Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

Journal Pre-proof

Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

Li-jun Ling InvestigationFormal analysisMethodologyData CurationSoftwareWriting - Original DraftWriting - Review Yan Lu Writing - Review & EditingProject administrationConceptualization , Yun-yi Zhang Writing - Review & EditingConceptualization , Hai-yan Zhu ResourcesMethodology , Peng Tu ValidationMethodology , Hong Li ResourcesWriting - Review & EditingProject administrationSupervisionConceptualization , Dao-feng Chen Funding acquisitionConceptualizationProject administrationSupervision PII: DOI: Reference:

S0944-7113(19)30466-0 https://doi.org/10.1016/j.phymed.2019.153150 PHYMED 153150

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

5 August 2019 3 December 2019 11 December 2019

Please cite this article as: Li-jun Ling InvestigationFormal analysisMethodologyData CurationSoftwareWriting - Orig Yan Lu Writing - Review & EditingProject administrationConceptualization , Yun-yi Zhang Writing - Review & Editin Hai-yan Zhu ResourcesMethodology , Peng Tu ValidationMethodology , Hong Li ResourcesWriting - Review & Ed Dao-feng Chen Funding acquisitionConceptualizationProject administrationSupervision , Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling, Phytomedicine (2019), doi: https://doi.org/10.1016/j.phymed.2019.153150

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier GmbH.

Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

Li-jun Linga, Yan Lua, Yun-yi Zhangb, Hai-yan Zhuc, Peng Tua, Hong Lib,*, Dao-feng Chena,* Affiliations: a

Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai

201203, China b

Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai

201203, China c

Department of Microbiological and Biochemical Pharmacy, School of Pharmacy,

Fudan University, Shanghai 201203, China

Corresponding authors Dao-feng Chen, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China E-mail address: [email protected] (D.-F. Chen)

Hong Li, Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China E-mail address: [email protected] (H. Li)

1

Abstract Background: Influenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata. Purpose: This study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice. Methods: Flavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. Results: HCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-β in lungs. HCF also 2

decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 μg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells. Conclusion: HCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. Among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.

Keywords: Houttuynia cordata; flavonoid glycosides; Influenza A virus; Acute lung injury; Antiviral activity; TLRs signalling

Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; CMC, caboxymethyl cellulose; CPE, cytopathic effect; DAB, 3,3N-diaminobenzidine tertrahydrochloride; DAPI, 4',6-diamidino-2-phenylindole; DEX, dexamethasone; DMEM, Dulbecco's Modified Eagle Medium; ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; HCF, Flavonoids from Houttuynia cordata; HPLC,

high-performance

liquid

chromatograph;

IFN-β,

interferon-β;

IHC,

immunohistochemistry; IL-8, interlekin-8; IVA, influenza virus A; MCP-1, monocyte chemotactic protein 1; MDA, malondialdehyde; MDCK, Madin-Darby canine kidney cells; MTT, tetrazolium salt 3-[4,5- dimethylthiazol -2-yl] -2,5- diphenyltetrazolium bromide; MUNANA, 2´-(4-methylumbelliferyl)-α-D-N-acetylneuraminic sodium salt hydrate; NF-κB p65(p), nuclear transcription factor-κB p65 phosphorylation; PBST, phosphate buffer solution with Tween; Rb, Ribavirin; RPMI 1640 medium, Roswell Park Memorial Institute 1640 medium; SARS, severe acute respiratory syndrome; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCID 50, 50% 3

tissue culture infective dose; TLRs, Toll-like receptors; TNF-α, tumor necrosis factor-α

Introduction Influenza A virus (IAV) is one of the most important human pathogens and causes significant morbidity and mortality worldwide (Herold et al., 2015). With the emergence of strain resistance in current anti-influenzal agents (M2 channel blockers and neuraminidase (NA) inhibitors), there is an urgent need to develop alternative therapeutic approaches with different anti-influenzal strategies (Behzadi et al., 2019). IAV-associated deaths are mainly attributed to acute respiratory distress syndrome (ARDS), which may be a result of the combination of intrinsic viral pathogenicity and a robust host innate immune response (Herold et al., 2015). An enhanced understanding of the pathogenesis of IAV suggests that targeting IAV-induced inflammation in combination with conventional antiviral therapies may be an alternative approach for the treatment of IAV infection (Herold et al., 2015; Behzadi et al., 2019). Toll-like receptors (TLRs) play key roles in IAV recognition and the immune response (Sandra et al., 2014). TLR activation stimulates the antiviral response (e.g., type-I interferons: IFN-α and IFN-β) and may result in the massive secretion of pro-inflammatory mediators (Lester and Li, 2014). A series of studies reveal that blocking TLR signalling prevents the over-activation of the immune system, which indicates a novel therapeutic strategy for influenza-induced inflammation (Le Goffic et al., 2006; Imai et al., 2008). Houttuynia cordata Thunb. (Saururaceae), often called fish mint in English and Yu-Xing-Cao in Chinese, is a perennial medicinal herb that is widely distributed in eastern and southern Asia (Shingnaisui et al., 2018). Traditionally, H. cordata is used for the treatment of pneumonia, bronchitis, and chronic obstructive respiratory diseases (Muluye et al., 2014). Modern pharmacology reveals that H. cordata has antiviral (Chiow et al., 2016), anti-inflammatory (Li et al., 2011), and anti-oxidative (Hsu et al., 2016) activities. Phytochemical investigations indicated that volatile oil 4

(Li et al., 2011), polysaccharides (Xu et al., 2015), alkaloids (Ahn et al., 2017), and flavonoids (Choi et al., 2012) are important bioactive constituents in the herb. The H. cordata

essential

oil,

purified

by

steam

distillation extraction,

exerts

anti-inflammatory activity and inhibits the activity of cyclooxygenase-2, which is a key regulator of the inflammatory process (Li et al., 2011). Polysaccharides extracted from the aqueous extract of H. cordata ameliorate influenza and lipopolysaccharide (LPS)-induced lung injury, possibly through anti-inflammatory or anti-complementary activities (Zhu et al., 2018; Xu et al., 2015). Alkaloids isolated from a methanolic extraction of H. cordata exert potent anti-inflammatory activity and inhibit LPS-stimulated NO production in RAW 264.7 cells (Ahn et al., 2017). Quercitrin and hyperin are two important flavonoid glycosides in H. cordata (Wu et al., 2009). Quercitrin, in a 70% ethanol extract of the herb, inhibits the total cell number in the bronchoalveolar fluid in LPS-induced lung inflammation in mice (Lee et al., 2015). It also decreased weight loss, mortality, and virus titers in influenza A/WS/33 virus-infected mice, with the mechanisms not fully elucidated (Choi et al., 2012). Hyperin also shows a good interaction with the NS1 protein in the influenza virus by molecular docking technology; however, this has not been further validated (Ahmad et al., 2015). Although quercitrin and hyperin present potential anti-influenzal activities, the overall therapeutic effect and related action mechanism of H. cordata flavonoids (HCF) in preventing influenza infection remains unclear. In this study, we evaluated the therapeutic effect of HCF on mice infected with H1N1 (a virus strain of IAV). To unveil the underlying mechanism, antiviral and TLR-inhibitory activities of HCF were explored in vivo and in vitro. Comparisons among HCF and four major compounds on in vitro antiviral and TLR-inhibitory activities were performed to further explore which flavonoid glycoside play key pharmacological roles in HCF.

Materials and methods Chemicals and reagents Four flavonoid glycosides reference substances (rutin, hyperin, isoquercitrin, and 5

quercitrin, purity > 98%) were obtained from Dalian Meilun Biotechnology Co., LTD (Dalian, China). Viral NA inhibitor oseltamivir was obtained from Yuanye Biotechnology Co., LTD (Shanghai, China). The primary antibodies of F4/80 (antibody to macrophage, ab100790), Ly6g (antibody to neutrophil, ab25377), influenza A virus (ab20841), TLR3 (ab62566), TLR4 (ab13556), TLR7 (ab45371), NF-κB p65(p) (ab86299), β-actin (ab179467), and secondary antibodies (Goat anti-Rabbit: ab6721, Goat anti-Rat: ab205720, Donkey anti-Goat: ab205723) were purchased

from

Abcam

(Cambridge,

UK).

A

3,3’-N-diaminobenzidine

tetrahydrochloride (DAB) kit was obtained from Beijing Zhongshan Biotechnology Co., LTD (Beijing, China). Haematoxylin solution was purchased from KeyGen Biotech Co., Ltd (Nanjing, China). Poly (I:C) (TLR3 agonist) was obtained from InvivoGen (Toulouse, France). LPS (TLR4 agonist), imiquimod (TLR7 agonist), and 2’-(4-methylumbelliferyl)- α-ᴅ-N- acetylneuraminic acid (MU-NANA) were obtained from Sigma-Aldrich (St Louis, MO, USA). The propofol injection was provided by Sichuan Guorui Pharmaceutical Co., Ltd (Sichuan, China). Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Shanghai Fuheng Biological Technology Co., Ltd (Shanghai, China). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit, electrophoresis buffer, and enhanced chemiluminescence (ECL) solutions were purchased from Beyotime Biotechnology Co., LTD (Nantong, China). Plant material and extraction The dried aerial part of H. cordata was purchased from Shanghai Lei Yun Shang Pharmaceutical Co., LTD (Guizhou, China; batch number: 20161023) and identified by Professor Lu Yan (Fudan University, Shanghai, China). The plant name was confirmed in http://www.theplantlist.org (access date: 2012-03-26). The voucher specimen (DFC-HC- 20161115) was deposited in the Herbarium of Materia Medica, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, China. HCF extraction was performed under optimal methods based on a previous report (Zhang et al, 2007). Briefly, the dried aerial part of H. cordata was extracted three 6

times in 70% ethanol. The extract was filtered, concentrated, dissolved in water, and applied to an AB-8 macroporous resin column (Nankai University Chemical Plant, Tianjin, China). The column was washed with 4BV deionized water and 4BV 10% ethanol respectively to remove impurity, then desorbed with 4BV 70% ethanol. The 70% ethanol eluent was dried to yield HCF. Quantification of HCF Total flavonoid glycoside content of HCF was determined by NaNO2-Al(NO3)3 -NaOH colorimetry as previously reported (Zhao et al., 2016), using quercitrin as the standard. The quantification of HCF was performed on an Agilent 1200 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA). The working conditions were similar to as previously described (Zhang et al., 2018) with minor modifications. Column: Phenomenex Luna C18 (2) (250 mm × 4.6 mm; 5 μm); mobile phase: 0.2% acetic acid (A) and acetonitrile (B) with a gradient mode; detection: UV, 256 nm; flow rate: 0.8 ml/min; injection volume: 20 μl; column temperature: 30 °C. The gradient profile was: 0–12 min, 85%–82.5% A; 12–27 min, 82.5%–81.5% A; 27–38 min, 81.5%–75% A; 38–42 min, 75%–0% A; 42–52 min, 100% B. HPLC was performed on HCF samples (60 µg/ml) and a diluted series of mixed standard. The mixed standard was prepared as follows: 0.562 mg rutin, 1.187 mg hyperin, 0.594 mg isoquercitrin and 2.225 mg quercitrin were transfered into a 25-ml volumetric flask and diluted into a series of concentrations (5.71-182.72 µg/ml). All solutions were filtered through a 0.45-μm membrane filter before HPLC. The regression equations of rutin, hyperin, isoquercitrin and quercitrin were obtained by plotting chromatogram peak area at λ = 256 nm versus corresponding concentrations. The linear range and value of correlation coefficient (R2) of each flavonoid glycoside was calculated. According to the calibration curves of four flavonoid glycosides, the concentration of rutin, hyperin, isoquercitrin and quercitrin in HCF (60 µg/ml) were obtained. Then, the content of each flavonoid glycoside in HCF was calculated. Three independent HCF samples were analyzed in parallel for repeatability evaluation. 7

Animals, cells, and virus Specific pathogen-free BALB/c mice (14–16 g, SPF II, Certificate No. SCXK2017-00 05/ Certificate No. SCXK2012-0002) were obtained from the SLACCAS-Shanghai Lab Animal Co. Ltd (Shanghai, China). The mice were housed at 22 ± 1 °C with a relative humidity of 50 ± 10% and a 12-h light/dark cycle. Mice had free access to food and water. All experiments were approved by the Animal Ethical Committee of the School of Pharmacy, Fudan University, Shanghai, China (Permit Number: 2015-10-SY-CDF-01). All animals were anaesthetised with propofol and euthanised. All efforts were made to minimize animal suffering. Madin-Darby canine kidney (MDCK) cells were obtained from Shanghai FuHeng Biology Co., Ltd (Shanghai, China). The murine macrophage cell line RAW 264.7 was gifted by the Department of Pharmacology in the School of Pharmacy, Fudan University. MDCK and RAW 264.7 cells were cultured at 37 °C under 5% CO2 in DMEM and medium 1640, respectively, and supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM D-glutamine, 50 U/ml penicillin, and 100 µg/ml streptomycin. Influenza virus H1N1 is a typical highly pathogenic IAV. In this study, a mouse-adapted strain of influenza virus H1N1 (A/FM/1/47) was used for animal model establishment and in vitro antiviral evaluation. The virus was supplied by the Shanghai Center for Disease Control and Prevention (Shanghai, China). Influenza virus infection and drug preparation The 50% lethal dose (LD50) of H1N1 virus was determined in accordance with a previous study (Zhu et al., 2018). HCF or Ribavirin (Rb) was dissolved in 0.5% carboxymethyl cellulose (CMC) solution for animal experiments. HCF was diluted to final concentrations of 5, 10, and 20 mg/ml. Rb was diluted to 10 mg/ml. Mice were intragastrically administered HCF (50, 100, and 200 mg/kg) or Ribavirin (100 mg/kg) with the volume of 0.1 ml/10 g of body weight. Survival experiment A total of 66 Balb/c mice were involved in the survival experiment and randomly 8

divided into six groups (n = 11 per group). Mice in the normal group were intranasally administered with 30 μl of solution without H1N1 virus and mice in the other groups were intranasally administered with 30 μl of virus solution (10 × LD 50). Mice were treated once daily for 7 d via gavage, beginning 2 h after viral infection: normal group (0.5% CMC), model group (0.5% CMC), HCF low-, middle-, and high-dose groups (HCF at 50, 100, and 200 mg/kg, respectively), Ribavirin group (Rb at 100 mg/kg). Clinical symptoms and mortality were continuously monitored for 14 d. ALI experiment A total of 48 Balb/c mice were involved in the ALI experiment and randomly allocated into six groups (n = 8 per group). The uninfected mice were intranasally administered with 0.5% CMC as a negative control (normal group). The other five groups were intranasally infected with 30 μl of H1N1 virus solution (3 × LD50). The dosage of each treatment was the same as mentioned above (survival experiment). Mice were treated once daily for 4 d via gavage, beginning 2 h after viral infection. The body weight of each mouse was recorded every day. On day 4 after infection, mice were anaesthetised and euthanised. Lungs were harvested and weighed to calculate lung indexes, which indicated oedema and inflammation in the lungs. Lung index = lung weight (mg) / body weight (g) (Wu et al., 2011). The left lung lobes were fixed in 4% formaldehyde, dehydrated, and embedded in paraffin for histopathological and immunohistochemical (IHC) examination. The right lung lobes were homogenized and stored at -80 °C. Histopathology and IHC evaluation Paraffin-embedded left lung tissues were cut into 4 μm thick sections and stained with haematoxylin and eosin (H&E) (Xu et al., 2015). Each lung slice was examined under a light microscope (CX41RF; Olympus, Japan). Images were acquired using Aperio ImageScope v10.2.2.2319 on an Aperio Scanscope XT image scanning system (Leica, Weztlar, Germany). The percentage of lung injury area was evaluated by Image J in H&E slices according to a previous report (Modepalli et al., 2016). Briefly, each image was adjusted using the colour threshold default method. The ‘Analysed Particles’ method was used to analyse the size of the inflammatory-infiltrated area and 9

the whole lung lobe. Percentage of lung injury area (%) = the size of inflammatory-infiltrated area / the size of whole lung lobe × 100% (n = 8 per group). The inflammatory cell infiltration, influenza virus load, expression of TLRs, and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) were evaluated using IHC according to Xu et al (2015). After antigen retrieval, elimination of endogenous peroxidase, and antigen blocking, the slices were incubated with anti-F4/80, anti-Ly6g, anti-IAV, anti-TLR3, anti-TLR4, anti-TLR7, or anti-NF-κB p65 (p) antibody overnight at 4 °C. Then, they were incubated with a secondary antibody at 37 ℃ for 30 min and visualised using DAB. The slices were counterstained with haematoxylin and observed under a microscope (Leica DMI 3000 B). Biomarkers determination in lung tissues The levels of monocyte chemoattractant protein 1 (MCP-1), interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), and interferon-β (IFN-β) in lung homogenates (n = 8) were detected by an enzyme-linked immunosorbent assay (ELISA), according to the manufacturers’ instructions (Boatman, Shanghai, China). Levels of MDA in lung tissues were evaluated by kits following the manufacturers’ instructions (Jiang Cheng, Nanjing, China) Determination of lung virus titers Viral titers in the homogenised lung samples were determined using the 50% tissue culture infective dose (TCID50) method according to a previous report (Zhi et al., 2019). Lung homogenates were serially diluted 10-fold and inoculated in corresponding wells of MDCK cells in rows. An MTT assay was used to evaluate cell viability in each well. TCID50 was calculated using the Reed-Muench formula (n = 8) (Ramakrishnan, 2016). Neuraminidase (NA) inhibition assay The NA inhibitory activity was evaluated as in a previous report (Zhi et al., 2019). Homogenized lung samples (50 µl) from each group (n = 8) were added into a 96-well plate with 50 µl of MU-NANA. After incubation at 37 °C for 30 min, the fluorescence was measured with the excitation at 360 nm and emission at 460 nm. Western blot analysis 10

Western blotting was used to further validate the expression of TLR3, TLR4, and TLR7 and levels of NF-κB p65 phosphorylation in the lung tissues. It was conducted as in a previous report (Ding et al., 2017). After SDS-PAGE and electrophoretic transfer, the membranes were incubated with primary anti-TLR3, anti-TLR4, anti-TLR7, or anti-NF-κB p65(p) antibody overnight at 4 ℃. Then, the membranes were washed three times with phosphate buffer solution with Tween (PBST) and incubated with the secondary antibody for 1 h at 37 °C. The membranes were visualised with a Universal Hood II system (Bio-Rad, Hercules, CA, USA) and analysed using Image Lab v5.1 (Bio-Rad). In vitro antiviral and NA inhibition evaluation In vitro antiviral evaluation was undertaken according to Zhi et al (2019). MDCK cells were cultured in 96-well plates for 24 h and infected with H1N1 virus for 2 h. Then, cells were treated with maintenance medium or HCF solution (50, 100, or 200 µg/ml) at 37 °C. MTT assays were performed and TCID 50 was calculated using the Reed-Muench formula (Ramakrishnan, 2016) (n = 3 replicates for each treatment). The in vitro NA inhibitory effect was evaluated based on 4-MU-NANA absorbance as described above (Zhi et al., 2019). HCF was diluted to concentrations of 50, 100 and 200 µg/ml in 2-(N-morpholino)ethanesulfonic acid NaOH buffer (pH 6.5) containing 4 mM CaCl2. Then, HCF solutions were mixed with H1N1 virus fluid and kept at room temperature for 10 min. 4-MU-NANA (1 mM) was added and incubated for another 30 min. The NA inhibitor oseltamivir was used as the positive control (working concentration: 2.5 µM). Fluorescence was measured at an excitation/ emission of 360/460 nm (n = 3 replicates for each treatment). Poly (I:C), LPS, and imiquimod stimulation on RAW 264.7 cells TLR agonists (TLR3: Poly (I:C); TLR4: LPS; TLR7: imiquimod) were used to activate the corresponding TLR pathway in RAW 264.7 cells (Zhou et al., 2013; Zhu et al., 2016; To et al., 2014). Cells were seeded in 96-well plates (1 × 106 cells/well) and incubated for 24 h in a humidified chamber (37 °C, 5% CO 2). Poly (I:C) (50 µM), 10 ng/ml LPS, or 25 µg/ml imiquimod were added to each well, along with the HCF (50, 100, or 200 µg/ml) solution. After incubation for 24 h, the levels of IL-6 and 11

IFN-β in cell supernatants were measured (n = 3 replicates for each treatment). Immunocytochemistry (ICC) was used to analyse NF-κB p65 phosphorylation and nuclear translocation as previously reported (Yang et al., 2016). RAW 264.7 cells were seeded in glass-bottom culture dishes and incubated for 24 h. Cells were then stimulated with TLR agonists as mentioned above and treated with 200 µg/ml of HCF for 24 h. After treatment, cells were fixed with 4% paraformaldehyde and incubated in 0.25% Triton X-100 for 10 min. After blockage by 1% BSA, cells were incubated with NF-κB p65(p) antibody overnight at 4 °C. Then, cells were incubated with a secondary antibody conjugated to Alexa Fluor 488 dye (green) in 1% BSA. The nucleus was stained by 4',6-diamidino-2-phenylindole (DAPI) for 15 min. Cells were observed at 630× magnification with a laser confocal microscope (LSM800; Zeiss, Oberkochen, Germany). In vitro antiviral and TLR-inhibitory effects of HCF and four major flavonoid glycosides In vitro experiments were performed to further explain the roles of four flavonoid glycosides (rutin, hyperin, isoquercitrin, and quercitrin) in HCF on its antiviral and TLR-inhibitory effects. In addition, a flavonoid glycoside mixture (MIX) was prepared as a reference control to further elucidate whether the activity of HCF was mainly due to the four flavonoid glycosides, rather than to other compounds in HCF. According to the HPLC analysis of the composition of HCF, HCF 100 µg/ml contained 8.8 µg/ml of rutin, 26.7 µg/ml of hyperin, 9.9 µg/ml of isoquercitrin, and 31.7 µg/ml of quercitrin. Four flavonoid glycoside standards (rutin, hyperin, isoquercitrin, and quercitrin, purity > 98% each) were mixed to obtain MIX solution (working solution: rutin 17.6 µg/ml, hyperin 53.4 µg/ml, isoquercitrin 19.8 µg/ml and quercitrin 63.4 µg/ml). The final solution of MIX on cells contained rutin 8.8 µg/ml, hyperin 26.7 µg/ml, isoquercitrin 9.9 µg/ml and quercitrin 31.7 µg/ml, which was equivalent to HCF (100 μg/ml). The inhibitory effect of HCF, MIX, rutin, hyperin, quercitrin and isoquercitrin (100 µg/ml) on virus proliferation and TLR signalling were evaluated according to the methods mentioned in the sub-section In vitro antiviral and NA inhibition evaluation and Poly (I:C), LPS, and imiquimod treatment 12

on RAW 264.7 cells.

Statistical analysis Data are expressed as mean ± standard deviation (SD) with statistical comparisons made using the one-way analysis of variance (ANOVA). All statistical tests were performed using GraphPad Prism (GraphPad Software Version 6.0, San Diego, CA, USA). P < 0.05 was considered significant.

Results HPLC analysis of HCF The total flavonoid glycoside content of HCF was 78.5% (flavonoid glycoside (g)/HCF (g)), calculated by the standard curve of quercitrin. The extraction rate of HCF from H. cordata was 1.64 g/100 g of raw material. Fig 1 shows the HPLC profile of HCF and the mixed standards. By comparing the retention times of HPLC peaks of the HCF sample and the mixed standards, four major flavonoid glycosides in HCF were identified as rutin, hyperin, isoquercitrin, and quercitrin. The proportions of major flavonoid glycosides in HCF were calculated based on the standard curves obtained. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8% ± 1.2%, 26.7% ± 8.5%, 9.9% ± 1.1%, and 31.7% ± 7.8%, respectively (Fig 1B; Table S1; Table S2). Effect of HCF on survival rate and life span of mice challenged with the lethal dose of the H1N1 virus The therapeutic effect of HCF was evaluated on mice challenged with the lethal dose of the H1N1 virus. Mice infected with 10 × LD50 of H1N1 virus presented clinical symptoms such as ruffled fur, huddling, lethargy, and poor appetite. Mice in the normal group (uninfected) remained active and were all alive during 14 d observation (survival rate: 100%, Fig 2A). Mice in the model group began to die on day 4 and were all dead by day 8 (survival rate: 0%). Treatment with HCF 50, 100, 200 mg/kg and Rb 100 mg/kg significantly increased the survival to 27%, 36%, 54%, and 82%, respectively (p < 0.05, Fig 2A). 13

Compared to the life span in the normal group (>14 d), virus infection decreased the life span to 6.64 ± 1.27 d in the model group (p < 0.001, Fig. 2B). Compared to the life span in the model group, HCF 50, 100, 200 mg/kg and Rb 100 mg/kg significantly prolonged the life span to 9.73 ± 2.87 d, 10.36 ± 3.16 d, 11.64 ± 2.91 d and 13.54 ± 1.08 (p < 0.05). These results indicated that HCF reduced the mortality induced by 10 × LD50 H1N1 virus challenge. Effect of HCF on ALI induced by H1N1 virus in mice The effects of HCF were further validated on H1N1-induced ALI in mice. As shown in Fig 2C, on Day 4, mice in the normal group increased 17.92% ± 4.55%, compared to day 0. Mice in the model group exhibited a considerable weight loss (12.98 ± 3.31% compared to day 0). Treatment of HCF 50, 100, 200 mg/kg and Rb 100mg/kg effectively prevented the loss and presented slight weight increase on day 4 (11.38% ± 8.51%, 11.22% ± 9.85%, 12.26% ± 8.84% and 5.48% ± 6.05%, respectively, compared to day 0). A significantly higher lung index was observed in the model group (13.30 ± 1.12) than that in the normal group (7.40 ± 0.43; p < 0.001; Fig 2D), which suggested that severe lung oedema occurred in virus-infected mice. Compared to lung index in the model group, treatment with HCF 50, 100, 200 mg/kg and Rb 100 mg/kg attenuated oedema in the lungs (lung indexes: 11.79 ± 0.87, 11.92 ± 0.89, 11.08 ± 0.56 and 8.48 ± 0.58, respectively), as shown in Fig 2D. The percentage of lung injury area was analysed. As shown in Fig 2E, H1N1 infection significantly elevated the percentage of lung injury area in the model group (45.77 ± 12.59%) compared to the normal group (p < 0.001). HCF 100, 200 mg/kg and Rb 100 mg/kg significantly decreased the percentage of lung injury (23.89 ± 3.95%, 17.11 ± 8.44% and 13.27 ± 6.38%, Fig 2E) compared to the model group (p < 0.001). Histopathological changes and inflammatory cell infiltration in the lungs were analysed by HE and IHC staining, respectively. As shown in Fig 2F, no histological change was observed in the normal group. Mice in the model group displayed severe pneumonia symptoms, such as the disappearance of pulmonary alveoli, extensive 14

consolidation, and massive macrophage and neutrophil infiltration (Fig 2F). The histological damages were relieved by HCF and Rb treatment. Compared to the model group, lung tissues from the HCF and Rb groups presented relieved tissue consolidation, more intact pulmonary alveoli and reduced macrophages or neutrophils infiltration, compared to model group (Fig. 2F). MCP-1 and IL-8 are important chemokines for macrophage and neutrophil recruitment; thus, contribute to fatal outcomes during H1N1 virus infection (Coates et al., 2018; Ito et al., 2015). As shown in Fig 2G, the levels of MCP-1 were higher in the model group (335.95 ± 27.17 pg/mg) than those in the normal group. HCF 50, 100, 200 mg/kg and Rb 100mg/kg significantly reduced MCP-1 secretion (241.99 ± 22.73, 213.14 ± 21.52, 159.27 ± 25.80 pg/mg and 136.41 ± 17.93 pg/mg, respectively, p < 0.05). The level of IL-8 was upregulated in the model group (21.20 ± 6.67 pg/mg) compared to the normal group (p < 0.001). Treatment with HCF 50, 100, 200 mg/kg and Rb 100 mg/kg significantly decreased the levels of IL-8 (14.91 ± 4.77, 13.11 ± 3.65, 12.14 ± 2.41 pg/mg and 10.09 ± 2.22 pg/mg, respectively, p < 0.05) in comparison to the model group (Fig 2H). Rb downregulated the levels of MCP-1 and IL-8 effectively compared to the model group (p < 0.05; Fig 2G and H). TNF-α is the prototypical proinflammatory cytokine in an influenza-induced cytokine storm. It promotes the severity of disease during infection (Guo and Thomas, 2017). Compared to the TNF-α levels in the normal group, the levels of TNF-α were increased to 256.07 ± 65.42 pg/mg in the model group by virus infection and reduced by treatment of HCF 50, 100, 200 mg/kg and Rb 100 mg/kg significantly (185.67 ± 45.98, 192.42 ± 40.21, and 165.82 ± 33.84 pg/mg and 142.18 ± 27.10 pg/mg, respectively, p < 0.05, Fig 2I). As a lipid peroxidation product, malondialdehyde (MDA) is a typical marker for the severity of oxidative damage and participates in the pathogenesis of influenza infection (Ng et al., 2014). As shown in Fig 2J, virus infection significantly increased the production of MDA in the model group (2.32 ± 0.21 nmol/mg, p < 0.001) compared to the normal group. Treatment of HCF 50, 100, 200 mg/kg and Rb 100 mg/kg decreased the levels of MDA to 1.84 ± 0.07, 1.58 ± 0.12, 1.30 ± 0.07 nmol/mg and 1.18 ± 0.12 nmol/mg, respectively (p < 0.05; Fig 2J). 15

Rb treatment significantly decreased the levels of TNF-α and MDA compared to the model group (p < 0.05; Fig 2I and J). These results indicated that HCF efficiently mitigated H1N1-induced ALI and pulmonary inflammation in mice. Antiviral effect of HCF on H1N1-infected mice To elucidate the underlying mechanisms, the antiviral effects of HCF in mice were explored. Virus titers of the lungs were measured in each group. Compared to the virus titres in the normal group, virus titers in the model group increased upon infection (TCID50: 6.54 ± 1.20). Oral administration of HCF 50, 100, 200 mg/kg and Rb 100 mg/kg significantly decreased the virus titers (TCID50: 3.73 ± 1.19, 2.87 ± 0.48, 2.12 ± 0.52 and 2.60 ± 0.32, respectively) in the lungs (p < 0.001, Fig 3A). NA is a major target for influenza antivirals (Behzadi et al., 2019). As shown in Fig 3B, NA activities in the model group were increased by virus infection (20716 ± 2205) compared to the normal group. They were significantly suppressed by treatment with HCF 50, 100, 200 mg/kg and Rb 100 mg/kg: 16780 ± 3224, 14113 ± 2625, 13752 ± 2273 and 14361.00 ± 1439.29, respectively) (p < 0.05, Fig. 3B). IFN-β, a typical type I interferon, plays key roles in restricting virus replication and spread during IAV infection (Killip et al., 2017). Compared to the level of IFN-β in the normal group (44.33 ± 4.29 pg/mg), virus infection significantly decreased the level of IFN-β in the model group (32.5 ± 3.83 pg/mg) (p < 0.001, Fig 3C). HCF treatment (100 and 200 mg/kg) increased IFN-β secretion (39.83 ± 2.83 and 42.16 ± 4.61 pg/mg, respectively) in comparison with the model group (p < 0.05, Fig 3C). In contrast, no increase in IFN-β secretion was observed in the Rb group (p > 0.05, Fig 3C). The viral antigens in the lungs were further detected by IHC analysis. As shown in Fig 3D, a large number of viral antigens were observed in lung tissues from the model group, whereas no positive staining was observed in the normal group. Mice treated with HCF (50, 100, and 200 mg/kg) and Rb (100 mg/kg) showed minimal viral antigen staining in lung slices (Fig 3D), which was consistent with the result of the viral titers of lungs (Fig 3A). 16

TLRs-inhibitory effect of HCF in H1N1-infected mice TLRs are important pattern recognition receptors (PRRs) during influenza infection (Lester and Li, 2014). A series of studies indicate that activation of TLRs may be attributable to the massive production of pro-inflammatory mediators during IAV infection (Le Goffic et al., 2006; Imai et al., 2008). The results in Fig 4A and 4B indicate that, compared to the expression in the normal group, the expression of TLR3, TLR4, TLR7, and NF-κB p65(p) in lung tissues were upregulated in the model group and effectively downregulated by HCF treatment (50, 100, and 200 mg/kg) and Rb (100 mg/kg) (Fig 4A and B). Antiviral effect of HCF in vitro Whether HCF had a direct antiviral effect was demonstrated in cells. MDCK cells were infected with the H1N1 virus. As shown in Fig 5A, compared to the untreated cells (Log10 TCID50: 0.03 ± 0.01), virus titers were increased upon infection in the virus group (Log10 TCID50: 5.95 ± 0.10) and decreased with treatment of HCF (50 μg/ml, 3.86 ± 0.08; 100 μg/ml, 2.78 ± 0.14; 200 μg/ml, 2.38 ± 0.19) and Rb (100 μg/ml, 1.56 ± 0.11) (p < 0.001). The NA inhibitory effect of HCF was further validated in vitro. Compared to the MU-NANA absorbance in the blank control (118 ± 53), MU-NANA absorbance (reflecting relative NA activity) in the virus group increased (16483 ± 543, Fig 5B). HCF treatment suppressed viral NA activity (50 μg/ml, 6899 ± 774; 100 μg/ml, 4239 ± 642; and 200 μg/ml, 2879 ± 1158, Fig 5B). Oseltamivir also suppressed NA activity (2.5 μM, 2513 ± 694). TLR-inhibitory effect of HCF in vitro Cell models were used to show whether HCF exerted direct TLR-inhibitory effects. Briefly, TLR3/4/7 pathways in RAW 264.7 cells were activated by related agonists (TLR3: Poly (I:C); TLR4: LPS; TLR7: imiquimod). The levels of downstream IL-6 and IFN-β were measured to indicate the status of activation. As shown in Fig 6A, compared to IL-6 secretion in untreated cells (166.74 ± 4.32 pg/ml), Poly (I:C) induced the increasing of IL-6 in RAW 264.7 cells (205.98 ± 12.39 pg/ml; p < 0.001). HCF treatment, especially 100 and 200 µg/ml, 17

downregulated the levels of IL-6 (100 µg/ml, 174.12 ± 14.75 pg/ml; 200 µg/ml, 125.05 ± 3.28 pg/ml) when compared to untreated cells (p < 0.001). On IFN-β secretion, Poly (I:C) induced the elevation of IFN-β secretion (158.33 ± 15.21 pg/ml), compared to cell group (121.12 ± 5.09 pg/ml; p < 0.001). HCF downregulated the levels of IFN-β (50 µg/ml: 138.21 ± 3.67 pg/ml; 100 µg/ml: 130.44 ± 4.14 pg/ml; 200 µg/ml:106.55 ± 8.11 pg/ml) when compared to untreated cells (p < 0.001). The positive control dexamethasone also downregulated the levels of IL-6 and IFN-β when compared to untreated cells (p < 0.001). On LPS stimulation, compared to the untreated cells (156.27 ± 7.89 pg/ml), the level of IL-6 in LPS treated cells were upregulated (241.34 ± 3.98 pg/ml). HCF decreased the levels of IL-6 (50 µg/ml, 183.55 ± 4.02 pg/ml; 100 µg/ml, 169.31 ± 4.89 pg/ml; 200 µg/ml, 167.44 ± 4.75 pg/ml) in comparison with the only LPS stimulated group (p < 0.001). Dexamethasone also decreased the level of IL-6 compared to untreated cells (p < 0.001). The secretion of IFN-β was affected neither by LPS, HCF nor dexamethasone treatment (Fig 6B). As indicated in Fig 6C, compared to IL-6 secretion in untreated cells (162.88 ± 4.53 pg/ml), imiquimod-stimulated TLR7 activation increased the level of IL-6 to 217.28 ± 3.11 pg/ml (p < 0.001). HCF treatment downregulated the level of IL-6 (50 µg/ml, 185.44 ± 3.72 pg/ml; 100 µg/ml, 156.77 ± 5.13 pg/ml; 200 µg/ml, 141.13 ± 4.65 pg/ml) in comparison with imiquimod treated cells (p < 0.001). Imiquimodstimulated TLR7 activation also increased IFN-β secretion (163.85 ± 3.18 pg/ml), compared to untreated cells (124.75 ± 5.21 pg/ml; p < 0.001). HCF treatment downregulated the level of IFN-β (50 µg/ml, 128.43 ± 4.15 pg/ml; 100 µg/ml, 124.22 ± 5.22 pg/ml; 200 µg/ml, 101.21 ± 4.65 pg/ml) in comparison with imiquimod treated cells (p < 0.001). The inhibitory effects of prednisone on IL-6 and IFN-β secretion were similar to HCF 200 µg/ml. NF-κB plays a central role in inflammatory regulation and is closely associated with TLR3/TLR4/TLR7 activation and the production of downstream inflammatory mediators such as IL-6 (Sandra et al., 2014). NF-κB p65 phosphorylation and nuclear translocation were measured using ICC. As shown in Fig 6D, upon the stimulation of 18

Poly (I:C), LPS, and imiquimod, the phosphorylation and nucleus accumulation of NF-κB p65 were increased in RAW 264.7 cells, which were strongly suppressed by HCF at 200 µg/ml. In vitro antiviral and TLR-inhibitory effects of four major flavonoid glycosides in HCF In vitro experiments were performed to further explain the roles of the four main flavonoid glycosides in HCF on its antiviral and TLR-inhibitory effects with MIX was the reference control. As shown in Fig 7A, TCID50 was significantly increased in virus-infected cells, compared to the uninfected cells. HCF, MIX and Rb significantly decreased TCID50 compared to the virus-infected group (p < 0.001). Treatment with 100 µg/ml of rutin, hyperin, and quercitrin significantly reduced TCID50 compared to the virus-infected cells (p < 0.001). Isoquercitrin showed no positive antiviral effects on cells. Fig 7B showed that HCF, MIX and oseltamivir decreased NA activity significantly, compared to untreated virus samples (p < 0.001). Rutin, hyperin, and quercitrin showed significant inhibitory effects on NA activity compared to untreated virus samples (p < 0.001, Fig 7B). Isoquercitrin did not suppress NA activity in vitro (Fig 7B). In TLR3-activated cells, the levels of IL-6 and IFN-β were significantly upregulated in Poly (I:C) stimulated cells when compared to the unstimulated cells (p < 0.001) (Fig 7C). As shown in Fig 7C, treatment with HCF and MIX significantly downregulated the levels of IL-6 and IFN-β compared to the Poly (I:C) stimulated group (p < 0.001). Hyperin and quercitrin, rather than rutin and isoquercitrin, showed significant inhibitory effects on TLR3-activated IL-6 and IFN-β secretion (p < 0.01) (Fig 7C). The positive control dexamethasone also downregulated the levels of IL-6 and IFN-β compared to the Poly (I:C) stimulated group (p < 0.001). In TLR4-activated cells, the levels of IL-6 were significantly upregulated compared to the unstimulated cells (p < 0.001) (Fig 7D). As shown in Fig 7D, HCF and MIX significantly downregulated the levels of IL-6 compared to LPS stimulated cells (p < 0.001). Among the four flavonoid glycosides, hyperin and quercitrin 19

inhibited TLR4-activated IL-6 secretion (p < 0.05). Dexamethasone also inhibited IL-6 secretion in cells. The level of IFN-β was affected by neither LPS, flavonoid glycoside nor dexamethasone treatment. In TLR7-activated cells, imiquimod stimulation significantly upregulated the levels of IL-6 and IFN-β compared to the unstimulated cells (p < 0.001) (Fig 7E). Treatment with HCF and MIX significantly suppressed the increase of IL-6 and IFN-β (p < 0.01). Hyperin and quercitrin inhibited TLR7-activated IL-6 and IFN-β secretion (p < 0.01), whereas rutin and isoquercitrin had little effect. Prednison significantly downregulated the levels of IL-6 and IFN-β compared to imiquimod stimulated group (p < 0.001). These results indicated that hyperin and quercitrin played crucial roles in the antiviral and TLR-inhibitory effects of HCF.

Discussion A series of findings indicated that H. cordata inhibited several viruses in vitro (Yang and Jiang, 2009). The steam distillate prepared from fresh plants of H. cordata had direct virucidal effects against influenza virus, human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 1 (HSV-1) in vitro (Yang and Jiang, 2009). Quercitrin, a flavonoid glycoside in H. cordata, possessed strong inhibitory effect on influenza A/WS/33 virus by reducing the formation of a visible CPE on virus-infected cells (Choi et al., 2009). Four flavonoid derivatives also displayed inhibitory activities against HSV-1 in vitro (Chen et al., 2013). In contrast, in vivo studies on antiviral activity of H. cordata were relatively few. Essential oil from H. cordata protected the chicken embryos against infectious bronchitis virus (IBV) challenge (Yin et al., 2011). Quercitrin decreased weight loss, mortality, and virus titers in influenza A/WS/33 virus-infected mice, with the in vivo mechanisms not fully elucidated (Choi et al., 2012). As was reported by Zhang et al., besides quercitrin, H. cordata also contained other flavonoid glycosides such as hyperin and rutin as well (Zhang et al, 2007). Therefore, the activity of quercitrin could not fully represent the overall therapeutic effect and action mechanism of HCF in preventing influenza 20

infection. Our study demonstrated for the first time that HCF effectively attenuated H1N1-induced ALI in mice. Investigation on action mechanism further revealed that HCF exerted dual inhibition of viral NA activity and TLR signalling both in vivo and in vitro. In addition, we also found that the effects of HCF were mainly due to four flavonoid glycosides, while hyperin and quercitrin were two major pharmacological components in HCF. In H1N1 lethally infected mice, HCF treatment significantly improved the survival rate and life span. In H1N1-induced ALI, HCF alleviated weight loss, decreased lung index and ameliorated IAV-induced lung pathological damages. Pulmonary inflammation were also attenuated by HCF, indicated by reduced recruitment and infiltration of macrophages and neutrophils, as well as downregulated expressions of inflammatory biomarkers (TNF-α and MDA) in lungs. These observations suggested that HCF exerted significant therapeutic effect on H1N1induced viral pneumonia in mice. Then, the antiviral mechanism of HCF was investigated both in vivo and in vitro. NA is an essential enzyme in the virus life cycle and is a major target for antiviral drugs (Behzadi et al., 2019). This study demonstrated that HCF effectively inhibited viral replication and suppressed NA activities in H1N1-infected mice. In vitro experiments validated that HCF, especially major flavonoid glycosides in it, had direct antiviral effects by the inhibiting viral NA activities. Further comparisons among HCF and four flavonoid glycosides revealed that rutin, hyperin, and quercitrin had similar inhibitory effects on virus proliferation and NA activity, indicated that they were the main antiviral compounds in HCF. Anti-inflammatory therapy has been gradually recognized as a novel strategy to curb IAV-induced pathological progress (Behzadi et al., 2019). TLR3, TLR4, and TLR7 are the most important PRRs for IAV recognition and antiviral response initiation (Sandra et al., 2014). However, TLR activation may play detrimental roles in IAV prevention by the stimulation of over-secretion of pro-inflammatory mediators during IAV infection (Le Goffic et al., 2006; Imai et al., 2008). The study on 21

anti-inflammatory mechanism of HCF indicated that HCF could inhibited TLR signalling both in vivo and in vitro. In H1N1 infected mice, treatment with HCF reduced the overexpression of TLR3, TLR4, and TLR7 and the levels of downstream NF-κB p65 phosphorylation in lungs. In TLR3, TLR4, and TLR7 agonist-stimulated cell models, HCF inhibited TLR agonist-activated cytokine secretion, as well as downstream NF-κB p65 phosphorylation and nuclear translocation. Further explorations were conducted among HCF and four major flavonoid glycosides on TLR-inhibitory effects in vitro. The results showed that hyperin and quercitrin exerted similar inhibitory potency on TLR signalling, whereas rutin and isoquercitrin had little effect. Therefore, hyperin and quercitrin were the major TLR-inhibitory components in HCF. It is increasingly evident that the clinical outcomes of IAV-induced lung injury is determined by both viral and host factors (Herold et al., 2015). This study demonstrated that flavonoid glycosides in H. cordata not only targeted viral replication machinery, but also interfered with influenza virus-related host TLR signalling. The action mode of HCF on reducing the severity of IAV-induced lung injury were consistent with the novel therapeutic strategy against IAV infection raised in recent years (Herold et al., 2015). In addition, besides quercitrin, which was previously discovered to possess potential anti-influenza virus activity (Choi et al., 2009; 2012), hyperin was also a very important bioactive compound with dual inhibitory effects on viral NA activity and host TLR signalling, which should not be ignored in H. cordata.

Conclusion We demonstrated for the first time that a mixture of flavonoid glycosides from H. cordata with rutin, hyperin, isoquercitrin and quercitrin as the markers, in the content of 8.8%, 26.7%, 9.9% and 31.7%, effectively alleviated H1N1-induced ALI in mice. The therapeutic effect of HCF were due to its dual antiviral and anti-inflammatory effects, possibly via inhibition of viral NA activity and host TLR signalling. Among the four major flavonoid glycosides, hyperin and quercitrin played key roles in the 22

effect of HCF.

Conflict of interest The authors have declared no conflict of interest.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant no. 81330089). We are also grateful to Mr Zhang Yongkang for providing a professional photographic work of H. cordata which is presented in the graphical abstract.

23

Li-jun Ling：Investigation, Formal analysis, Methodology, Data Curation, Software, Writing - Original Draft, Writing - Review & Editing, Visualization Yan Lu：Writing - Review & Editing, Project administration, Conceptualization Yun-yi Zhang: Writing - Review & Editing, Conceptualization Hai-yan Zhu：Resources, Methodology Peng Tu：Validation, Methodology Hong Li: Resources, Writing - Review & Editing, Project administration, Supervision, Conceptualization Dao-feng Chen: Funding acquisition, Conceptualization, Project administration, Supervision

References: Ahmad, A., Ahad, A., Rao, A.Q., Husnain, T., 2015. Molecular docking based screening of neem-derived compounds with the NS1 protein of Influenza virus. Bioinformation 11, 359-365. Ahn, J., Chae, H., Chin, Y., Kim, J., 2017. Alkaloids from aerial parts of Houttuynia cordata and their anti-inflammatory activity. Bioorg Med Chem Lett 27, 2807-2811. Behzadi, M.A., Leyva-Grado, V.H., 2019. Overview of current therapeutics and novel candidates against influenza, respiratory syncytial virus, and middle east respiratory syndrome coronavirus infections. Front Microbiol 10, 1327. Chen, S.D., Li, T., Gao, H., Zhu, Q.C., Lu, C.J., Wu, H.L., Peng, T., Yao, X.S., 2013. Anti HSV-1 flavonoid derivatives tethered with houttuynin from Houttuynia cordata. Planta Med 79, 1742-1748. Chiow, K.H., Phoon, M.C., Putti, T., Tan, B.K., Chow, V.T., 2016. Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercitrin

24

and cinanserin on murine coronavirus and dengue virus infection. Asian Pac J Trop Med 9, 1-7. Choi, H.J., Song, J.H., Kwon, D.H., 2012. Quercetin 3-rhamnoside exerts antiinfluenza A virus activity in mice. Phytother Res 26, 462-464. Choi, H.J., Song, J.H., Park, K.S., Kwon, D.H., 2009. Inhibitory effects of quercetin 3-rhamnoside on influenza A virus replication. Eur J Pharm Sci 37, 329-333. Coates, B.M., Staricha, K.L., Koch, C.M., Cheng, Y., Shumaker, D.K., Budinger, G., Perlman, H., Misharin, A.V., Ridge, K.M., 2018. Inflammatory monocytes drive influenza A virus-mediated lung injury in juvenile mice. J Immunol 200, 2391-2404. Ding, X., Jin, S., Tong, Y., Jiang, X., Chen, Z., Mei, S., Zhang, L., Billiar, T.R., Li, Q., 2017. TLR4 signalling induces TLR3 up-regulation in alveolar macrophages during acute lung injury. Sci Rep 7, 34278. Guo, X.J., Thomas, P.G., 2017. New fronts emerge in the influenza cytokine storm. Semin Immunopathol 39, 541-550. Herold, S., Becker, C., Ridge, K.M., Budinger, G.R., 2015. Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J 45, 1463-1478. Hsu, C., Yang, H., Ho, J., Yin, M., Hsu, J., 2016. Houttuynia cordata aqueous extract attenuated glycative and oxidative stress in heart and kidney of diabetic mice. Eur J Nutr 55, 845-854. Imai, Y., Kuba, K., Neely, G.G., Yaghubian-Malhami, R., Perkmann, T., van Loo, G., Ermolaeva, M., Veldhuizen, R., Leung, Y.H., Wang, H., Liu, H., Sun, Y., Pasparakis, M., Kopf, M., Mech, C., Bavari, S., Peiris, J.S., Slutsky, A.S., Akira, S., Hultqvist, M., Holmdahl, R., Nicholls, J., Jiang, C., Binder, C.J., Penninger, J.M., 2008. Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 133, 235-249. Killip, M.J., Jackson, D., Perez-Cidoncha, M., Fodor, E., Randall, R.E., 2017. Single-cell studies of IFN-beta promoter activation by wild-type and NS1-defective influenza A viruses. J Gen Virol 98, 357-363. 25

Le Goffic, R., Balloy, V., Lagranderie, M., Alexopoulou, L., Escriou, N., Flavell, R., Chignard, M., Si-Tahar, M., 2006. Detrimental contribution of the Toll-like receptor (TLR) 3 to influenza A virus-induced acute pneumonia. PLoS Pathog 2, e53. Lee, J.H., Ahn, J., Kim, J.W., Lee, S.G., Kim, H.P., 2015. Flavonoids from the aerial parts of Houttuynia cordata attenuate lung inflammation in mice. Arch Pharm Res 38, 1304-1311. Lester, S.N., Li, K., 2014. Toll-Like receptors in antiviral innate immunity. J Mol Biol 426, 1246–1264. Li, W., Zhou, P., Zhang, Y., He, L., 2011. Houttuynia cordata, a novel and selective COX-2 inhibitor with anti-inflammatory activity. J Ethnopharmacol 133, 922-927. Modepalli, V., Hinds, L.A., Sharp, J.A., Lefevre, C., Nicholas, K.R., 2016. Marsupial tammar wallaby delivers milk bioactives to altricial pouch young to support lung development. Mech Develop 142, 22-29. Muluye, R.A., Bian, Y., Alemu, P.N., 2014. Anti-inflammatory and antimicrobial effects of heat-clearing Chinese Herbs: a current review. J Tradit Complement Med 4, 93-98. Ng, M.P., Lee, J.C., Loke, W.M., Yeo, L.L., Quek, A.M., Lim, E.C., Halliwell, B., Seet, R.C., 2014. Does influenza A infection increase oxidative damage? Antioxid Redox Signal 21, 1025-1031. Ramakrishnan, M.A., 2016. Determination of 50% endpoint titer using a simple formula. World J Virol 5, 85. Shingnaisui, K., Dey, T., Manna, P., Kalita, J., 2018. Therapeutic potentials of Houttuynia cordata Thunb. against inflammation and oxidative stress: A review. J Ethnopharmacol 220, 35-43. Tian, L., Wang, Z., Wu, H., Wang, S., Wang, Y., Wang, Y., Xu, J., Wang, L., Qi, F., Fang, M., Yu, D., Fang, X., 2011. Evaluation of the anti-neuraminidase activity of the traditional Chinese medicines and determination of the anti-influenza A virus effects of the neuraminidase inhibitory TCMs in vitro and in vivo. J 26

Ethnopharmacol 137, 534-542. To, E.E., Broughton, B.R., Hendricks, K.S., Vlahos, R., Selemidis, S., 2014. Influenza A virus and TLR7 activation potentiate NOX2 oxidase-dependent ROS production in macrophages. Free Radic Res 48, 940-947. Wu, L., Si, J., Yuan, X., Shi, X., 2009. Quantitive variation of flavonoids in houttuynia cordata from different geographic origins in China. Chin J Nat Medicines 7, 40-46. Wu, X.N., Yu, C.H., Cai, W., Hua, J., Li, S.Q., Wang, W., 2011. Protective effect of a polyphenolic rich extract from Magnolia officinalis bark on influenza virus-induced pneumonia in mice. J Ethnopharmacol 134, 191-194. Xu, Y.Y., Zhang, Y.Y., Ou, Y.Y., Lu, X.X., Pan, L.Y., Li, H., Lu, Y., Chen, D.F., 2015. Houttuynia cordata Thunb. polysaccharides ameliorates lipopolysaccharideinduced acute lung injury in mice. J Ethnopharmacol 173, 81-90. Yang, L., Jiang, J., 2009. Bioactive components and functional properties of Hottuynia cordata and its applications. Pharm Biol 47, 1154-1161. Yang, N., Dong, Z., Tian, G., Zhu, M., Li, C., Bu, W., Chen, J., Hou, X., Liu, Y., Wang, G., Jia, X., Di, L., Feng, L., 2016. Protective effects of organic acid component from Taraxacum mongolicum Hand.-Mazz. against LPS-induced inflammation: Regulating the TLR4/IKK/NF-κB signal pathway. J Ethnopharmacol 194, 395-402. Yin, J., Li, G., Li, J., Yang, Q., Ren, X., 2011. In vitro and in vivo effects of Houttuynia cordata on infectious bronchitis virus. Avian Pathol 40, 491-498. Zhang, X.X., Wu, Q.F., Yan, Y.L., Zhang, F.L., 2018. Inhibitory effects and related molecular mechanisms of total flavonoids in Mosla chinensis Maxim against H1N1 influenza virus. Inflamm Res 67, 179-189. Zhang, Y., Li, S., Wu, X., Zhao, X., 2007. Macroporous resin adsorption for purification of flavonoids in houttuynia cordata thunb. Chinese J Chem Eng 15, 872-876. Zhao, C., Zhao, X., Zhang, J., Zou, W., Zhang, Y., Li, L., Liu, J., 2016. Screening of bacillus strains from sun vinegar for efficient production of flavonoid and 27

phenol. Indian J Microbiol 56, 498-503. Zhi, H.J., Zhu, H.Y., Zhang, Y.Y., Lu, Y., Li, H., Chen, D.F., 2019. In vivo effect of quantified flavonoids-enriched extract of Scutellaria baicalensis root on acute lung injury induced by influenza A virus. Phytomedicine 57, 105-116. Zhou, Y., Guo, M., Wang, X., Li, J., Wang, Y., Ye, L., Dai, M., Zhou, L., Persidsky, Y., Ho, W., 2013. TLR3 activation efficiency by high or low molecular mass poly I:C. Innate Immun 19, 184-192. Zhu, H., Lu, X., Ling, L., Li, H., Ou, Y., Shi, X., Lu, Y., Zhang, Y., Chen, D., 2018. Houttuynia cordata polysaccharides ameliorate pneumonia severity and intestinal injury in mice with influenza virus infection. J Ethnopharmacol 218, 90-99. Zhu, T., Zhang, W., Feng, S.J., Yu, H.P., 2016. Emodin suppresses LPS-induced inflammation in RAW264.7 cells through a PPARgamma-dependent pathway. Int Immunopharmacol 34, 16-24.

28

Fig 1

Fig 2

29

Fig 3 30

Fig 4

31

32

Fig 5

Fig 6

33

fig. 7

34

35

Legends Fig 1. HPLC chromatogram of HCF. (A) HCF sample (60 μg/ml) and (B) A diluted standards mixture. 1 - Rutin; 2 - Hyperin; 3 - Isoquercitrin; 4 - Quercitrin. (C) Structures of the four standard compounds.

Fig 2. Effect of HCF on H1N1 virus-induced lethal infection or ALI in mice. (A-B) Effect of HCF on survival rate and life span of mice challenged with lethal dose of H1N1 virus (10 × LD50): (A) Survival rate of mice; (B) Life span. (C-J) Effect of HCF on acute lung injury induced by H1N1 virus in mice (3× LD50): (C) Body weight loss; (D) Lung index; (E) Percentage of lung injury area in mice; (F) Histopathological injury and inflammatory infiltration of representative lung sections in each group with 400× magnification (Bar = 50μm); (G-J) Levels of MCP-1, IL-8, TNF-α and MDA in lungs. Data were presented as mean ± SD, n = 8 for each group. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to model group (ANOVA and Fisher’s PLSD).

Fig 3. Antiviral effect of HCF in H1N1-infected mice.(A) Viral titers in lungs. (B) Viral NA activities in lungs. (C) IFN-β levels in lungs. Data were presented as mean ± SD. n=8 for each group in A-C. *p < 0.05,

**

p < 0.01,

***

p < 0.001, as compared to model group (ANOVA and Fisher’s PLSD). (D)

Immunohistochemical staining of influenza A virus antigen in representative lung sections. 400× magnification (Bar = 50μm). n = 3 for each group in D.

Fig 4. TLR-inhibitory effect of HCF in H1N1-infected mice. (A) IHC analysis of TLR3, TLR4, TLR7 and NF-κB p65(p) in lungs. 400× magnification (Bar = 50μm). (B) Western blot detection of TLR3, TLR4, TLR7 and NF-κB p65(p) in lung homogenates. β-actin was used as the internal control. n = 3 for each groups.

Fig 5. In vitro antiviral effect of HCF. (A) Effect of HCF on H1N1-infected MDCK cells. (B) In vitro inhibitory effect of HCF on viral NA activity. Data were presented as mean ± SD. n = 3 replicates for each treatment. * P < 0.05,

**

P < 0.01,

***

P < 0.001 compared to the virus or blank group (ANOVA

36

and Fisher’s PLSD).

Fig 6. In vitro TLR-inhibitory effect of HCF. TLR3/4/7 pathways in RAW 264.7 cells were stimulated by related TLR agonist respectively (TLR3: Poly (I:C); TLR4: LPS; TLR7: imiquimod). (A-C) Inhibition of HCF on TLR agonists stimulated IL-6 and IFN-β secretion. Levels of IL-6 and IFN-β upon (A) Poly (I:C) stimulation; (B) LPS stimulation; (C) imiquimod stimulation. Data were presented as mean ± SD. n=3 replicates for each group.

*

p < 0.05,

**

p < 0.01,

***

p < 0.001, as compared to

stimulated (untreated) group (ANOVA and Fisher’s PLSD). (D) Inhibition on TLR activated NF-κB p65 phosphorylation and nuclear translocation in RAW 264.7 cells. Representative cell samples from each group. White arrows indicate the location of NF-κB p65(p). Bar = 50μm. n = 3 for each group, 630× magnification.

Fig 7. In vitro antiviral and TLR-inhibitory effects of major flavonoid glycosides in HCF. (A-B) In vitro antiviral effects of major flavonoid glycosides: (A) Inhibition on virus proliferation in MDCK cells; (B) Inhibition on viral NA activity. Data were presented as mean ± SD of 3 independent replicates.

*

p < 0.05,

**

p < 0.01,

***

p < 0.001 compared to the model group (ANOVA and Fisher’s

PLSD). (C-E) TLR-inhibitory effects of four major flavonoid glycosides in RAW 264.7 cells. (C) Poly (I:C) stimulation; (D) LPS stimulation; (E) imiquimod stimulation. Data were presented as mean ± SD. n = 3 replicates for each treatment.

*

p < 0.05,

**

p < 0.01,

stimulated group (ANOVA and Fisher’s PLSD). # p < 0.05, HCF group.

37

***

##

p < 0.001, as compared with agonist

p < 0.01,

###

p < 0.001, as compared to

Graphical Abstract

38