Chrysin, as illustrated by the chemical structure in Figure 1A, is a natural flavone abundant in many plant extracts, honey, and propolis that possesses a variety of biological actions. To investigate the toxicity of chrysin on cells, MDCK cells and A549 cells, two commonly utilized influenza virus susceptible cell lines, were treated with various concentrations of chrysin, followed by the cell counting kit 8 (CCK8) assay. The results demonstrated that chrysin had no cytotoxicity in both cell lines at 12 μM (Figures 1B, C). However, chrysin impacted cell viability up to 14 μM (Figures 1B, C), suggesting that subsequent studies at concentrations less than 12 μM chrysin are required.

Influenza virus infection results in typical cell cytopathic effects (CPE) (1). To determine the antiviral activity of chrysin, a combination of chrysin and the influenza A virus (IAV) strain A/Jilin/SW1182/2013 (H1N1) was inoculated into MDCK cells and supplemented with chrysin for 24 hours. Here, we found that chrysin strongly diminished H1N1 virus-induced CPE formation on MDCK cells in a dose-dependent manner (Figure 2A). Moreover, plaque reduction assays the viral titers in the MDCK cell supernatant, indicating that chrysin can reduce H1N1 replication in the MDCK cell (Figure 2B). By using another IAV strain, A/PR/8/34 (H1N1), we found that 8 μM chrysin can similarly inhibit cytopathic effect (CPE) generation (Figure 2C) and viral titers (Figure 2D) in MDCK cells. These results reveal that chrysin has substantial antiviral efficacy against the H1N1 influenza virus.

Given that chrysin can dampen IAV infection in vitro (18), we sought to see if the viral mRNA and protein levels of HA and NP were affected in cells supplemented with chrysin. To verify this hypothesis, MDCK cells were infected at an MOI of 0.5 with two IAV strains, A/PR/8/34 and A/Jilin/SW1182/2013, in the presence or absence of 8 μM chrysin for six hours before RT-qPCR analysis. Our results showed that the levels of viral HA and NP mRNA in MDCK cells were dramatically increased upon IAV infection, whereas the upregulated expression of viral HA and NP mRNA was abrogated in the presence of chrysin (Figures 3A, B). To test whether the effect of chrysin on viral mRNA expression also affect viral protein expression, the accumulation of viral HA and NP proteins was then examined in MDCK cells and A549 cells in the presence of chrysin following A/PR/8/34 infection by western-blot analysis of cell lysates, using antibodies for the HA and NP. When the IAV infection was supplemented with 8 μM chrysin in MDCK cells, HA and NP proteins decreased about 2-fold compared with the IAV control group (Figure 3C). Likewise, the production of HA and NP proteins was also significantly reduced in chrysin-treated A549 cells after IAV infection compared with IAV-infected control cells (Figure 3D). Consistent with the viral RNA and protein results, immunofluorescence analysis on IAV strain A/PR/8/34-infected MDCK cells to assess HA and NP levels found that HA and NP production were increased in IAV-infected cells but decreased in chrysin-treated infected cells (Figures 3E, F).

Since type I and type III interferon (IFN) systems are essential in the innate immune response against viruses (19), we intend to examine whether the inhibitory action of chrysin in response to IAV infection involves inducing type I and type III interferons as well as IFN-stimulated genes (ISG) expression. RT-qPCR was used to evaluate the innate antiviral gene expression of IFN (IFNα, IFN-β1, and IFN-λ) (Supplementary Figure 1A) and ISGs (Mx1, Isg15, and Oas2) (Supplementary Figure 1B) in IAV strain A/PR/8/34 infected A549 cells in the presence of chrysin, and it was determined that chrysin had no benefit in boosting mRNA expression of IFNα, IFN-β1, IFN-λ, Mx1, Isg15, and Oas2 after IAV infection (Supplementary Figure 1). Taken together, these results demonstrate that chrysin suppresses IAV HA and NP production in an antiviral innate immunity-independent manner.

To investigate the antiviral effect mechanism of chrysin, we executed multiple models to evaluate the effectiveness of chrysin on influenza lifecycles, including premixed administration, prophylactic administration, and therapeutic administration. When chrysin was premixed with A/PR/8/34 (H1N1) for 30 minutes (Figure 4A) or 2 hours (Figure 4B), it failed to hinder H1N1 virus-induced CPE development (Figures 4A, B, upper panel) and limit virus replication (Figures 4A, B, lower panel), indicating that chrysin exerts its antiviral activity by indirect interaction with IAV. To confirm this hypothesis, we measured the binding capacity of chrysin and IAV related proteins by biolayer interferometry (BLI). As a result, BLI analyses revealed a poor affinity between chrysin and the HA, NA, M1, NS1, and NP, respectively (Supplementary Figure 2). In accordance with the findings of premixed administration, chrysin had no effect on depressing A/PR/8/34 virus-induced CPE generation (Figure 4C, upper panel) and viral titers (Figure 4C, lower panel) during prophylactic administration, implying that chrysin is ineffective throughout the early stages of the IAV lifecycle. It is worth noting that the inhibitory impact of chrysin on viral infection could be identified in therapeutic administration since the chrysin persists during the whole cycle of A/PR/8/34 virus replication (Figure 4D). Overall, these results suggest that chrysin confers an antiviral effect through indirectly interacting with IAV particles, and that chrysin has therapeutic potential when confronted with IAV infection.

Since influenza viruses can regulate the cell cycle, particularly at the G1/S transition, we wondered if the viral inhibition elicited by chrysin involves modulating cell cycle events. To address this question, A549 cells were infected with the influenza virus in the presence or absence of chrysin for flow cytometry investigation of cell cycle distribution. As expected, we were able to see an apparent accumulation of cells in the G0/G1 phase in A/PR/8/34 virus-infected cells, accompanied by reduced cell numbers in the S and G2/M phases (Figure 5A). The quantitative analysis showed that A/PR/8/34 virus-infected cells had a 21% increase in cell population in the G0/G1 phase as compared to mock-infected cells (Figure 5B). Surprisingly, chrysin can dampen the accumulation of cells in the G0/G1 phase induced by A/PR/8/34 virus infection, with a 15% reduction (Figure 5B). However, chrysin has a minor effect on S and G2/M phases (Figures 5A, B). These data indicate that chrysin can inhibit cell cycle arrest in the G0/G1 phase with IAV infection.

To further dissect the mechanism by which chrysin prevents G0/G1 cell cycle arrest, we investigated the DNA damage-mediated p53/p21 signaling pathway and checkpoint Cyclin/CDK complexes that govern the cell cycle. Western blot results revealed that IAV infection highly activated P53/P21 signaling pathways, resulting in elevated P53 and P21 protein levels, whilst chrysin clearly downregulated A/PR/8/34 virus-induced P53 and P21 protein expression (Figures 5C, D). It should be emphasized that A/PR/8/34 virus infection appears to selectively restrict cellular CDK4 and Cyclin D1 protein expression while having no influence on the expression of other checkpoint factors, including CDK6, CDK2, and Cyclin E1 (Figures 5C, D). Notably, treatment of chrysin in A/PR/8/34 virus-infected cells leads to increased expression of the proteins CDK4, Cyclin D1, and Cyclin E1 (Figures 5C, D). As a result of these findings, chrysin appears to effectively block G0/G1 phase cell cycle arrest by depressing P53/P21 signaling while promoting Cyclin D1/CDK4 and Cyclin E1/CDK2 activity.

Influenza viruses can induce cell apoptosis (20). To determine whether IAV infection caused cell apoptosis can be reversed by chrysin, we performed Annexin V staining, a classic approach for detecting apoptosis. FACS analysis showed that A/PR/8/34 virus infection massively boosts the apoptotic cell population compared with the uninfected control in A549 cells (Figures 6A, B). Importantly, these early and late apoptotic events are obviously disrupted in A/PR/8/34 virus-infected A549 cells when chrysin is supplemented (Figures 6A, B). Previous studies have demonstrated that mitochondrial pathway-mediated intrinsic apoptosis is one of the most frequent types of apoptosis, involving the release of mitochondria-related signal factors. Cells with poor mitochondrial membrane potential (MMP) have been identified as apoptotic in general. Following A/PR/8/34 virus infection, A549 cells were treated with or without chrysin, and the changes in the MMP were examined utilizing the fluorescent mitochondrial membrane potential probe JC-1. FACS analysis showed a considerable loss in MMP after A/PR/8/34 virus infection, as evidenced by accumulations of cells stained with green fluorescence after exposure to furanodienone (Figures 6C, D). By contrast, chrysin can effectively abolish A/PR/8/34 virus-induced MMP reduction (Figures 6C, D).

To support the notion that the mitochondrial apoptotic pathway is responsible for chrysin suppressing A/PR/8/34 virus-mediated apoptosis, we investigated the expression of apoptosis-related proteins, particularly Bax and Bcl-xl. We found that chrysin dramatically reduced the level of pro-apoptotic Bax protein and markedly elevated the level of anti-apoptotic Bcl-xl protein in A549 cells after A/PR/8/34 virus infection (Figures 6E, F). In A/PR/8/34 virus-infected A549 cells, western blot analysis of caspase-9 and caspase-3 activation revealed that chrysin could restrain the levels of cleaved caspase-9 and cleaved caspase-3 (Figures 6E, F). These results suggest that chrysin-restrained apoptosis occurs in response to IAV infection via a mitochondrial-dependent apoptosis mechanism featuring Bcl-xl upregulation and caspase-dependent pathway activation as well as Bax downregulation.

The ROS produced by mitochondria plays a crucial role in the control of apoptosis and cell cycle arrest. We thus investigated if the anti-apoptotic and cell cycle regulatory activities of chrysin were a result of ROS modulation within IAV-infected cells. The DCFH-DA fluorescent probe was used to measure the change in intracellular ROS levels in chrysin-treated A549 cells and MDCK cells after A/PR/8/34 virus infection. As anticipated, A/PR/8/34 virus infection markedly increased ROS production in A549 cells compared with uninfected control (Figure 7A). Likewise, the ROS level was significantly elevated in A/PR/8/34 virus-infected MDCK cells when compared to that of the uninfected control (Figure 7B). Of note, chrysin treatment remarkably reversed the A/PR/8/34 virus-induced ROS level in A549 cells and MDCK cells (Figures 7A, B). These results reveal that intracellular ROS generation is involved in the inhibitory effects of chrysin on IAV-induced apoptosis and cell cycle arrest.

To assess the protective efficacy of chrysin treatment on influenza-infected mice, C57BL/6 mice were challenged intranasally with a lethal dose of A/PR/8/34 virus before receiving an intraperitoneal or intragastric dosage of 100 mg/kg chrysin daily, while body weight and survival rate were monitored. It is disappointing that intraperitoneal chrysin treatment did not reverse IAV-induced weight loss and did not extend the survival period or improve the survival rate of mice post IAV infection (Supplementary Figures 3A, B). Consistent with these results, intragastric administration of chrysin also failed to protect mice against IAV challenge (Supplementary Figures 3C, D). We next sought to investigate whether the antiviral role of chrysin was limited to the upper respiratory tract rather than the lower respiratory tract, failing to face the challenge of IAV. To verify this hypothesis, C57BL/6 mice were administered 100 mg/kg chrysin intragastrically every day after intranasal A/PR/8/34 virus infection, and viral titers in the snout and lungs were measured at days 1 and 3 post-infection. We found at least 10-fold less infectious IAV in the snouts of chrysin-treated mice compared with untreated control between days 1 and 3 post-infection (Figure 8A), but the viral tiers in the lungs were not affected by chrysin at the indicated time points (Figure 8B). These results indicate that chrysin treatment potently suppressed IAV replication in the upper airways, which might be beneficial for restraining viral transmission.

Madin-Darby canine kidney (MDCK) cells and Adenocarcinomic human alveolar basal epithelial cells (A549 cells) from ATCC were cultured at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco: C11995500BT) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco: 10270106) and 1% penicillin/streptomycin (Gibco: 15140-122).

Influenza A viruses, A/PR/8/34 and A/Jilin/SW1182/2013, were propagated in MDCK cells and titrated as previously described (35). Viruses were stored at -80 °C until they were used in subsequently experiments.

Female C57BL/6 mice (6-8 weeks) were purchased from GemPharmatech Co., Ltd. All mice were bred at the Shenzhen University animal facility under specific pathogen-free (SPF) conditions. The institutional Animal Care and Use Committee at Shenzhen University authorized all animal experiments.

MDCK and A549 cells were seeded in 96 well culture plates at 1.0 × 10⁴ cell/well and incubated at 37°C overnight with 5% CO₂. Chrysin (Meryer, F21744-5G) was applied to the cells and incubated for 24 hours at the indicated concentrations (2, 4, 6, 8, 10, 12, 14 μM). Then the CCK-8 reagent (Dojindo, CK04) was added at 10 μl per well for 2 hours. The solution absorbance was measured at 450 nm by a microplate reader (Rayto, China).

MDCK cell were plated in 12-well plates at a density of 2.0 ×10⁵ cells/well and incubated overnight at 37°C with 5% CO₂. The next day, the various concentrations of chrysin and A/Jilin/SW1182/2013 (H1N1) (MOI = 0.5) or A/PR/8/34 (H1N1) (MOI = 0.5) were mixed at 4°C for 30 min before being added to MDCK cells at 37°C for 2 hours. The supernatant was then discarded, and the cells were washed twice with PBS (Solarbio, P1010). Finally, different quantities of chrysin were applied to the cells and they were cultured at 37°C for 24 hours. The cytopathic effect was observed under a microscope.

MDCK cells and A549 cells were treated with a combination of A/PR/8/34 (H1N1) (MOI = 0.5) and chrysin (8 μM) for 2 hours at 37°C. Then, the medium was removed, and the cells were treated with chrysin (8 μM) alone for a total of 10 hours at 37°C. Flow cytometric analysis was performed using the following kits: mitochondrial membrane potential assay kit with JC-1 (Beyotime, C2006); Reactive Oxygen Species (ROS) Assay Kit (Beyotime, S0033S); Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, 556547); Cell Cycle and Apoptosis Analysis Kit (Beyotime, C1052). Cells were collected, which were then stained according to the manufacturer’s instructions. Flow cytometric analysis was performed using an Attune™ NxT Acoustic Focusing Cytometer (Thermo Scientific, America) and the data was analyzed with Flowjo software.

MDCK cells (2.0 × 10⁵ cells) were treated with the mixture of A/PR/8/34 (H1N1) (MOI = 0.5) and Chrysin (8 μM) for 2 hours at 37°C. The medium was discarded and treated with fresh medium containing 8 μM chrysin at 37°C for 4 hours. Cells were fixed in a 4% paraformaldehyde solution (Beyotime, P0099) for 15 minutes and permeabilized in 0.2% Triton X-100 (Sigma, X100) for 15 minutes. After blocking with 3% bovine serum albumin (Sigma, A1933) for 1 hour, cells were incubated with anti-HA antibody (Thermofisher, PA5-34929) or anti-NP antibody (Thermofisher, MA5-35899) overnight at 4°C. Cells were incubated with an Alexa Fluor 488 tagged anti-rabbit IgG antibody (Cell signaling technology, ab150077) or an Alexa Fluor 647 tagged anti-mouse IgG antibody (Cell signaling technology, ab150115) at 37°C for 1 hour. After washing with PBS, the cells were stained with 1 µg/ml DAPI (Beyotime, C1006) for 20 minutes and analyzed using ZEISS ZEN digital microscope software.

Confluent MDCK cells seeded into 12-well plates were washed with PBS and inoculated with a gradient dilution of virus solution [Opti-MEM (Thermo Fisher Scientific, 31985070) with 0.3% BSA and 2.5 μg/ml TPCK-treated trypsin (Sigma, T1426)] for 2 hours. Cell monolayers were overlaid with 1.5 ml of semisolid medium (1.5% CMCNa: DMEM = 1:1, with 0.3% BSA and 2.5 μg/ml TPCK-treated trypsin) and incubated at 37°C for 30 hours after the virus inoculums were removed, followed by fixation of the cell monolayers with 4% paraformaldehyde and staining with 0.2% (w/v) crystal violet (Sigma, C0775). The size of plaques was counted and measured using Image J software.

Cells were harvested for extraction of total RNA using Trizol reagent (Takara, 9109) according to the manufacturer’s protocol. cDNA synthesis was carried out using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, K1622). After reverse transcription, qPCR amplification was performed using Tip Green qPCR SuperMix (Transgen, China, AQ141-01) on a CFX96 Real-Time PCR System (Bio-Rad, USA). The sequences of the primers are as follows: 18s: forward: 5’-CCCCTCGATGCTCTTAGCTG-3’, reverse: 5’-CTTTCGCTCTGGTCCGTCTT-3’; Ha: forward: 5’-TATTCGTCT

CAGGGAGCAAAAGCAGGGG-3’, reverse: 5’ ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3’; Np: forward: 5’-TATTCGTCTCAGGGAGCAAAAGCAGGGTA-3’, reverse: 5’-ATATCGTCTCGTATTAGTAGAAAC

AAGGGTATTTTT-3’; Ifnα: forward: 5’-AGAAGGCTCCAGCCATCTCTGT-3’, reverse: 5’-TGCTGGTAGAGT

TCGGTGCAGA-3’; Ifnβ1: forward: 5’-CTTGGATTCCTACAAAGAAGCAGC-3’, reverse: 5’-TCCTCCTTCTG

GAACTGCTGCA-3’; Ifnλ: forward: 5’-AACTGGGAAGGGCTGCCACATT-3’, reverse: 5’-GGAAGACAGGA

GAGCTGCAACT-3’; Mx1: forward: 5’-GGCTGTTTACCAGACTCCGACA-3’, reverse: 5’-CACAAAGCCTGG

CAGCTCTCTA-3’; Isg15: forward: 5’-CTCTGAGCATCCTGGTGAGGAA-3’, reverse: 5’-AAGGTCAGCCAG

AACAGGTCGT-3’; Oas2: forward: 5’-GCTTCCGACAATCAACAGCCAAG-3’, reverse: 5’-CTTGACGATTTT

GTGCCGCTCG-3’. PCR programs consist of 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 30 seconds at 61°C. The mRNA expression of target genes in each group was determined by the 2^−ΔCt method relative to the expression of 18S.

MDCK cells were seeded into 12-well plates and cultured at 37°C under 5% CO₂ for 24 hours.

For premixed administration, chrysin (8 μM) and A/PR/8/34 (H1N1) (MOI = 0.5) were mixed at 4°C for 30 min or 2 hours before being added to MDCK cells for 2 hours of treatment at 37°C. The supernatant was discarded and washed twice with PBS. Cells were cultured for 24 hours at 37°C. The supernatant was collected for a plaque assay to determine the viral titer.

For prophylactic administration, chrysin (8 μM) was added to stimulate MDCK cells for 2 hours, then the cells were infected by A/PR/8/34 (H1N1) (MOI = 0.5) for 2 hours. The medium was removed and replaced by fresh medium at 37°C for 24 hours. The supernatant was collected, and the virus titer was detected by plaque assay.

For therapeutic administration, MDCK cells were infected with A/PR/8/34 (H1N1) (MOI = 0.5) at 37°C for 2 hours. The medium was removed and replaced by fresh medium containing 8 μM chrysin at 37°C for 24 hours. The supernatant was collected, and the virus titer was determined by plaque assay.

The affinity of chrysin and His-tagged protein of influenza A virus (IAV) was determined using Ni-NTA biosensors (Forte Bio, 18-0029), onto which 1 μM of protein of influenza virus was in running buffer (PBS, 0.02% Tween-20, and 0.1% BSA) was loaded. IAV HA protein (Sino Biologicol, 40673-V08H), NA protein (Sino Biologicol, 40197-V07H), M1 protein (Sino Biologicol, 40010-V07E), NP protein (Sino Biologicol, 11675-V08B), and NS1 protein (Sino Biologicol, 40011-V07E). Chrysin was diluted to 500 μM, 250 μM, and 125 μM in the running buffer. First, the Ni-NTA sensor tips were hydrated in the buffer for 10 minutes prior to use. Then sensor baselines were equilibrated in the running buffer for 30 seconds (the initial baseline). Next, the protein was loaded until saturated for 240 seconds (the loading phase), and the sensor was washed for 120 seconds in the running buffer (baseline phase). The sensors were immersed in wells containing chrysin for 240 seconds (the association phase), followed by immersion in running buffer for another 240 seconds (the dissociation phase). Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software.

A549 cells were washed with ice-cold PBS, and lysed with RIPA buffer (Solarbio, R0010) for protein extraction, and then quantified using the BCA assay kit (Beyotime, P0012) according to the manufacturer’s instructions. They then added 5 × protein loading buffer (Meilunbio, MA0003-D) to sample and were boiled at 95°C for 5 minutes. An equivalent quantity of protein samples was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Beyotime, P0012AC) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, IPVH15150). Then on-specific binding was blocked with 5% skimmed milk (BioFroxx, 1172GR500), and it was incubated with primary antibodies against P53 Polyclonal antibody, P21 Rabbit Polyclonal antibody (Proteintech, 10355-1-AP), Cyclin D1 Rabbit Polyclonal antibody (Proteintech, 60186-1-lg), CDK4 Polyclonal antibody (Proteintech, 11026-1-AP), CDK6 Polyclonal antibody (Proteintech, 14052-1-AP), Cyclin E1 Rabbit Polyclonal antibody (Proteintech, 11554-1-AP), CDK2 Polyclonal antibody (Proteintech, 10122-1-AP), Bax antibody (Cell signaling technology, 2772), Bcl-xl rabbit polyclonal antibody (Proteintech, 26967-1-AP), Caspase 3 antibody (Cell signaling technology, 9662), Caspase 9 polyclonal antibody (Proteintech, 10380-1-AP), H1N1 NP Monoclonal Antibody (Thermo Fisher, GT1236), H1N1 HA Polyclonal Antibody (Thermo Fisher, PA5-34929), β-actin antibody (Abcam, ab8226) overnight at 4°C. After washing with Tris-buffered solution containing 0.05% Tween 20, the blots were incubated with secondary antibodies (Abcam, ab6721/ab6728) for two hours at room temperature. After five additional washes, the protein bands were visualized using an ECL reagent kit (Solarbio, PE0010) based on the manufacturer’s instructions (Carestream, USA).

Female C57BL/6 mice (6-8 weeks old) were acclimated for 7 days before experiments. Mice were intranasally infected with 18 PFU A/PR/8/34 (H1N1) viruses in a total volume of 40 μl for 2 hours before receiving chrysin (100 mg/kg) daily by either gavage or intraperitoneal injection. Weight loss and survival were monitored on a daily basis until the animals were scarified after they had lost 25% of their initial body weight. Snout and lung samples were collected on days 1 and 3 following infection for plaque assays in several repeats.

Statistical analysis was performed using GraphPad Prism 8.0. The experimental results were conducted out in at least three replicates. All data are shown as means with standard deviations (SEM). The data were statistically analyzed using unpaired two-tailed Student’s t-test or one-way ANOVA with Dunnett’s multiple comparisons test. P values are considered to indicate a significant difference, as shown in the figures as follows: *p<0.05; **p<0.01; ***p<0.001; ****P<0.0001.