王晓霞/徐帅/单超团队：阔叶山麦冬皂苷 C 对甲型流感病毒和新型冠状病毒均展现抗病毒潜力

摘要 

阔叶山麦冬皂苷 C（又名 DT-13）是从阔叶山麦冬块茎中提取的主要活性成分，具有多种药理活性，包括抗肿瘤、抗血栓、心肌保护及抗炎。然而，其抗病毒潜力尚未被研究。本研究旨在探究阔叶山麦冬皂苷 C 对甲型流感病毒和新型冠状病毒的抑制活性及其潜在作用机制。体外实验结果表明，阔叶山麦冬皂苷 C 可呈剂量依赖性地抑制甲型流感病毒在犬肾细胞（MDCK）和人肺腺癌细胞（A549）中的复制。药物添加时间实验显示，阔叶山麦冬皂苷 C 作用于流感病毒进入细胞后的复制阶段，破坏病毒核糖核蛋白（vRNP）的功能。病毒聚合酶活性受抑制、病毒 RNA 合成减少以及病毒核糖核蛋白的核输出延迟，均验证了这一作用机制。本研究进一步利用甲型 H1N1 流感病毒致死性小鼠模型验证了该化合物的体内抗病毒效果。结果显示，预防性和治疗性给予阔叶山麦冬皂苷 C 均能呈剂量依赖性地显著提高感染小鼠的存活率，降低肺组织中的病毒载量，并减轻病毒诱导的肺部炎症。除甲型流感病毒外，阔叶山麦冬皂苷 C 还可有效抑制新型冠状病毒的感染，其通过干扰病毒刺突蛋白与人血管紧张素转换酶 2（ACE2）受体的结合、抑制刺突蛋白介导的膜融合，从而阻断病毒入侵过程。综上，本研究证实阔叶山麦冬皂苷 C 对甲型流感病毒和新型冠状病毒均具有抗病毒潜力，有望成为治疗呼吸道病毒感染的候选药物。

Fig. 1. Inhibitory activity of Saponin C against H1N1 infection in vitro. A Chemical structure of Saponin C. B, C Dose-dependent effects of Saponin C on H1N1-HiBiT (MOI = 0.01) replication and cell viability in MDCK and A549 cells. D The effect of Saponin C on NP expression, as assessed by immunofluorescence assay (green: NP; blue: nucleus; 200×; Scale bar = 100μm). The data was obtained from 2–3 independent experiments performed in triplicate. *P < 0.05; **P < 0.01.

Fig. 2. The antiviral activity of Saponin C against different IAV strains. A, B The inhibitory effect of Saponin C on PR8 virus (MOI = 0.01) was evaluated in MDCK (A) and A549 (B) cells. Virus titers (TCID50/mL) were measured at the indicated time points. C–E The antiviral activity of Saponin C against H3N2 (C), H6N6 (D), and H9N2 (E) strains, respectively. Virus titers were measured at 48 h post-infection. All experiments were performed in triplicate, and data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3.  Saponin C blocked IAV replication at post-entry stages. A, B Effects of Saponin C on the attachment (A, adsorption) and entry (B, internalization) of H1N1-HiBiT virus. C Effect of Saponin C on IAV-induced aggregation of chicken erythrocytes was evaluated using a HI assay with PR8 virus (4 HAU/25 μL). D Schematic of time-of-addition assay. E Time-of-addition assay in H1N1-HiBiT-infected MDCK cells (MOI = 0.05) with Saponin C presented at indicated time periods. Data was obtained from 2–3 independent experiments performed in triplicate. *P < 0.05, ****P < 0.0001, ns means not significant vs. control group.

Fig. 4. Effects of Saponin C on viral RNA synthesis and vRNP. A–C Effects of Saponin C on viral RNA synthesis in MDCK cells infected with PR8 (MOI = 0.5). Intracellular levels of viral vRNA (A), mRNA (B), and cRNA (C) were quantified by qRT-PCR. D Effect of Saponin C on viral RNA polymerase activity was detected in 293T cells via a mini-replicon assay. E Effect of Saponin C on nucleocytoplasmic shuttling of vRNP complex. MDCK cells infected with PR8 (MOI = 0.5) were treated with or without Saponin C (10 μM) and stained with anti-NP antibody (green) and DAPI (blue) at the indicated time points. vRNP localization was observed by confocal microscopy (scale bar = 20 μm). The data are presented from 2–3 independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5. Saponin C had no significant effect on NP subcellular distribution, NP-CRM1 interaction, or protein thermal stability. A Saponin C does not alter the subcellular distribution of NP. 293T cells transfected with an HA-NP expression plasmid. At 12 hours post-transfection (p.t.), cells were treated with either DMSO or Saponin C (5–20 μM) and fixed at 36 hours p.t. Immunofluorescence was performed using an anti-HA antibody (green). Scale bar = 20 μm. B Effect of saponin C on the NP-CRM1 interaction. 293T cells were co-transfected with HA-NP- and Myc-CRM1 expression plasmids. At 12 hours p.t., cells were treated with Saponin C (0–10 μM). At 48 hour p.t., cells were collected and lysed for immunoprecipitation using anti-Myc magnetic beads. The co-precipitated HA-NP was detected by western blotting with an anti-HA antibody. C, E CETSA binding assays of NP (C) and CRM1 (E) in the presence or absence of 40 μM Saponin C at indicated temperatures. D, F Temperature melting curves of NP (D) and CRM1 (F). Relative chemiluminescent intensities at different temperatures were used to generate temperature-dependent melting curves. The apparent aggregation temperature (Tagg) was calculated by nonlinear regression. Data are representative of three independent experiments.

Fig. 6. Dose-dependent antiviral efficacy of Saponin C against PR8 virus in mice. A Schematic diagram of the experimental design. B, C The body weights (B) and survival rate (C) were monitored daily for 14 days (n = 8/group). D Histopathologic changes in lung tissues were examined by H&E staining on days 3 and 5 post-infection (n = 3/group). Arrows indicate edema, hemorrhage, and inflammatory infiltration 100×. E Viral titers in lungs were determined by EID50 on days 3 and 5 post-infection (n = 3/group). Data are presented as mean ± SEM. ** P < 0.01; ****P < 0.0001.

Fig. 7. Preventive and therapeutic effects of Saponin C against PR8 virus in mice. A Schematic diagram of the experimental design. B, C BALB/c mice were treated with Saponin C (50 mg/kg, i.p.) 24 h before or 2 h after PR8 virus (105 EID50) infection and monitored daily for 14 days for weight loss (B) and survival rate (C) (n = 6/group). D, E Histopathologic changes (D) and viral titers (E) in lung tissues were examined on days 3 and 5 post-infection (n = 3/group). Arrows indicate edema, hemorrhage, and inflammatory infiltration 100×. The data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8. Antiviral activity of Saponin C against SARS-CoV-2. A Inhibitory effect of Saponin C on SARS-CoV-2 Omicron (XBB.1.16) replication in Vero E6 cells (MOI = 0.01) was assessed by qRT-PCR at 24 h post-infection. B, C Inhibition of SARS-CoV-2 pseudovirus entry in Vero E6-hACE2 (B) and 293T-hACE2 (C) cells treated with Saponin C were determined by luciferase activity as measured at 48 h post-infection. The data represent mean ± SEM from 2–3 independent experiments performed in triplicate.

Fig. 9. Mechanistic effects of Saponin C on SARS-CoV-2 entry and fusion. A Time-of-addition assay for SARS-CoV-2 pseudovirus. 293T-hACE2 cells were infected with SARS-CoV-2 pseudovirus. Saponin C (10 μM) were added either during (0–48 h) or 2 h after infection (2–48 h), and antiviral activity was determined at 48 h post-infection. B Inhibition of Spike–hACE2 binding by Saponin C was assessed using a Spike–ACE2 binding inhibitor screening kit. C Inhibition of hACE2 enzymatic activity by Saponin C was evaluated with an ACE2 inhibitor screening kit. D Inhibition of SARS-CoV-2 S-mediated cell–cell fusion. 293T/S/GFP effector cells were co-cultured with 293T-hACE2 target cells in the presence of Saponin C. Syncytia were imaged at 6 h post-treatment (scale bar = 100 μm). E Fusion rate was quantified using Image-Pro Plus from three fields per condition from two independent experiments. All data represent means ± SEM from two to three independent experiments performed in triplicate. ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Fig. S1. Dose-dependent effects of oseltamivir phosphate on H1N1-HiBiT (MOI = 0.01) replication in MDCK cells.

Supplementary Fig. S2. Molecular interactions of Saponin C with the polymerase subunits PA (A), PB1 (B) and PB2 (C) of IAV. Saponin C was shown in cyan. Purple and blue represent bound amino acid residues and red represents hydrogen bonds.

Supplementary Fig. S3. The inhibition activity of Saponin C against SARS-CoV-2 and VSV-G pseudoviruses entry. 293T-hACE2 cells were infected with pseudoviruses harboring SARS- CoV-2-S or VSV-G in the presence of increasing concentrations of Saponin C. Pseudovirus infection was analyzed at 48 hpi by detecting luciferase activity in cell lysates. The data were collected from two independent experiments performed in triplicate and expressed as the mean ± SEM. ns: not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Fig. S4. Inhibitory Effect of saponin C on the Mpro Enzyme in the FRET Assay. A Protease inhibition activity of saponin C was evaluated using the FRET assay. Nirmatrelvir (1 μM) and DMSO were used as positive and negative controls, respectively. B Dose-response curve of Saponin C against the Mpro enzyme. The corresponding IC50 value is shown.

Supplementary Fig. S5. Molecular interactions of Saponin C with the SARS-CoV-2 spike proteins and the host protein ACE2. Saponin C was shown in cyan. Purple and blue represent bound amino acid residues and red represents hydrogen bonds. A Binding pattern of Saponin C with SARS-CoV-2 receptor-binding domain (RBD). Orange represents the region that binds to human ACE2. B Binding pattern of Saponin C with SARS-CoV-2 S1 subunit. Light purple for the S1 subunit and yellow for the RBD. C Binding pattern of Saponin C with SARS-CoV-2 fusion peptide 1 of the S2 subunit; Orange represents fusion peptide 1 of the S2 subunit. D Binding pattern of Saponin C with ACE2 (human receptor). Orange represents motifs interacting with the SARS-CoV spike glycoprotein.

Supplementary Fig. S6. The action of Ruscogenin on H1N1 infection. A Chemical structure of Ruscogenin. B Dose-dependent effect of Ruscogenin on H1N1-HiBiT replication (MOI = 0.01) and viability in MDCK cells. Data are presented as mean ± SEM from 2–3 independent experiments performed in triplicate.

本文亮点

皂苷 C 靶向病毒入胞后阶段，通过抑制聚合酶活性、阻碍病毒核糖核蛋白核输出，强效抗甲型流感病毒

在 H1N1 致死感染小鼠模型中，皂苷 C 可提高存活率、降低肺部病毒载量并减轻肺部炎症

皂苷 C 靶向刺突蛋白阻断新型冠状病毒入侵，兼具抗多种呼吸道病毒的双重活性