of SARS-CoV-2 S protein to HEK293-hACE2-EGFP cell membranes was blocked by myricetin (Fig. 3 A and Suppl. Fig. 3). The SARS-CoV-2 RBD^WT and mutant proteins (RBD^N501Y, RBD^N439K, RBD^Y453F, and S-D614G) on HEK293-hACE2-EGFP cell surfaces showed a dose-dependent decrease in immunoreactive signal after myricetin treatment (Fig. 3B), indicating that myricetin might engage in a crosstalk between the host cells and wide-type and mutant SARS-CoV-2.

Fig. 3.

The inhibitory effects of myricetin on the interaction between the SARS-CoV-2 S protein and ACE2. (A) The inhibitory of myricetin on binding of the SARS-CoV-2 S protein to HEK293-hACE2-EGFP cells; (B) The inhibitory effect of myricetin on the binding of the SARS-CoV-2 S protein to HEK293-hACE2-EGFP cells; The RBD^WT or mutant proteins are co-incubated with HEK293-hACE2-EGFP cells after mixed with 0, 100, 200, or 300 μM myricetin. The nucleic acid stain DAPI is used to label the cell nuclei. (C) Inhibitory effect of myricetin on the SARS-CoV-2 lentiviral pseudo virus infectivity in HEK293-hACE2-mCherry cells; (D) The inhibitory effect of myricetin against SARS-CoV-2 lentiviral pseudo viruses (RBD^WT, S-D614G, RBD^N501Y, RBD^N439K and RBD^Y453F) by flow cytometry. (E) Inhibitory effect of myricetin on the binding of SARS-CoV-2 lentiviral pseudo viruses to HEK293-hACE2-mCherry cells; SARS-CoV-2 pseudo viruses including those expressing RBD^WT, RBD^N501Y, RBD^N439K, RBD^Y453F, and S-D614G, are mixed with 0, 100, 200, and 300 μM myricetin. The colocalization of SARS-CoV-2 and ACE2 is confirmed by confocal and FCM. Data are expressed as the mean ± SEM. *p<0.05, **p<0.01 vs. the control group.

Furthermore, to confirm whether myricetin acts as a competitive inhibitor of the S protein during the binding of SARS-CoV-2 to ACE2 in HEK293-hACE2-mCherry cells, we incubated myricetin at different concentrations with SARS-CoV-2 pseudo typed lentivirus in tubes for 0.5 h at room temperature. Then, the pre-treated myricetin-pseudo virus solutions were added to HEK293-hACE2-mCherry cells. Myricetin repressed the binding of the SARS-CoV-2 pseudo typed lentivirus to HEK293-hACE2-mCherry cells in a dose-dependent manner, as shown by a decrease in the fluorescence signal (Fig. 3C) and these results were also confirmed by confocal analysis (Fig. 3E, Suppl. Fig. 4 ). Moreover, further validation that myricetin effectively inhibited the entry of the pseudo virus into HEK293-hACE2-mCherry cells was obtained by FCM. The mutant (RBD^N501Y, RBD^N439K, RBD^Y453F, S-D614G) pseudo viruses were more infectious than SARS-CoV-2 RBD^WT in HEK293-hACE2-mCherry cells, and myricetin effectively inhibited infections with these pseudo viruses (Fig. 3D). These results above suggest that myricetin was able to inhibit wide-type SARS-CoV-2 and its variants.

Fig. 4.

Myricetin downregulates NF-κB signaling pathway in THP1 cells. (A-G) mRNA expression levels of NF-κBp65, RIPK1, RIPK3, FADD, MLKL, Caspase8, and JNK. (H) Western blot analysis of NF-κBp65, p-NF-κBp65, RIPK1, and p-RIPK1. THP1 cells are incubated with 0, 100, 200, and 400 μM myricetin followed by treatment with the LPS. NF-κBp65 is nuclear factor-κB p65; RIPK1 is receptor interacting serine/threonine kinase 1; RIPK3 is receptor-interacting serine-threonine kinase 3; FADD is fas-associating protein with a novel death domain; MLKL is Mixed lineage kinase domain like; Caspase 8 is cysteinyl aspartate specific proteinase 8; JNK is c-Jun N-terminal kinase. Data are expressed as the mean ± SEM. **p<0.01, *p<0.05 vs. the model group.

Myricetin suppresses the expression levels of RIPK1 and inhibits NF-κB signaling

It was reported that SARS-CoV-2 had a strong capability of inhibiting the receptor-interacting serine/threonine protein kinase 1 (RIPK1) -mediated host defense response to promote its propagation. Inhibition of RIPK1 may be a therapeutic option for treating COVID-19 (Xu et al., 2021). We first established an LPS-induced hyperinflammatory cell model in THP1 macrophages to simulate a state with high RIPK1 levels. The results showed that myricetin significantly reduced RIPK1 expression levels, as well as the mRNA levels of NF-κBp65, RIPK3, RIPK1, MLKL, FADD, Caspase8 and JNK (Figs. 4A-G). Additionally, we evaluated whether myricetin can affect the canonical LPS-induced RIPK1/NF-κB pathway by western blotting. Treatment of THP1 macrophages with LPS in the absence and presence of myricetin demonstrated that the RIPK1, phosphorylation of NF-κBp65 and RIPK1 induced by LPS stimulation was significantly inhibited by myricetin, but the abundance of the NF-κBp65 protein among the total proteins was unaltered (Fig. 4H). NEC-1 is a well-established RIPK1 inhibitor that blocks RIPK1 and inhibits the interaction between RIPK1 and RIPK3. Our results demonstrated that myricetin has a similar suppressive effect on RIPK1 like NEC-1, suggesting the property of myricetin is comparable to the RIPK1 inhibitor.

Myricetin ameliorates acute inflammation in rats and mice

To elucidate the potential of myricetin for treating COVID-19, the animal models of carrageenan-induced paw edema, auricle edema, LPS-induced acute lung injury (ALI) were established for the study. We sensitized BALB/c mice by painting DNFB to provoke the sensitization phase of DTH and myricetin was used against DTH inflammation. As seen in Fig. 5 A, the H&E staining showed that myricetin inhibited inflammation by reducing the infiltration of lymphocytes and edema in the dermis of mouse ears, and spongiosis in the epidermis of the ear was also improved markedly after treatment (Fig. 5A). The mouse ear thickness was decreased by myricetin treatment at 20, 40, 60 and 80 min later after modeling (Fig. 5B) without any organ damage (Suppl. Fig. 5A). We also validated the anti-inflammatory effect of the drug on the model of dimethylbenzene-induced auricular edema. The results showed that myricetin significantly suppressed the extensive edema formation in the dermis (Suppl. Figs. 5B, C) without immune organ toxicity (Suppl. Fig. 5D). In the rat model of carrageenan-induced paw edema, myricetin could significantly reduce inflammatory activity at the 2nd-5th h after injection (Figs. 5C, D, E). Moreover, we established LPS-induced acute lung injury model in mice, and found that pre-treatment with myricetin (200, 300, and 400 mg/kg body weight) not only alleviated lung edema but also decreased the alveolar inflammation in the ALI mice (Fig. 5E). Consistent with the in vitro results, the mRNA levels of TNFα, IL-6 and NF-κBp65 in the lung tissues were significantly downregulated by myricetin (Fig. 5G, H). In addition, the acute toxicity of myricetin was previously examined in mice and no exposure-related clinical symptoms were observed during the administration of myricetin at 6000 mg/kg (Suppl. Fig. 7), suggesting that myricetin is a druggable compound with low toxicity and good safety.

Fig. 5.

The anti-inflammatory effect of myricetin in vivo. (A) Histological analysis of ear skin sections obtained from BALB/c mice; (B) Ear thickness is monitored during the DTH modeling period in BALB/c mice; (C) The carrageenan-induced change in hind paw volume in rats; (D) Representative photographs of the rats in each group after carrageenan sensitization; (E) Histological analysis of lungs obtained from C57BL/6 mice 24 h after LPS induction; (F) The serum level of TNFα; (G, H) The expression levels of IL-6 and NF-κBp65 in the lung tissues. Data are expressed as the mean ± SEM. **p<0.01, *p<0.05.

Discussion

Infectious respiratory diseases caused by viruses have posed a great challenge to public health since the Spanish influenza pandemic in 1918 till today. The current COVID-19 pandemic is caused by SARS-CoV-2 and has once again posed a tremendous challenge to global health. However, standard drugs to target SARS-CoV-2 are still not available. As drug discovery research usually takes years or even decades, the repurposing of existing drugs, mainly small compounds with antiviral and anti-inflammatory effects from natural sources, has been thought as a good alternative for coping the COVID-19 infection. Similar to the coronaviruses SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), the SARS-CoV-2 virion contains structural proteins (the nucleon capsid (N), membrane (M), envelope (E) and spike (S) proteins) (Brown et al., 2021), and ACE2 is the obliged receptor by which the RBD of the SARS-CoV-2 (S glycoprotein) enters the cell (Walls et al., 2020). Recent studies have revealed that the binding of the RBD with the host cell acceptor ACE2 is a vital step for virus infection (Cao et al., 2021), so antiviral drugs would ideally inhibit the interaction between RBD and ACE2 at the early stage.

Herbal medicines derived from Chinese plants have long been used for treating various infections and diseases. Flavonol, which are found in Chinese herbs, have been suggested as potential protective agents against COVID-19 due to their anti-inflammatory and antiviral properties (Khazdair et al., 2021). Several computational and experimental studies indicated flavonols, such as luteolin, quercetin and kaempferol, may target SARS-CoV-2 proteases (3CLpro and PLpro), S protein, RNA-dependent RNA polymerase (RdRp) and ACE2 to exert potential anti-COVID-19 effects (Mouffouk et al., 2021). Myricetin is a natural flavonol that has previously shown inhibitory properties of SARS-CoV-2 Mpro or 3CL-Pro (Xiao et al., 2021). but its effect on viral-entry facilitators remains still unclear. The current study demonstrates that myricetin has a good affinity for the SARS-CoV-2 RBD^WT protein and can inhibit the interaction between RBD^WT and ACE2, indicating that myricetin might be a druggable compound.

Mutations frequently occur in the evolution of viruses, and SARS-CoV-2 is no exception to this phenomenon. In this study, we also examined SARS-CoV-2 strains with different S proteins due to missense mutations. Interestingly, myricetin showed much better affinity for the S-D614G, RBD^N501Y, RBD^N439K and RBD^Y453F mutants, in which detailed, and precision molecular mechanisms need to be expounded further. In addition, we have also investigated the role of myricetin in the interaction of SARS-CoV-2 mutations with ACE2, which is helpful for us to understand the property of myricetin as a candidate drug against SARS-CoV-2.

Many reports have evidenced that COVID-19 pathogenesis is closely associated with exaggerated immune responses and cytokines storm. Clinical signatures, such as a sharp increases in the levels of chemokines (CXCL1, CXCL2, CXCL8, CXCL9, CXCL10 etc.) (Lucas et al., 2020), proinflammatory cytokines (TNF-α, IL-1β, IL-6 etc.) (Coperchini et al., 2021), the neutrophil-to T-cell ratio (Mann et al., 2020) and CD4⁺ and CD8⁺ T-cell proliferation (Mathew et al., 2020) are reliable predictors of COVID-19 severity and mortality (Valle et al., 2020). Moreover, RIPK1 kinase activity plays an essential role in apoptosis, necroptosis and inflammation. Clinical studies have demonstrated the up-regulation of RIPK1 in upper respiratory epithelial cells from COVID-19 patients, while systemic inflammation in severe COVID-19 is even regarded as a biomarker for RIPK1 activation (Xu et al., 2021). The inflammatory cytokines storm caused by SARS-CoV-2 was shown to be inhibited by the RIPK1 inhibitor Nec-1 s, and the in vivo viral load also subsequently decreased (Xu et al., 2021). In summary, RIPK1 inhibitors may be a considerable strategy to treat severe COVID-19 that improves the driving inflammatory response and viral load in host. Myricetin is a natural compound that have anticancer, hepatoprotective, cardiovascular protection and anti-inflammatory effects with low toxicity (Agraharam et al., 2022). Besides, previous studies indicated that the anti-inflammatory mechanisms of myricetin were associated with the inhibition of the TLR4-MyD88-NF-κB signaling pathway in the ALI rat model and the NF-κB/AKT/p38/MAPK in the ALI mouse model (Mao and Huang, 2017; Wei et al., 2018). Dihydromyricetin, the flavanonol analogue of myricetin, also exerted its anti-inflammatory effect by inhibiting p38 MAPK signaling pathway (Wang et al., 2018). Our in vivo study demonstrated that myricetin had suppressive action on the RIPK1-driven inflammation and NF-kappa B signaling in THP1 macrophages and had significant beneficial effects in LPS-induced ALI in mice through NF-κB. Taken together, myricetin is a valuable candidate for the development of a new drug against inflammation and SARS-CoV-2 infection.

Here, we employed several animal models to simulate the multisystem inflammatory illness after SARS-CoV-2 infection. Myricetin was also confirmed to have significant inhibitory effects on inflammation via NF-κB signaling. In the future, the in vivo protective role of myricetin against SARS-CoV-2 infection should be assessed.

However, the current research has several limitations. The mutant Alpha, Delta and Omicron strains were only validated using pseudo viruses due to the experimental conditions. Moreover, since the bioavailability of myricetin by oral administration, and absorption by passive diffusion in vivo, is low, the oral administration of myricetin is usually at high doses (Guo et al., 2015; Hu et al., 2018). We also conducted in vivo toxicity assays and results indicated no exposure-related clinical signs after the administration of myricetin at 6000 mg/kg. Therefore, a dosage ranging from 50 to 400 mg/kg was reasonable to observe the anti-inflammatory efficacy of myricetin in our study.

Conclusion

Here, we show the antiviral effect of myricetin on HCoV-229E and SARS-CoV-2 replication in vitro. Myricetin inhibits the interaction between the SARS-CoV-2 spike protein and its receptor ACE2, blocking the viral entry facilitators of SARS-CoV-2. Moreover, the administration of myricetin results in a reduction in cytokine expression and inhibits the RIPK1/NF-κB pathway, which may be used to relieve the acute inflammation reactions after virus infection. Thus, myricetin has the potential to be developed as a therapeutic agent against COVID-19 either alone or as part of a combined treatment against COVID-19.

Editor conflict of interest statement

Given their role as Associate Editor, Liang Liu had no involvement in this article and no access to information regarding its peer-review.

CRediT authorship contribution statement

Hudan Pan: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft. Jinlian He: Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft. Zifeng Yang: Validation. Xiaojun Yao: Validation. Han Zhang: Validation. Runfeng Li: Validation. Yao Xiao: Validation. Caiping Zhao: Validation, Visualization. Haiming Jiang: Validation. Yuntao Liu: Validation. Zhanguo Li: Investigation, Project administration, Writing – review & editing. Bin Guo: Validation. Chuanhai Zhang: Validation, Visualization. Run-Ze Li: Writing – review & editing. Liang Liu: Conceptualization, Investigation, Project administration, Supervision, Writing – review & editing.

Declaration of Competing Interest

All authors declare no competing interest.