Thuja orientalis folium protects against Influenza A virus via inhibition of hemagglutinin and viral binding at early stage

doi:10.21203/rs.3.rs-1948239/v1

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Thuja orientalis folium protects against Influenza A virus via inhibition of hemagglutinin and viral binding at early stage

https://doi.org/10.21203/rs.3.rs-1948239/v1

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Background: Morbidity and mortality as a result of influenza is an ongoing global health issue. Macrophages are part of the immune defense mechanism, as they play a role in the inflammatory response that blocks viral replication. Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this investigation, we have assessed the anti-viral properties of TOF water extract on the infectivity of the influenza A (H1N1) virus in RAW 264.7 macrophages.

Method: We assessed the ability of GFP-tagged influenza A virus (A/PR/8/34-GFP) to infect murine macrophages. To determine the potential of antiviral activity using Fluorescence-activated cell sorting (FACS), fluorescent microscopy with green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus. Hemagglutination inhibition assay, Western blotting, and Immunofluorescence analyses were performed to find the target protein. To investigate which stage of viral infection is associated with the TOF antiviral impact, we evaluated viral infection at

three stages: attachment, entry, and virucidal in the presence of TOF.

Results: FACS and immunofluorescence analyses results showed that TOF significantly inhibited viral infection and 100 μg/mL of TOF repressed that up to 98% of untreated control. Consistently, Western blot and immunofluorescence analyses against the influenza viral protein confirmed the TOF extract reduced the influenza viral protein expression in a dose-dependent manner. Time of addition and hemagglutination assays elucidated that TOF suppresses influenza A viral infection at early stages and interfere with HA protein.

Conclusion: The TOF extract was thus found to exhibit inhibitory effects on influenza virus infection by inhibiting hemagglutination, blocking viral attachment and entry, and through virucidal effects. Based on our results, TOF may be a potential natural resource that could be utilized to develop an antiviral reagent.

anti-influenza viral activity

herbal medicine

hemagglutinin (HA)

Thuja orientalis Folium

Influenza A(H1N1) viruses are a major cause of respiratory infection in humans and are responsible for a significant amount of morbidity and mortality worldwide[1].The most common circulating subtypes are the influenza A (H1N1) viruses that cause seasonal epidemics,, which are responsible for 290,000–650,000 deaths annually [2]. Many vaccines and treatments have been developed to prevent the spread of the influenza virus, and these provide the backbone for the current preventive treatment strategy. These vaccines must be modified annually due to the

viral tendency to mutate to escape detection in the immune system[3]. However, the development of new vaccine strains is difficult to predict, which means that it takes time to provide a virus pandemic losses[4]. The most fundamental countermeasure against the outbreak and pandemic preparation is to develop antiviral drug for the suppression and treatment of viral infections. Influenza treatments include M2 ion channel blockers (e.g., admantanes, rimantadine), which inhibit virus proliferation by interfering with the process of removing the skin of host cell infection, and neuraminidase inhibitors (e.g., oseltamivir, zanamivir), which inhibit the spread of virus particles by reducing their release from host cells and increasing the formation of virus aggregates[5, 6]. Oseltamivir (Tamiflu), an NA inhibitor, is a successful example of an anti-influenza drug synthesized from natural products, as it was researched and developed using quinic acid and sikmic acid, which were obtained from plants[7]. However, the 2008–2009 seasonal H1N1 influenza virus strain showed almost complete resistance to oseltamivir[8]. Baloxavir marboxyl was appoved for influenza treatment in 2018. but recent studies have shown that virus resistance remains a concern[9]. It is thus necessary to develop a new agent to treat influenza A with a clear mechanism of action.

Natural products provided unlimited opportunities for new drug discoveries because of their inherent chemical diversity, which has prompted a continuous search for plant with medical value.

The dried leaf of TOF is a traditional herb, widely used for the treatment of Bronchiti, psoriasis, pancystitis, and diarrhea [10, 11]. TOP contains a lot of chemical components including amentoflavone, quercitrin, myricetin, afzelin and Kaempferol. Several studies have reported that TOF has a variety of biological functions such as antioxidant[12], anti-inflammatory action[13], anticancer activity,[14] as well as reducing fever[15]. The essential oil, in particular, has been used for mycotic infection and the treatment of roundworm[15]. The previous studies showed that TOF contains the antiviral activity against influenza virus A/PR8/34[16], but the report only demonstrated the inhibitory effect on cytopathic effect and viral M2 gene expression.

In this study, we elucidate the underlying mechanism of antiviral properties of TOF using RAW 264.7 macrophages infected with GFP-expressing H1N1 influenza virus to confirm the potential as an antiviral drug capable of inhibiting the proliferation of influenza viruses.

2.1.1 Materials and chemicals

The High-Performance Liquid Chromatography (HPLC) system, Thermo Dionex UltiMate 3000, equipped with a binary pump, an auto-sampler, a column oven, and a diode array UV/VIS detector (Thermo Fisher Scientific, San Jose, CA, USA). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA) and ACS reagent grade formic acid was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Ultrapure water used for the HPLC analysis was purified by Puris-Evo UP Water system with Evo-UP Dio VFT and Evo-ROP Dico20 (Mirae ST Co., Ltd., Anyang, Gyeonggi-do, Korea). Reference standard compounds, amentoflavone and quercetin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Quercitrin was obtained from ChemFaces (Wuhan, China). The purity of all reference standard was above 95%.

2.1.2Preparation of standards and sample solution

Stock solutions of amentoflavone, quercetin and quercitrin were prepared by dissolving in methanol at a concentration of 1000 µg/mL. Then, three stock solutions were diluted to a final concentration of 100 µg/mL of each compound to prepare a mixed working solution. The water extract of Thuja orientalis Folium (TOF), an analytical sample, was prepared by dissolving it in methanol at a concentration of 10 mg/mL. All solutions of standards and sample used for analysis were filtered using 0.22 µm PTFE membrane filters (Whatman International ltd., Maidstone, UK), and were stored at 4°C until analysis.

2.1.3 HPLC-UV/Vis analysis

In order to achieve identify three components, amentoflavone, quercetin and quercitrin in Thuja orientalis Folium (TOF), HPLC-UV/Vis analysis was performed via HPLC system, Thermo Dionex UltiMate 3000. The analysis conditions were conducted by referring to and modifying the method of the reported literature[17]. Chromatographic separation was carried out on a Thermo Acclaim® 120 C18 column (250 × 4.6 mm, 5 µm), and the mobile phase composition was consisted of 0.1% formic acid (v/v) in water (A) and acetonitrile (B). To improve chromatographic separation capacity, the gradient elution system was programmed at a flow rate of 1.0 mL/min as follows: 10% B, 0.0 min; 10–60% B, 0.0–40.0 min; 60% B, 40.0–45.0 min; 60–100% B, 45.0–50.0 min; 100% B, 50.0–55.0 min; 100 − 10% B, 55.0-55.5 min; 10% B, 55.5–65.0 min. The temperature of the column was maintained at 30 ^oC, and the injection volume of each sample was 5 µL. The detection wavelengths for all analytes, amentoflavone, quercetin and quercitrin were set at 365 nm. Data acquisition and analysis were performed via Dionex Chromelon software (Thermo Fisher Scientific).

2.2 Cells and Viruses

RAW 264.7 cells (Mouse Leukemic Monocyte Macrophage cell line; ATCCTIB-71) were acquired originally ATCC and were passaged in Le Roswell Park Menorial Institute medium (RPMI; Hyclone, Logan, UT), and cell culture flasks supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and antibiotic/ antimycotic solution (Gibco) at 37℃ and 5% CO². Green fluorescent protein (GFP)-tagged Influenza A (A/PR8/34(H1N1))-GFP and A/PR/34/(H1N1) viruses were provided by Dr. Jong-Soo Lee (Chungnam National University, Daejeon, South Korea). Stock viruses were prepared in the allantoic flu;id of 10-d-old chicken embryos.

2.3 Cell Cytotoxicity

The Cell counting kit-8 (CCK-8) assay was used to evaluate the cyototoxicity of TOF in the cells, according to the manufacturer’s recommendation (Dojindo, Rockville, MD, USA). RAW 264.7 cells were seeded into 24-well plates at a density of 1 x 10⁵ cells/well and cultured overnight before the TOF treatment. TOF was added to the wells at various concentrations (0, 10, 50 ,100, 200, 500 and 1000 µg/mL). After 24hr of incubation, 10 µLCCK-8 was added to the cells, and they were then incubated for 2 h at 37℃, after which the absorbance at 450 nm was measured using spectrophotometer (BioTek, Winooski, VT, USA).

2.4 Anti-viral activity against influenza viruses

The influenza PR8-GFP and H1N1 viruses were mixed with TOF and incubated for 1 h at 4 ℃ and the mixtures were then added to RAW 264.7 cells for 2 h at 37 ℃. After washing with PBS, the cells were further incubated for 24 h until GFP expression. To investigate the effects of TOF, the levels of GFP expression in the cells infected with PR8-GFP IAV were observed under fluorescence microscopy (Nikon ECLOPSE Ti-U, Nikon Co., Japan) and photographed or assessed using fluorescence-activated cell sorting (FACS) analysis.

2.5 Time of addition Assay

Time of addition assays were used to assess the attachment, entry, and virucidal stages. In the attachment stages, RAW 264.7 cells were infected with TOF 200 µg/mL and PR8-GFP IAV at 4℃ for 30 min. After washing, the cells were incubated at 37℃ for 24 h. In the virucidal stages, PR8-GFP IAV and TOF were incubated at 4℃ for 30 min, and then the mixture was added to the cells and incubated at 37℃ for 30 min, and after washing. The cell were incubated at 37℃ for 24 h. All stages were analyzed using a fluorescence microscope and FACS.

2.6 Fluorescence-activated cell sorting (FACS) analysis

TOF was incubated with PR8-GFP IAV for 1 h at 4℃ and then treated with the RAW 264.7 cells for 24 h at 37℃. The cells were harvested by centrifugation at 15 000 rpm for 10 min at 4℃, washed thrice with cold PBS, and fixed with 4% paraformaldehyde. The cells were resuspended in PBS and analysed for GFP expression using a CytoPLEX flow cell counter (Beckman Coluter Inc., Pasadena, CA, USA)

2.7 Western Blotting analysis

Western blotting analysis was performed to determine the induction of viral protein (NP, NS1, PA, HA, M2, M1, NA, PB1, PB2) with a bicinchoninic acid reagent (Thermo Scientific). Equal protein (approximately 20µg) amounts were resolved on sodium dodecyl sulfate gels using polyacrylamide gel electrophoresis and transferred to 0.45-mm polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% BSA for 1 h, the membranes were reprobed with each primary antibody overnight at 4℃. The membranes were then washed three times using 0.05% PBST and incubated for 1 h with the respective HRP-conjugated secondary antibody. Western blots were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific), and GAPDH was used as the control.

2.8 Immunofluorescence staining

Immunofluorescence staining was performed as previously described. RAW 264.7 cells (1 × 10⁵) grown on 4-well tissue culture slides, were incubated at 37℃ for 12 h. The medium was then removed, and the cells were washed with cold PBS three times and infected with A/PuertoRico/8/34 (MOI 1) for 2 h. The virus and medium were then removed and the cells were washed with PBS three times. Complete medium was then added to the cells, and the cells were incubated at 37℃ with 5% CO² for 24 h, cells were washed with cold PBS and fixed with cold absolute methanol for 10 min and 4% paraformaldehyde for 30 min at RT and permeabilized with 1% Triton X-100 in TBS for 15 min at RT. After washing with PBS containing 0.05% Tween 20 (PBST), the cells were subjected to blocking with 1% BSA-PBS for 30 min and incubated with antibodies against influenza viral protein for 1 h at room temperature. After washing with PBS containing 0.05% Tween 20 (PBST), the cells were incubated with Alexa Fluor 594-tagged secondary antibody in PBST for 1 h in the dark, followed by incubation with Hoechst 33342 for 5 min. The images of the red viral proteins and blue nuclei were visualized under fluorescent microscopy.

2.9 Hemagglutination(HA) assay

The influenza A (H1N1) viruses were incubated with TOF for 1 h at 4℃, and the mixture was added to RAW 264.7 cells for 2 h at 37℃. After washing with PBS they were incubated for another 24 h, then the supernatants were collected for the HA assay. Briefly, the supernatant was subjected to a serial two-fold dilution and added to a round-bottom 96-well-plate. The blank medium was used as a negative control. Each sample was mixed with 1% chicken RBCs (Innovative Research, Inc., Southfield, MI, USA) in PBS buffer. After incubation for 1 h at room temperature, the incubated plates were photographed.

2.10 Statistical analysis

All data are expressed as the mean ± standard deviation (SD). Statistically significant differences among the groups were analyzed by one-way ANOVA followed by post-hoc multiple comparisons with Fisher’s LSD t-test using IBM SPSS statistics 20.0 (SPSS Inc. Chicago, IL). Differences at p < 0.05 were considered statistically significant.

3.1 HPLC-UV-Vis analysis of TOF

HPLC-UV-Vis analysis was performed to identify amentoflavone, quercetin and quercitrin in the TOF (Fig. 1). Three components were identified and determined by comparing their retention times (t_R) and chromatogram patterns with those of references compounds. In addition, the UV wavelengths for the three components in the TOF were optimized according to the maximum absorption UV spectrum when compared to the UV wavelengths of the standards, and all analytes were detected at 365 nm. The results showed that the quercitrin (1, 16.91 min), quercetin (2, 23.62 min), and amentoflavone (3, 30.05 min) all have good selectivity with minimal interference from other analytes, and were well separated within 50 min.

3.2 Determination of the effective cytotoxicity of TOF in vitro

The toxic effects of the TOF on RAW 264.7 cells were evaluated using cell viability with the enhanced Cell Counting Kit-8. The cells were treated with TOF and CCK-8 was added 24h after the treatment to determine the optimal concentration that would provide anti-viral activity with minimum toxicity. The results showed that ≤ 200 \({\mu }\)g/ml of TOF had no effect on cell viability, indicating that it was not toxic for the RAW 264.7 cells. Therefore, subsequent experiment were performed using 100 µg/mL TOF. (Figure. 2)

3.3 TOF suppressed influenza A(H1N1) virus infection of the RAW 264.7 cells

Influenza A viral replication of the GFP-tagged PR8 (PR8-GFP influenza A(H1N1) showed significant increases in GFP expression in the RAW 264.7 cells. To explore the effect of TOF on the GFP-tagged PR8 (PR8-GFP influenza A(H1N1) viruses) virus infection, RAW 264.7 cells were co-infected with GFP-tagged PR8 influenza A virus (PR8-GFP IAV) and TOF and cultured for 24 h. The inhibitory impact of the TOF on the viral infection was evaluated by measuring the amount of GFP expression in the cells infected with the virus and treated with 10, 25, 50, and 100 µg/mL of TOF. GFP expression was substantially decreased in the cells infected with TOF in a dose-dependent manner. GFP expression was detected in the control cells without TOF when infected with PR8-GFP IAV (Fig. 3A). The antiviral activity of TOF on the PR8-GFP IAV infection was then verified by FACS analysis (Fig. 3B-C). The cells were fixed with paraformaldehyde and GFP-expressing cells were counted by measuring FITC levels in uninfected cells, those infected with the PR8-GFP virus alone, and those infected with PR8-GFP and TOF extract in the given concentrations (Fig. 3C.) The levels of GFP expression for each group were compared with the PR8-GFP virus-infected control and graphed as a relative intensity. GFP expression levels in the virus only group were higher than those of the mock group, whereas TOF significantly reduced the GFP expression levels of the influenza A (H1N1) virus (p < 0.01 for 10, 25, 50, and 100 µg/mL, Fig. 3B-C). The TOF significantly suppressed viral infection, which was consistent with the results presented in Fig. 3. These findings indicate that the TOF had a potent inhibitory impact on the PR8-GFP influenza virus infection.

3.4 TOF reduced influenza viral protein expression

The effect of the TOF on influenza virus Protein expression was examind by Western blot analysis and immunofluorescence. TOF and the PR-8 influenza A (H1N1) virus were co-incubated 4℃ for 1h before infection the RAW 264.7cells and they were then incubated at 37℃ for 24h until viral protein expression was observed. TOF decreased the immunofluorescence of the HA, M2, and NP proteins (Fig. 4A). Western blotting was then conducted to confirm the inhibitory effect of the TOF on the influenza A(H1N1) virus protein expression. All virus proteins were significantly suppressed in the presence of 100 µg/mL TOF (Fig. 4B).

3.5 TOF inhibits viral infection at the attachment, entry and virucidal stages

To investigate which stage of viral infection is associated with the TOF antiviral impact, we evaluated viral infection at three stages: attachment, entry, and virucidal in the presence of TOF. Figure 5 represents TOF inhibits all three stages of infection.

3.6 Effect of TOF on HA activity

Influenza virus can cause RBC hemagglutination (HA) during infection at early stage. Because co-treatment results and time of addition assay indicated that TOF directly affected the influenza A infection and viral binding into the cells, we investigated whether TOF affects HA activity of IAV. As shown in Fig. 6, in the absence of TOF, HA units of IAV were 4 units, but TOF at 10 ug/mL reduced 2-fold lower HA units than virus only and 50 or 100 ug/mL of TOP completely blocked HA. These findings confirm that TOF limits influenza A (H1N1) virus infection by blocking viral attachment to the cells via inhibiting HA of IAV.

TOF has been widely used as a botanical medicine herb to treat regular seasonal influenza [15]. It has been demonstrated to have antibacterial activity against methicillin-resistant Staphylococcus aureus[18], antiviral action on Fig leaf mottle-associated virus 1(FLMaV-1)[19], especially fungi toxic activity in essential oils[20], as well as anti-cancer and neurotoxicity effects.[21].

In this study, TOF was found to significantly inhibit influenza A (H1N1) virus infection. We investigated the anti- viral effects of TOF using GFP-tagged influenza A (PR8-GFP) virus replication system. Fluorescence microscopy and FACS analysis demonstrated that TOF inhibited IAV infection. The immunofluorescence staining and Western blot analysis for the IAV protein confirmed that TOF substantially reduces IAV protein expression. Based on the inhibitory effects of the TOF on IAV infection, we investigated whether TOF could prevent viral attachment to and entry into cells. HA is responsible for viral binding to the main target and cell surface and thus a target for antiviral therapies. HA inhibition tests showed that TOF inhibits IAV HA. Many herbal extracts and chemicals have been shown to prevent hydroxyacetone binding to the target membrane, such as Curcumin, New Zealand conferencing Podocarpus totara, and Dacrydium cupressinum which have been shown to prevent influenza virus attachment to cell membranes through HA inhibition[22].

A time addition assay revealed that TOF reduces viral binding and penetration into the cells. Recently, it was reported that the Epimedium Koreananum Nakai (EKN) water extract directly inhibits influenza virus binding to cells and has virucidal effects before entering the cell. The virucidal effects of EKN are due to specific components of the extract which have the ability to directly interact with the virus, and this inhibits viral particles from adhering to the cells. There are several recent reports on nature extracts such as Broccoli leaves ethanol extract[23], Eupatorium Perfoliatum[24], cranberry extract[25], and jatropha curcas Linn. Leaf extracts[26], which were shown to have inhibit HA activity and viral attachment into the cells. Viral invasion inhibitors have attracted considerable attention from synthetic chemists and biochemists as a new type of antiviral drug that can be used to successfully prevent viruses from attaching to host cells. [27]. Zhou et al. found that plant-derived pentacyclic triterpenes act as highly potent antiviral agents by efficiently preventing viral attachment to the host cells.[28].

Amentoflavone, one of the components of TOF, reportedly has anti-viral effects on HSV-1[29], Coxackie virus B3[30], and respiratory syncytial virus (RSV)[31]. In contrast, Wedelolactone has been reported to have inhibitory effects on hepatitis virus[32], cytomegalovirus[33] and HIV[34]. TOF contains quercitrin and myricetin, which both exhibit antiviral activity against IAV.

The results of this investigation show that TOF inhibits IAV infection in its early stages, including attachment, entry, and its virucidal effects, especially HA, thus leading to strong antiviral effects. In this regard, TOF could thus be used in combination treatments with oseltamivir to protect against IAV infection. It will be necessary, however, to confirm the antiviral effects of TOF through in vivo experiments, and further research is also required to identify which major components have antiviral effects.

Author Contributions: Conceptualization, W.-K.C. and J.Y.M; Data curation, N.-H, Y, W.-K.C. and M.-M.L.; Funding acquisition, JY M; Investigation; M.-M.L. and H.-J.Y.; Methodology, , N.-H, Y, W.-K.C. and M.-M.L.; Project administration, J.Y.M.; Resources, J.Y.M.; Supervision, W.-K.C. and J.Y.M.; Writing-original draft, M.-M.L. and H.-J.Y.; Writing-review & editing, W.-K.C. and J.Y.M. All authors read and approved the final manuscript.

Funding: This work was supported by Grant numbers KSN1812101 and KSN2022230 from KIOM (Korea Institute of Oriental Medicine), provided by the Ministry of Science and ICT, Republic of Korea.

Acknowledgments:We thank Dr. Jong-Soo Lee (Chungnam National University, Republic of Korea) for providing GFP-tagged Influenza A/PR/8/34 and H1N1 influenza viruses.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data in this study are available on request from the corresponding author.

Conflicts of Interest: The authors declare no conflict of interest.

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No competing interests reported.

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Thuja orientalis folium protects against Influenza A virus via inhibition of hemagglutinin and viral binding at early stage

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Abstract

Figures

1. Background

2. Materials And Methods

2.1.1 Materials and chemicals

2.1.3 HPLC-UV/Vis analysis

2.2 Cells and Viruses

2.3 Cell Cytotoxicity

2.4 Anti-viral activity against influenza viruses

2.5 Time of addition Assay

2.6 Fluorescence-activated cell sorting (FACS) analysis

2.7 Western Blotting analysis

2.8 Immunofluorescence staining

2.9 Hemagglutination(HA) assay

2.10 Statistical analysis

3. Results

3.1 HPLC-UV-Vis analysis of TOF

3.2 Determination of the effective cytotoxicity of TOF in vitro

3.3 TOF suppressed influenza A(H1N1) virus infection of the RAW 264.7 cells

3.4 TOF reduced influenza viral protein expression

3.5 TOF inhibits viral infection at the attachment, entry and virucidal stages

3.6 Effect of TOF on HA activity

4. Discussion

Declarations

References

Additional Declarations

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