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. 2025 Apr 17;18(2):262–271. doi: 10.1016/j.chmed.2025.04.004

Combined network pharmacology and biological verification to investigate anti-influenza A virus effect and potential mechanism of Pudilan Xiaoyan Oral Liquid

Hualing Li a,1, Wenlei Wang a,1, Zhihui Zheng a, Yuqian Zhang a, Danting Li b, Qin Su a, Hailin Wei a, Zehua Wang a, Rui Min a, Xinyue Zhang a, Zihan Chen a, Xiaoquan Wang c, Pinghu Zhang a,d,⁎
PMCID: PMC13069621  PMID: 41971578

Abstract

Objective

Influenza is a common respiratory disease affecting human health and life. Pudilan Xiaoyan Oral Liquid (PDL) has frequently been used to treat respiratory infections such as COVID-19, pharyngitis, and tonsillitis. However, its efficacy against influenza A virus (IAV) infection remains unclear. This study investigates the anti-IAV activity and potential mechanism of action of PDL.

Methods

The in vitro anti-IAV activity was investigated in a virus-infected MDCK (Madin-Darby canine kidney) cell model. The in vivo protective effect on AIV infection was evaluated in a virus-infected mice model. qRT-PCR was performed to examine the anti-inflammatory effects of the PDL. A network pharmacology method was used to investigate the underlying mechanism of PDL against IAV. Western blotting was performed to confirm the antiviral mechanism of PDL against IAV.

Results

Our findings indicated that PDL had a broad-spectrum anti-IAV effect in vitro and exhibited an excellent protective effect against IAV infection by significantly increasing the survival rate, extending the survival time, and reducing pathological lung tissue damage. Moreover, PDL effectively inhibited the mRNA expression of inflammatory factors in lung tissue. GO analysis revealed that the potential targets were primarily associated with defense response regulation, positive regulation of cytokine production, and positive regulation of responses to external stimulation. PPI analysis indicated that Sarcoma (SRC), signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase1 (MAPK1), phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1), and epidermal growth factor receptor (EGFR) might be potential PDL targets against IAV infection. Mechanistically, inhibition of the Toll-like receptor 3 (TLR3)/myeloid differentiation primary response gene (88) (MyD88) signaling pathway to attenuate an excessive inflammatory response might be one of the primary mechanisms of PDL against IAV.

Conclusion

PDL efficiently suppressed in vitro IAV replication and provided protection against lethal influenza virus infection in vivo. Network pharmacology analysis revealed that the key PDL targets against influenza might be highly associated with biological processes, such as regulation of defense responses, cytokine production, and the Toll-like receptor signaling pathway. Mechanistically, regulating the TLR3/MyD88 signaling pathway may be one of the primary mechanisms of PDL against influenza virus infection. These findings indicate that PDL could be a potential therapeutic option for the clinical treatment of influenza viral infections.

Keywords: inflammatory factor, influenza A virus, network pharmacology, Pudilan Xiaoyan Oral Liquid, TLR3/MyD88 signaling pathway

1. Introduction

Influenza is a common human respiratory disease caused by influenza A virus (IAV), which can lead to pandemic outbreaks worldwide (Liu et al., 2023, Long et al., 2019, Brüssow, 2022). Due to the high mutation rate of IAV and its weak cross-protective effect, vaccination against IAV has a relatively limited protective effect (Elliott, Olufemi, & Daly, 2023). Furthermore, emerging avian influenza viruses, such as H5N6, H7N9, and H10N3, which can directly infect humans without adaptation, pose significant threats to human health (Zhang et al., 2022, He et al., 2021, Liu et al., 2022, Qi et al., 2022). Numerous studies have demonstrated that acute lung injury resulting from an excessive inflammatory response is the leading cause of high mortality in IAV-infected patients (Gu et al., 2019, Liu et al., 2024). Consequently, regulating excessive inflammatory responses may be an effective therapy for IAV infection.

Pudilan Xiaoyan Oral Liquid (PDL), composed of Taraxaci Herba (Pugongying in Chinese, dried whole grass of Taraxacum mongolicum Hand.-Mazz.), Scutellariae Radix (Huangqin in Chinese, dried whole roots of Scutellaria baicalensis Georgi), Corydalis Bungeanae Herba (Kudiding in Chinese, dried whole grass of Corydalis bungeana Turcz.), and Isatidis Radix (Banlangen in Chinese, dried whole roots of Isatis indigotica Fort.), has been frequently used in clinics to treat inflammatory diseases such as COVID-19, pharyngitis, and tonsillitis (Cheng et al., 2019, Tian et al., 2021, Tian et al., 2020, Liu et al., 2022, Feng et al., 2018). Several clinical studies have reported that PDL primarily treats these diseases due to its anti-inflammatory effects (Cheng et al., 2019, Liu et al., 2022). However, whether PDL has a therapeutic effect against IAV infection remains unknown.

This study is the first to investigate the effects and potential mechanisms of PDL against IAV. Our findings indicate that PDL can effectively inhibit the replication of IAV in vitro. Moreover, PDL effectively increased mouse survival, prolonged survival time, and improved IAV-induced lung injury. The potential mechanism of PDL against IAV was investigated using network pharmacology. Our findings provide a scientific basis for treating and preventing influenza with PDL.

2. Materials and methods

2.1. Reagents

Dulbecco's modified Eagle's medium (DMEM) was obtained from Jiangsu Kaiji Biotechnology Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) was purchased from Biological Industries (TlavivYafo, Israel). Primary rabbit antibodies (TLR7, MyD88, TRAF3, TRAF6, IRF7, and ACTB) were acquired from Beyotime Biotechnology (Shanghai, China). The qRT-PCR reagents were used as previously described (Zhang et al., 2021). PDL (lot 19022581) was provided by Jumpcan Pharmaceutical Co., Ltd. (Taizhou, China) and diluted with DMEM (2% FBS, 2 μg/mL TPCK-treated trypsin, 100 U/mL penicillin, and streptomycin). To further confirm the active components of PDL, its fingerprint was analyzed using high-performance liquid chromatography (HPLC) (Tian et al., 2020, Feng et al., 2018). Oseltamivir phosphate (S2597) was purchased from Selleck Chemicals Co., Ltd. (Shanghai, China) and prepared as previously described (Zhang et al., 2021). Ribavirin (RBV, 100 mg/mL) was received from Jiangsu Lianshui Pharmaceutical Co., Ltd. (Lianshui, China) and prepared as previously described (Zhang et al., 2021).

2.2. Virus and cells

Madin-Darby canine kidney (MDCK) and A549 cell lines and IAV strains were previously described (Zhang et al., 2021). The 50% tissue culture infective dose (TCID50) for MDCK cells and 50% lethal dose (LD50) for Institute of Cancer Research (ICR) mice were measured as previously described (Zhang et al., 2021). All experiments were conducted in the BSL-2 or BSL-3 biosafety laboratory at Yangzhou University, China.

2.3. MTT assay

The cytotoxicity of the drugs in MDCK and A549 cells was determined using the MTT assay as previously described (Zhang et al., 2017). Briefly, MDCK or A549 cells (2 × 104 cells/well) were plated in 96-well plates for 12 h and then exposed to drugs for 72 h. After treatment, 20 μL MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium was removed, and 150 μL DMSO was added to each well to dissolve the formazan crystals. The optical density of each well was measured at 570 nm using a multi-wavelength microplate reader (BioTek, Winooski, USA). The 50% cytotoxic concentration (TC50) for each drug was determined.

2.4. Anti-influenza virus activity of PDL in vitro

The antiviral effect of PDL was determined as previously described (Zhang et al., 2021). Briefly, MDCK cells (2 × 104 cells/well) were plated in 96-well plates for 12h and then inoculated with 100 TCID50 viruses for 1 h. After inoculation, the plates were exposed to various concentrations of drugs for 72 or 96 h at 35°C under 5% CO2. Then, 30 µL of the cell supernatant per well was transferred to a 96-well V-type hemagglutination plate to examine the virus titers using a hemagglutination test. As previously described, the plates were fixed and stained with crystal violet (Zhang et al., 2021). The Reed-Muench method was used to calculate the TC50 and IC50 values of the drugs.

2.5. Anti-influenza virus effects of PDL in vivo

Sixty ICR female mice [(14–16) g] were purchased from the Comparative Medical Center for Yangzhou University and raised in IVC at (22 ± 2) °C on a 12 h light/dark cycle. All animal experiments were approved by the Animal Ethics Committee of Yangzhou University (No. 202010002) and complied with the Chinese Animal Protection Act and Jiangsu Animal Welfare regulations. Sixty mice were divided into five groups: control, virus-infected group, PDL high-dose group [5.5 g/(kg·d)], PDL low-dose group [4.0 g/(kg·d)], and RBV [80 mg/(kg·d)] group, with 10 mice in each group. All mice anesthetized with isoflurane were inoculated intranasally with five LD50 of influenza virus suspension and then intragastrically administered PDL [5.5 or 4.0 g/(kg·d)], RBV [80 mg/(kg·d)], or saline for 7 d. Weight loss, food consumption, and mortality in each group were recorded daily for 15 d.

2.6. Anti-inflammatory effect of PDL in vivo

In total, 24 ICR female mice (14–16 g) were divided into four groups: control, virus-infected group, PDL high-dose group [5.5 g/(kg·d)], and RBV group [80 mg/(kg·d)]. They were then infected with five LD50 of H1N1 virus and treated with PDL, RBV, or saline for 6 d. All mice (n = 6, each group) were sacrificed after anesthesia with Zoletil®50, and lung tissues were collected for H&E staining, as previously described (Zhang et al., 2024; Baranovich et al., 2016). The injury score was assessed based on the observed pulmonary lesions, according to the following scale: 0, no lesions; 1, minimal, rare, or inconspicuous lesions; 2, mild multifocal, small focal, or widely separated but conspicuous lesions; 3, moderate, multifocal, and prominent lesions; 4, marked, extensive-to-coalescing lesions or areas of inflammation, with some loss of structure or lobar appearance; and 5, severe, extensive, and diffuse lesions with multilobar consolidation.

2.7. Network pharmacological analysis

The active chemical components in PDL identified using HPLC were imported into the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to search for their corresponding SMILES numbers (Kim et al., 2021). Subsequently, the SMILES numbers were imported into the Swiss Target Prediction database (http://swisstargetprediction.ch/) to retrieve their potential targets (select “homo sapiens” as the study species and “probability”> 0 as effective active ingredient targets (Wang et al., 2024). The influenza-associated targets were retrieved from the GeneCards database (Wang et al., 2024). The potential targets of PDL against influenza were identified from the component targets of PDL and potential influenza-associated targets using protein–protein interaction (PPI) analysis (more than twice the average value were recognized as core targets), thus performing gene ontology (GO) analysis, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, as previously described (Zhang et al., 2024; Chen et al., 2024).

2.8. RT-qPCR analysis

Total RNA from 50 mg of lung tissue from each group was extracted with Isolater (Vazyme, Nanjing, China), and cDNA was synthesized by reverse transcription of the Hiscript II 1st strand enzyme, according to the manufacturer's instructions (Vazyme, Nanjing, China). RT-qPCR was performed using Light Cycler 480 (Roche, Santa Clara, USA). The relative expression levels of TNF-α, IL-6, IP10, IL-10, IFN-γ, TLR3, TLR7, TLR8, TLR9, MyD88, IRF7, IRAK4, and TRAF3 mRNA were examined by RT-qPCR using GAPDH as an internal reference, as previously described (Zhang et al., 2021). The primer sequences used for qPCR are listed in Table S1.

2.9. Western blotting analysis

Western blotting was performed as previously described (Zhang et al., 2024). Briefly, A549 cells were infected with IAV and subsequently exposed to PDL (0.8 mg/mL or 0.4 mg/mL) or RBV for 24 h. After treatment, cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) to obtain total protein lysates. Protein samples were separated on 10% SDS-PAGE gels before being transferred onto a nitrocellulose (NC) membrane (0.45 µm, Millipore, Billerica, USA) using a tank transfer system (Bio-Rad, Hercules, CA, USA). After blocking with 5% BSA for 1 h, the membranes were successively incubated with primary and secondary antibodies and exposed using an enhanced chemiluminescence kit (Millipore, Billerica, USA) with a Molecular Imager SH-523 System (ShenHua, Hangzhou, China). Relative expression levels were measured using Quality One software (Bio-Rad, Hercules, CA, USA).

2.10. Statistical analysis

All data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) or Student's t-test was used to determine statistical differences between groups. Differences between groups were analyzed using the Log-rank test. P-values ≤ 0.05 were considered statistically significant.

3. Results

3.1. HPLC profile of PDL

To confirm whether PDL contains the main bioactive ingredients of four traditional Chinese medicines (Taraxaci Herba, Scutellariae Radix, Corydalis Bungeanae Herba and Isatidis Radix), UPLC-MS analysis was performed as previously described (Feng et al., 2018). Nine effective constituents of PDL, including adenosine (0.307 4 mg/mL), (R, S)-epigoitrin (0.083 0 mg/mL), chlorogenic acid (0.034 1 mg/mL), caffeic acid (0.099 5 mg/mL), cichoric acid (0.360 6 mg/mL), scutellarin (0.078 6 mg/mL), baicalin (7.762 8 mg/mL), oroxylin A (0.340 2 mg/mL), and wogonoside (0.317 3 mg/mL) were identified and quantified as previously described (Feng et al., 2018) (Fig. 1). This result is consistent with the literature (Tian et al., 2020, Feng et al., 2018), suggesting that the components in the PDL are stable and repeatable.

Fig. 1.

Fig. 1

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HPLC chromatograms (A) and structures (B) of nine active constituents of PDL. (1), Adenosine; (2), (R, S)-Epigoitrin; (3), Chlorogenic acid; (4), Caffeic acid; (5), Cichoric acid; (6), Scutellarin; (7), Baicalin; (8), Oroxylin A; (9), Wogonoside.

3.2. In vitro anti-IAV effect of PDL

All anti-IAV activities of PDL were performed under nontoxic dose conditions (TC50 of PDL was 7.94 mg/mL). PDL significantly inhibited the replication of influenza H1N1 (FM1, PR8, and WSN), H3N2, H5N1, H7N9, and H9N2 viruses, indicating a protective effect against IAV-induced cell pathogenicity. The IC50 values of PDL on FM1, PR8, WSN, H3N2, H5N1, H7N9, and H9N2 were 0.55, 1.10, 0.98, 1.88, 0.98, 1.67 and 1.82 mg/mL, respectively.

3.3. In vivo anti-IAV effect of PDL

As presented in Fig. 2A, compared with the model group, PDL treatment was superior to RBV treatment in preventing weight loss in virus-infected mice. Additionally, the model group began to die on the ninth day after infection, with a median survival time of (9.8 ± 1.0) d and 100% mortality. However, PDL therapy effectively increased the survival rate and prolonged the survival time of virus-infected mice (Fig. 2B and C). The survival rates in the PDL (H)- and PDL (L)-treated groups were 67.5% and 50.0%, respectively. The mean survival periods of the PDL (H)- and PDL (L)-treated groups were (13.5 ± 2.3) and (12.8 ± 2.4) d, respectively. RBV provided 80% protection against lethal IAV infection. Moreover, the lung index significantly increased in the model group. However, PDL and RBV treatment significantly reduced this effect in virus-infected mice (Fig. 2D). The lung index level of the PDL (H) group was similar to that of the positive drug RBV. Collectively, PDL exhibited good protective effects against lethal IAV infection.

Fig. 2.

Fig. 2

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In vivo protective effects of PDL against lethal H1N1 virus (mean ± SD, n = 10). Weight loss (A), survival rate (B), survival time (C) and lung index (D) in each group were recorded. *P < 0.05, **P < 0.01, ***P < 0.001 vs model group. ###P < 0.001 vs control group.

3.4. PDL effectively reduced IAV-mediated lung injury

Pathological analysis was performed to explore the protective effects of PDL against viral lung injury. Compared to the control group, the model group showed severe pathological injury, including different alveolar sizes, severe damage and thickening of the alveolar wall, inflammatory cell infiltration, and bleeding in the alveolar cavity (Fig. 3A). The lung index of the model group was significantly higher than that of the control group. However, a dose-dependent reduction in the lung index was observed in the PDL treatment groups. H&E staining and pathological scoring confirmed that the PDL treatment reduced lung tissue injury in a dose-dependent manner. These results suggest that PDL treatment effectively prevented IAV-induced lung injury (Fig. 3B).

Fig. 3.

Fig. 3

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In vivo protective effect of PDL against acute lung injury caused by influenza H1N1 virus (mean ± SD, n = 8). Lung histological changes analyzed by H&E staining and photographed (A) (×200, Scale bar = 100 µm) and lung injury scores (B) in each group were recorded. ###P < 0.001 vs control group; **P < 0.01, ***P < 0.001 vs model group.

3.5. Network pharmacological prediction results

Ninety-four common targets were predicted to be strongly associated with PDL and influenza (Fig. 4A). PPI network analysis revealed that STAT3, SRC, MAPK1, PIK3R1, and EGFR were the primary targets of PDL in treating influenza-associated pneumonia (Fig. 4B). GO enrichment analysis indicated that PDL targets against influenza were predominantly involved in biological processes such as regulation of defense response, positive regulation of cytokine production, and positive regulation of response to external stimuli (Fig. 4C). KEGG pathway enrichment analysis demonstrated that the potential top targets of PDL treatment for influenza were enriched in the C-type lectin receptor signaling pathway, TNF signaling pathway, coronavirus disease-COVID-19, VEGF signaling pathway, and influenza signaling pathway (Fig. 4D). Component-target-pathway network analysis indicated that oroxylin A, caffeic acid, chlorogenic acid, chicoric acid, and scutellarin were the most active anti-influenza compounds in the PDL (Fig. 4E). In addition, KEGG analysis indicated that the Toll-like receptor (TLR) signaling pathway, which has been demonstrated to be highly associated with viral lung injury mediated by IAV, was a potential target for PDL treatment of influenza. Consequently, the potential mechanism by which PDL regulates the TLR signaling pathway against influenza was further verified in vitro and in vivo.

Fig. 4.

Fig. 4

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Network pharmacological analysis of PDL. (A) Intersection targets of PDL and influenza. (B) PPI network of core targets. (C) GO enrichment analysis. (D) KEGG enrichment analysis. (E) Component-target-pathway network.

3.6. In vivo inhibitory effect of PDL on inflammatory cytokines

It has been reported that pro-inflammatory cytokines play an important role in IAV-induced lung injury and Acute Respiratory Distress Syndrome (ARDS) (Lobo et al., 2019). Therefore, the mRNA levels of pro-inflammatory factors were investigated. PDL treatment significantly reduced the transcription of TNF-α, IP10, IL-10, IL-6, and IFN-γ mRNA compared with the model group (Fig. 5). These findings indicated that PDL treatment effectively suppressed the expression of inflammatory cytokines caused by IAV.

Fig. 5.

Fig. 5

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In vivo inhibitory effect of PDL on inflammatory cytokines (mean ± SD, n = 6). qRT-PCR was used to quantify TNF-α (A), IL-6 (B), IP10 (C), IL-10 (D), and IFN-γ (E) mRNA levels. Relative fold-increases of these cytokines were measured using the 2−ΔΔCt method, with GAPDH as a reference. *P < 0.05, **P < 0.01 vs model group. #P < 0.05, ##P < 0.01vs control group.

3.7. Potential mechanism of PDL against influenza A virus infection

As the host's first line of defense against pathogenic microbial infections, TLRs are crucial in viral clearance and suppression of excessive inflammatory responses (Arora et al., 2019, Elshabrawy, Essani, Szekanecz, Fox, & Shahrara, 2017). Moreover, TLR-mediated signaling pathways are widely involved in regulating the inflammatory responses of the host to IAV (Zhang et al., 2022, Huo et al., 2018). Because network pharmacology analysis indicated that the TLR signaling pathway might be one of the primary targets of PDL against IAV infection, qRT-PCR was used to evaluate the mRNA expression levels of TLR3, TLR7, TLR8, TLR9, MyD88, IRF7, IRAK4, and TRAF3 in the lung tissues. As demonstrated in Fig. 6, the PDL-treated group had significantly decreased mRNA levels of TLR3, MyD88, IRF7, and TRAF3 compared with the model group. Conversely, no significant differences were observed in the mRNA levels of TLR7, TLR8, and TLR9 (data not presented), suggesting that the TLR3-mediated signaling pathway may be the most important signaling pathway of PDL against influenza virus infection.

Fig. 6.

Fig. 6

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Potential mechanism of action of PDL against viral pneumonia (mean ± SD, n = 6). Relative transcription levels of inflammation-related signaling pathways, including TLR3 (A), MyD88 (B), IRF7 (C), IRAK4 (D), and TRAF3 (E) mRNA, were quantified by qRT-PCR using 2−ΔΔCt method, with GAPDH as a reference. *P < 0.05, **P < 0.01 vs model group; ##P < 0.01 vs control group.

To further confirm these findings, A549 cells were used to investigate the potential mechanism by which PDL protects against IAV infection by regulating the TLR3 signaling pathway. As presented in Fig. 7, a significant increase in the protein levels of TLR3, MyD88, IRF7, TRAF6, and TRAF3 was observed in the model group, whereas PDL effectively reduced the expression of these proteins. Furthermore, compared with the model group, the expression levels of viral nucleoprotein (NP) were significantly decreased. These results suggest that regulating the TLR3/MyD88 signaling pathway may be one of the primary mechanisms of PDL for treating influenza.

Fig. 7.

Fig. 7

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Potential mechanism of PDL against IAV infection (mean ± SD, n = 6). Expression levels of viral NP, TLR3, MyD88, IRF7, TRAF6, and TRAF3 analyzed by immunoblotting (A). Relative expression levels of NP (B), TLR3 (C), MyD88 (D), IRF7 (E), TRAF6 (F), and TRAF3 (G) measured using quality one software. ##P < 0.01 vs control group; *P < 0.05, **P < 0.01 vs model group. PDL (H), 0.5 mg/mL; PDL (L), 0.25 mg/mL.

4. Discussion

IAV can trigger global pandemics and epidemics. Influenza remains a significant public health concern because of the high mortality and morbidity rates associated with IAV infection. Although the preventive administration of antiviral drugs such as oseltamivir and zanamivir could improve patient outcomes, no effective strategies are found for severe seasonal and highly pathogenic avian influenza (Elliott, Olufemi, & Daly, 2023, Globenko et al., 2023). It has been demonstrated that the “cytokine storm” is responsible for its high mortality and morbidity (Lobo et al., 2019, Ferreira et al., 2023). Therefore, inhibiting excessive inflammatory responses may be an effective treatment for this disease. Traditional Chinese medicines, such as Lonicerae Japonicae Flos (Jinyinhua in Chinese), Forsythiae Fructus (Lianqiao in Chinese), Arisaematis Rhizoma (Tiannanxing in Chinese), Taraxaci Herba (Pugongying in Chinese), are frequently used to treat infectious diseases. In addition, many traditional Chinese medicines have been used extensively to treat the common cold, influenza, and upper respiratory tract infections (Cheng et al., 2023, Law, Yang, Lau, & Chan, 2017, Liu et al., 2024, Ma et al., 2020, Zhang et al., 2021). Due to its significant anti-inflammatory action, PDL, a well-known Chinese patent medicine, has been frequently used to treat pharyngitis, tonsillitis, and upper respiratory tract infections (Deng et al., 2020; Cheng et al., 2019, Tian et al., 2021, Tian et al., 2020, Liu et al., 2022, Feng et al., 2018). In the present study, our results indicate that PDL effectively inhibited the in vitro replication of IAV. Furthermore, our data indicated that PDL exhibited a protective effect against IAV infection in vivo by decreasing mortality, extending survival time, and alleviating pulmonary edema and pathological scores.

Network pharmacology is a novel strategy to investigate potential drug targets or drug-target-disease associations. It is commonly used to predict multi-target mechanisms of the complex components of TCM (Chen et al., 2024). Based on the enrichment analysis of PDL-related targets, we identified that PDL targets were primarily associated with biological processes such as regulation of the defense response, the C-type lectin receptor signaling pathway, and the TLR signaling pathway. PPI network analysis indicated that the key target of PDL against influenza virus infection may be STAT3, which may play an unanticipated role in IAV pathogenesis (Liu et al., 2023). Biological validation suggested that the anti-influenza effect of PDL in vivo may be strongly associated with the inhibition of inflammatory cytokines such as TNF-α, IL-6, IP-10, IFN-γ, and IL-1β via the TLR3/MyD88 signaling pathway.

Emerging studies have indicated that an excessive inflammatory response is the primary cause of high clinical mortality associated with IAV infection. For instance, high levels of pro-inflammatory cytokines were commonly identified in patients with severe IAV (Gu et al., 2019, Ferreira et al., 2023). Consequently, controlling excessive inflammatory responses could improve the prognosis of IAV infection. Recently, it was demonstrated that PDL significantly inhibits the expression of the pro-inflammatory factors TNF-α and IL-6 in an LPS-induced respiratory injury model (Feng et al., 2018). Similar to previous studies, we identified that PDL treatment significantly inhibited the expression of pro-inflammatory cytokines, including TNF-α, IP-10, IFN-γ, IL-6, and IL-10.

TLRs are critical for triggering host antiviral responses and are key pattern recognition receptors for identifying viruses. TLR3, 7, 8, and 9 primarily recognize viral nucleic acids in mammals (Arora et al., 2019, Duan et al., 2024, Elshabrawy, Essani, Szekanecz, Fox, & Shahrara, 2017, Huo et al., 2018, Zhang et al., 2022). TLR3, an important receptor that recognizes viral double-stranded RNA (dsRNA), is essential for defense against viral infections (Huo et al., 2018). However, it has been observed that activation of the TLR3 signaling pathway may be detrimental in certain viral infections. For example, TLR3-deficient mice survive IAV infection better than wild-type mice because of the decreased levels of inflammatory mediators, indicating that TLR3 plays a detrimental role in TLR3-mediated inflammatory responses (Le Goffic et al., 2006). Recently, Huo et al. revealed that lethal IAV preferentially activates TLR3 and induces severe inflammatory responses (Huo et al., 2018).

Furthermore, network analysis suggested that the TLR signaling pathway may be one of the primary PDL targets against IAV infection. Therefore, we investigated whether PDL inhibits the expression of TLRs. In vitro and in vivo results indicated that PDL inhibited the TLR3-mediated signaling pathway but had no effect on other TLRs, confirming that regulating the TLR3/MyD88 signaling pathway may be one of the primary mechanisms of PDL against influenza virus infection (Fig. 8).

Fig. 8.

Fig. 8

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Potential mechanism model of PDL against IAV infection. PDL, Pudilan Oral Liquid; IAV, influenza A virus; TLR-3, Toll-like receptor 3; MyD88, myeloid differentiation primary response gene (88); TRAF3, TNF receptor-associated factor 3; TRAF6, TNF receptor-associated factor 6; IRF7, interferon regulator factor 7; IL-10, interleukin (IL)-10; IL-6, interleukin (IL)-6; IP10, chemokine CXCL10; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ.

5. Conclusion

In summary, our findings demonstrate that PDL effectively inhibited IAV replication in vitro and protected against lethal influenza virus infection in vivo. Network pharmacology analysis reveals that the key targets of PDL against influenza are highly associated with biological processes, including the regulation of defense responses, positive regulation of cytokine production, and the TLR signaling pathway. Mechanistically, regulation of the TLR3/MyD88 signaling pathway may be one of the primary mechanisms of PDL against influenza virus infection. These findings provide a scientific basis for the clinical application of PDL in the treatment of IAV-related respiratory infections.

CRediT authorship contribution statement

Hualing Li: Conceptualization, Methodology, Writing – original draft. Wenlei Wang: Investigation, Methodology, Validation. Zhihui Zheng: Investigation, Methodology, Validation. Yuqian Zhang: Investigation, Methodology, Validation. Danting Li: Investigation, Methodology, Validation. Qin Su: Investigation, Methodology, Validation. Hailin Wei: Investigation, Methodology, Validation. Zehua Wang: Investigation, Validation. Rui Min: Investigation, Validation. Xinyue Zhang: Investigation, Validation. Zihan Chen: Investigation, Validation. Xiaoquan Wang: Investigation, Methodology, Resources. Pinghu Zhang: Project administration, Supervision, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by Jiangsu Province Traditional Chinese Medicine Technology Development Plan Project (No. MS2022155), the Jiangsu Students’ Innovation and Entrepreneurship Training Program (NO. 202111117111Y), and Open Project from Jiangsu Key Laboratory of Human Zoonosis (No. R1707). We thank Mr. Mohammed Salem for language improvement.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chmed.2025.04.004.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Supplementary Data 1
mmc1.docx (31.3KB, docx)

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