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. 2025 Jan 27;18(2):282–293. doi: 10.1016/j.chmed.2025.01.007

Exploring possible mechanisms of antiviral effect of Shufeng Jiedu Capsule on viral pneumonia: A comprehensive analysis

Shan Cao a, Lei Bao a, Zihan Geng a, Yingli Xu a, Bo Pang a, Jingsheng Zhang a, Qiang Zhu b, Jun Pan b, Yaxin Wang a, Mengping Chen a, Yu Zhang a, Yanyan Bao a, Jing Sun a, Ronghua Zhao a, Xiaolan Cui a,⁎, Shanshan Guo a,⁎
PMCID: PMC13069657  PMID: 41971587

Abstract

Objective

Influenza A virus (IAV) is a major cause of respiratory tract infections. Shufeng Jiedu Capsule (SFJD) has a broad-spectrum antiviral effect, however, it is not clear whether SFJD acts by targeting the immune-inflammatory pathway. The aim of this study was to elucidate the therapeutic effect and the mechanism of antiviral action of SFJD in mice with influenza virus pneumonia.

Methods

In this study, a mouse model of influenza virus pneumonia was used to evaluate the efficacy of SFJD in the treatment of influenza virus pneumonia. Next, the potential pathways of action by which SFJD exerts its antiviral effects were analysed in conjunction with network pharmacology and and untargeted metabolomics. Finally, in vivo experimental validation was conducted through protein immunoblotting, transmission electron microscopy, and immunofluorescence experiments.

Results

SFJD significantly reduced lung index, lowered viral load, attenuated lung tissue lesions, modulated inflammatory metabolism in lung tissue, reduced macrophage inflammatory infiltration and protected lung epithelial cells in mice. The results of the network pharmacological analysis and metabolomics analysis suggested that SFJD might act through the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/ TNF receptor associated factor (TRAF6)/nuclear factor kappa-B(NF-κB) signalling pathway. SFJD was able to down-regulate the expression of TLR4, MyD88, TRAF6, and NF-κB proteins in lung tissues.

Conclusion

SFJD could effectively attenuate lung injury induced by influenza virus infection and provide a scientific basis for the protection of respiratory mucosal epithelial cells. SFJD could effectively alleviate lung injury by regulating the TLR4/MyD88/TRAF6/NF-κB signaling pathway, inhibiting the levels of TNF-α, IL-6, which may be the key mechanism for its antiviral effects.

Keywords: influenza A virus, mucosal epithelial cells, Shufeng Jiedu Capsules, toll-like receptors, metabonomics

1. Introduction

Evolving viruses put the globe at risk of a pandemic. The respiratory tract is the main gateway for viruses to enter and cause infection. In humans and animals, influenza viruses, which are RNA viruses, induce influenza and function as respiratory viruses causing acute infections in the upper respiratory tract. They have the ability to disseminate swiftly through the air, resulting in global pandemics happening periodically (Braciale, Sun, & Kim, 2012). There are still many challenges in the prevention and control of influenza. In order to prevent influenza infection and its complications, the most effective way is to get vaccinated against influenza (Braciale et al., 2012, Smith and Ribeiro, 2010). The immune system faces a distinct challenge when dealing with respiratory infections caused by influenza viruses, as it must swiftly eliminate the virus while also managing tissue inflammation to prevent further deterioration. In recent years, respiratory mucosal defence mechanisms have received much attention in antiviral drug and immunological research. The respiratory mucosa is both an invasion site for influenza viruses but also an important site of the body’s immune defense. Among these, the respiratory epithelium is thought to be central to the coordination of inflammatory and immune responses in the lungs and key to tissue remodeling. Vaccination serves as the primary defense against a wide range of airborne pathogens and plays a crucial role in regulating both innate and adaptive immune responses to these pathogens (Hiemstra, McCray Jr, & Bals, 2015).

Respiratory epithelial cells are the main target cells of the influenza virus. In the early stage of viral infection, the body can recognize pathogen-associated molecular patterns (PAMPs) on the surface of the virus through pattern recognition receptors (PRRs), which activates the body’s intrinsic immune response and induces the body to produce cytokines, such as type I interferons, and chemokines, to curb the replication of the virus (Ardain et al., 2020, Weitnauer et al., 2016). The innate immune system recognizes influenza viruses through multiple types of pattern recognition receptors (PRRs), such as Toll Like Receptor 2(TLR2), TLR4, TLR7, and TLR3. Among these, TLR4 has been found to play a significant role in the infection process of influenza viruses. Studies have demonstrated that TLR4 expression is upregulated in lung tissues following virus infection, leading to the activation of myeloid differentiation primary response protein 88(MyD88), nuclear factor kappa-B (NF-κB), and other downstream signaling pathways (Dai et al., 2018, Root-Bernstein, 2021, Wang et al., 2008, Zhu et al., 2023). Therefore, from the perspective of respiratory mucosal immunity, reducing the damage of viral infection to lung epithelial cells by regulating the TLR4-mediated signaling pathway provides some ideas for antiviral therapy.

The medical profession widely recognizes the advantages of Chinese medicine in preventing and treating viral pneumonia, particularly in the clinical treatment of H1N1 and COVID-19 (Dai et al., 2020). Early herbal intervention is important in mitigating disease progression. Traditional Chinese medicine (TCM) utilizes combinations of herbs, which can be seen as mixtures of various active ingredients (Xia et al., 2021). Thus, the combination of active ingredients with different targets can affect different signaling pathways and produce synergistic effects, for example in the treatment of viral respiratory infections (Li et al., 2017, Xia et al., 2021). Shufeng Jiedu Capsule (SFJD) is a herbal combination consisting of eight medicinal plants that is used for treating various viral respiratory infections (Xu et al., 2024). SFJD is a first-line herbal medicine recommended by the government for the prevention and treatment of influenza symptoms. Through rational and organic combination, SFJD has brought into play the unique advantages of compound Chinese medicine. Studies have been conducted to analyse the identification and characterisation of the herbs, the finished product, and the orally ingested blood components to elucidate the chemical composition of the SFJD. Previous studies have shown that SFJD can have a protective effect on the lungs by activating the mitogen-activated protein kinase (MAPK)/NF-κB signaling pathway (Mei et al., 2023, Tao et al., 2014). Additionally, it has been found to have synergistic antiviral effects when used in combination with oseltamivir against acute exacerbation of chronic obstruction caused by influenza A virus (Ji et al., 2020). The respiratory mucosal defence system is the body’s first line of defence against viral infections. Whether SFJD is able to exert its antiviral effects through the respiratory mucosal defence mechanism needs to be investigated urgently. With the development of network pharmacology and molecular biology, it provides more possibilities for us to study the mechanism of antiviral action of SFJD.

In this study, we used a series of experimental methods such as Micro CT (μCT), histopathological (H&E) staining, real-time polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) to evaluate the immunomodulatory role of SFJD in influenza virus pneumonia. Further metabolomics and network pharmacology were combined to explore its potential antiviral pathway and the anti-inflammatory protective and antiviral effects of SFJD on lung epithelial cells were validated in in vivo experiments.

2. Materials and methods

2.1. Medicine and reagents

SFJD was purchased from Jiren Pharmaceutical Co., Ltd. (3220411; Anhui, China). Oseltamivir phosphate capsules (abbreviated as OP) were provided by Yichang HEC Changjiang Pharmaceutical Co., Ltd. (0221903034; Dongguan, China). The protein extraction kit was provided by Beijing Solarbio Science & Technology Co., Ltd. (20220224; Beijing, China). Mouse tumor necrosis factor-α (TNF-α) ELISA kit and mouse interleukin-6 (IL-6) ELISA kit were provided by BIORIGIN (BN50578; BN50553; Beijing, China). Influenza virus A real time RT-PCR kit was provided by Shanghai ZJ Bio-Tech Co., Ltd. (20230301; Shanghai, China). Anti epithelial cell adhesion molecule (EpCAM) antibody was provided by Serrvicebio (GB11274; Wuhan, China). Anti-F4/80 antibody was provided by HUABIO (RT1212; Zhejiang, China); anti-TLR4 antibody, anti-MyD88 antibody and anti-NF-κB antibody were provided by Proteintech (10005089, 10022637, 66535; Wuhan, China); anti-NF-κB p65 antibody was provided by Abcam (ab76302; Cambridge, UK); anti- TNF receptor associated factor (TRAF6) antibody was provided by Santa Cruz (sc-8409; Santa Cruz, USA,).

2.2. Virus and animals

Mouse-adapted influenza virus A/FM/1/47 (H1N1) were purchased from American Type Culture Collection (ATCC) (Manassas, American). The virus was stored in the ABSL-2 laboratory, Institute of Traditional Chinese Medicine, Chinese Academy of Sciences, in an environment at −80 °C. SPF healthy male and SPF healthy female ICR mice (13–15 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) (Certificate number: SCXK Jing 2021-0006). All mice were housed under standard laboratory conditions and fed SPF-quality food and purified water. The Animal Ethics Committee of the Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences ratified animal experimentation and the corresponding protocol (2022D052).

2.3. Infection and treatment

Thirty-six ICR mice were randomized into the following groups: control group (control), control + SFJD-high-dose group (control + SFJD-H), model group (FM1), OP group (OP), SFJD high-dose group (SFJD-H), and SFJD low-dose group (SFJD-L), half male and half female in each group. Apart from the control group and control + SFJD-H group, mice were infected with 35 μL of 100TCID50 FM1 virus solution through the nose after light anesthesia. Treatment was initiated 1 h after infection. Each group of mice received the drug via gavage and were continuously administered for 4 d. The dosage was calculated as 0.2 mL per 10 g of body weight (Fig. 1A). The doses in the SFJD-H, SFJD-L and OP groups were 2.288 g/kg/d, 0.572 g/kg/d and 27.5 mg/kg/d, respectively. Survival rate and body weight loss rate were calculated for each group of mice. All mice were observed at the end of each day’s gavage. On day 4 post-infection, all mice were weighed and dissected, and the lungs were removed. The lung index was calculated based on the equations: lung index = W0 / W1 × 100 (W0: lung wet weight; W1: body weight) (Xia et al., 2021). Survival rate was calculated by the following formula: survival rate = (number of surviving mice / number of test mice) × 100. Weight loss rate was calculated by the following formula: weight loss rate = [weight (control) − weight (infection)] / weight (control) × 100.

Fig. 1.

Fig. 1

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SFJD protected against influenza virus-infected mice. (A) Influenza virus pneumonia model in mice (Drawn by FigDraw). (B) Results of μCT scans and representative 3D rendering of lung image. (C) Arated lung volume in mice (n = 3). (D) Lung index of mice in each group. (n = 6). (E) Virus load of mice in each group. (n = 6). Data were shown as mean ± SD. ##P < 0.01, ###P < 0.001 vs control group and control + SFJD-H group; *P < 0.05, **P < 0.01, ***P < 0.001 vs model group (FM1).

2.4. Micro CT scanning (μCT) and histopathological (H&E)

Each group of mice was scanned with a μCT scanner (PE Quantum GX2). On day 4 of infection, mice were anesthetized with isoflurane inhalation, and CT scans were performed. The parameter settings are as follows: 90 kV, 88 mA, and 36 mm, scanning time of 2 min per mouse. The pulmonary left lung was immersed in 4% formaldehyde solution for a minimum of 7 d to undergo fixation. Followed by gradual alcohol dehydration and subsequent paraffin embedding, individual tissue samples were sectioned and subjected to a 20-minute immersion in xylene I, a subsequent 20-minute immersion in xylene II, 5 min of exposure to 100% ethanol I, an additional 5 min of exposure to 100% ethanol II, a 5-minute incubation in 75% ethanol, and finally rinsed consecutively with tap water. The sections were then treated with haematoxylin differentiation solution and haematoxylin-Scott’s blue. The slides were sealed with neutral gum and viewed under a microscope for inspection, image capture and analysis.

2.5. Enzyme-linked immunosorbent assay (ELISA)

Tissue homogenization was performed on 100 mg of lung tissue frozen in a −80 °C refrigerator. The supernatant of each group was taken and tested for the expression of inflammatory factors TNF-α and IL-6 in lung tissues according to the kit instructions.

2.6. Western blotting (WB) and real-time polymerase chain reaction (RT-PCR)

Equal amounts of protein (60 μg/lane) were separated by electrophoresis in 10% polyacrylamide gels and subsequently transferred to Millipore PVDF membranes. The membranes were then incubated overnight at 4 °C with appropriately diluted antibodies targeting Tubulin, TLR4, Myd88, TRAF6, and NF-κB. After three washes with tris buffered saline tween (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG diluted 1:5 000 in TBST for 1 h at 37 °C. Following three additional washes with TBST, the protein bands were detected using a super enhanced chemiluminescence (ECL) plus kit according to the manufacturer’s instructions. Scanned images of the target bands were analyzed using ImageJ software. Total RNA samples from lung tissue were extracted using TRIzol® reagent according to the product instructions. The assay was performed according to the H1N1-FM1 Real-Time RT-PCR kit instructions.

2.7. Immunofluorescence (IF)

Paraffin sections of lung tissue were subjected to a series of treatments including dewaxing, antigen repair, endogenous enzyme blockade and serum closure. The primary antibodies (TLR4, MyD88, and NF-κB p65 antibodies) were diluted to a specific concentration using an antibody diluent and incubated overnight at 4 °C. Subsequently, an HRP enzyme-labeled secondary antibody was added and incubated at 37 °C for 1 h. Tyramide signal amplification (TSA)-670 was then dropwise added to the tissue and incubated at 37 °C for 30 min. The tissue was washed three times with phosphate buffer saline (PBS) for 5 min each. The slides were immersed in 1× citric acid and microwaved on high for 5 min for repair. After cooling to room temperature, the slides were ready for further analysis. Serum from the same source as the secondary antibody was used for containment and incubated at 37 °C for 30 min. The operation was repeated for multiple fluorescence staining. 4′,6-Diamidino-2′-phenylindole (DAPI) was added dropwise to the tissue and incubated at room temperature for 5 min. Afterward, the tissue was washed with PBS three times, each time for 5 min. The liquid was then shaken dry, and anti-fluorescent sealer was added dropwise to the tissue. Finally, coverslips were placed on the tissue, and the finished fluorescent slides were stored at 4 °C away from light.

2.8. Network pharmacology research

The TCMSP system was used to search for SFJD components, setting oral availability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, and after obtaining the active ingredient, we searched for the target of action of a single drug component through its MOL.ID number. A search for “Influenza A virus pneumonia” was performed in five databases of GenGards, OMIM, etc. to identify candidate targets. The Venny software was utilized to identify the overlapping targets between active compounds in traditional Chinese medicines (TCMs) and diseases. These overlapping targets were considered as potential key targets for the treatment of diseases using TCM compound ingredients. The network topology of the overlapping targets was analyzed using Cytoscape 3.9.0 software, and a “component-target-disease”network diagram was constructed. The intersecting targets were then imported into the String platform and visualized using Cytoscape 3.9.0. To analyze the functional enrichment of biological processes, the clusterProfiler, Stringin, and Pathview packages within the R software were installed and run using the bioinformatics open-source software Bioconductor. The results were visualized and displayed through the microbiology platform.

2.9. Liquid chromatograph mass spectrometer (LC-MS) metabolomics analysis

The mice were anaesthetised for perfusion of the heart to remove the effects of blood, and the lung tissue was removed and frozen in liquid nitrogen. A 50 mg lung tissue sample was added to a 2 mL centrifuge tube, along with a 6 mm diameter grinding bead. The samples were ground using the Wonbio-96c frozen tissue grinder (Shanghai Wanbo Biotechnology Co., Ltd., Shanghai, China) for 6 min at a temperature of −10 °C and a frequency of 50 Hz. Subsequently, low-temperature ultrasonic extraction was performed for 30 min at a temperature of 5 °C and a frequency of 40 kHz. Afterward, the samples were kept at −20 °C for 30 min, followed by centrifugation for 15 min at a temperature of 4 °C and a speed of 13 000 × g. The resulting supernatant was then transferred to an injection vial for LC-MS/MS analysis. The LC-MS/MS analysis of the sample was carried out at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 μm; Waters, Milford, USA).

2.10. Statistical analysis

All data processing and analysis were performed using GraphPad Prism 9.5 (GraphPad, San Diego, CA, United States). The data were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for the significance of differences between multiple group comparisons. The significance of the difference between groups was tested by Student’s t-test. P < 0.05 indicates that the data has significant differences.

3. Results

3.1. SFJD protected against influenza virus infection-induced viral pneumonia in vivo

To validate the therapeutic efficacy of SFJD for the treatment of influenza virus pneumonia, we established a mouse model of viral pneumonia infected by transnasal drip and evaluated the disease state of the mice after drug treatment (Fig. 1A). The survival rate of mice in all groups was 100%. The weight loss rate of FM1 group, OP group, SFJD-H group and SFJD-L group were 17.7%, 10.79%, 16.32% and 17.92%. The μCT results showed uniform lung density in the control group, and the corresponding 3D reconstruction maps showed intact lung tissue structure without areas of tissue defects. Compared with the control group, diffuse infiltrating shadows and dense enhancing shadows were present in the lung tissues of the model mice, and their 3D reconstruction maps showed the presence of localized defects in the lung tissues, and the volume of the lung tissues was significantly reduced compared with that of the normal group, indicating the presence of localized lesions. After the treatment of the positive drug and SFJD, the lung density was reduced, the dense-enhancing shadow was reduced, and the 3D reconstruction map construct showed a reduction of localized defects in the lung tissue and a reduction of the lesion area (Fig. 1B–C). Influenza virus infection caused viral pneumonia in mice, and inflammatory exudation increased lung weight; the greater the pulmonary index value was, the more severe the degree of pneumonic lesion was. Compared with the control group and the control + SFJD-H group, the lung index of mice in the model group was distinctly increased with a significant difference (P < 0.001). In comparison to the model group, the positive drug OP group and SFJD-H group had significantly lower lung indexes (P < 0.001, P < 0.05), and the SFJD-L group had lower lung indexes than the model group, but the difference was not statistically significant (Fig. 1D). The viral load represents that the number of viruses replicating in the body. We used real-time fluorescence quantitative PCR to assess viral load in lung tissue. The viral load in lung tissues of the model group was significantly higher (P < 0.001); compared with the model group, the viral load in the OP, SFJD-H, and SFJD-L groups was significantly lower (P < 0.001), suggesting that SFJD could reduce viral replication in lung tissues (Fig. 1E).

3.2. Network pharmacology target prediction results

Based on TCMSP and UniProt databases, a total of 122 SFJD targets were identified. By searching for disease targets in databases such as GeneCards and OMIM, a total of 1 911 targets related to influenza virus pneumonia were obtained. The Venny software was used to identify the overlapping targets between the active compounds of TCMs and diseases, which could potentially serve as key targets for the treatment of diseases using TCM compound ingredients (Fig. 2A). Cytoscape 3.9.0 software was used to analyze the topology of the network and construct a network diagram of “TCM component-target-disease” (Fig. 2B). The intersecting genes were imported from the String database, where a confidence level of 0.900 was set and the free gene nodes were hidden. Subsequently, the core targets were selected for a protein–protein interaction PPI (PPI) analysis (Fig. 2C). The Gene Ontology (GO) analysis is divided into three parts, namely biological process (BP), cellular component (CC), and molecular function (MF). These parts help in annotating the role of target proteins in gene function for the treatment of diseases. The major biological processes predicted include cellular response to chemical stress, response to lipopolysaccharide, response to oxidative stress, and regulation of apoptotic signaling pathways. The major cellular components involved are membrane region and vesicle lumen. Lastly, the major molecular functions identified are cytokine receptor binding, cytokine activity, inhibition of transcription factor binding, and protein phosphatase binding (Fig. 2D). The kyoto encyclopedia of genes and genomes (KEGG) prediction results suggested that the mechanism of pneumonia against influenza virus may be related to Toll-like receptor signaling pathway, IL-17 signaling pathway, TNF signaling pathway, C-type lectin receptor signaling pathway, apoptosis and other pathways (Fig. 2E). Based on the network pharmacological analysis, the mechanism of SFJD for the treatment of influenza virus pneumonia may be related to inflammation, and IL-6 and TNF-α are potential drug targets of SFJD, with Toll-like receptor signaling pathway and TNF signaling pathway as the main pathways of action.

Fig. 2.

Fig. 2

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Pharmacologic analysis of SFJD-influenza A virus pneumonia network. (A) Venny figure. (B) Core ingredient target of SFJD. (C) PPI protein interactions. (D) GO enrichment analysis (contain BP, CC, MF). (E) KEGG enrichment analysis.

3.3. Metabonomic analysis results of mouse lung tissue

Mouse lung tissues were analysed for metabolomics and the data matrices were analysed by principal component analysis (PCA) and orthogonal least partial squares discriminant analysis (OPLS-DA). PCA visualisation analysis showed significant differences between metabolite communities (Fig. 3A). Venn diagrams were created to show the metabolites that are shared or unique across metabolism sets. The visualization shows different metabolites for different comparison groups, with each group represented by a different color. Overlaps in the visualization indicate the number of metabolites that are common to multiple metabolism sets. The non-overlapping parts represent the number of metabolites that are specific to each metabolism set. The numbers in the visualization indicate the corresponding number of metabolites (Wang et al., 2023). The number of metabolites common to the control, control + SFJD-H, FM1 and SFJD-H groups was 49 (Fig. 3B). Metabolites with VIP > 1 and P < 0.05 were selected as significantly different metabolites. Compared with the control group, there were 132 metabolites with up-regulated expression and 117 metabolites with down-regulated expression in FM1 group (Fig. 3C); Compared with the FM1 group, a total of 69 metabolites were up-regulated and 214 metabolites were down-regulated in SFJD-H group (Fig. 3D). Hierarchical clustered heat maps showed the extent to which metabolite changes are produced. Metabolite expression between groups showed significant changes in alpha-D-glucose 1,6-bisphosphate, glutathione and oxiglutatione, and contignasterol (Fig. 4A–E). Differential pathways were mainly enriched to T helper type 1 (Th1), and T helper type 2 (Th2) differentiation, and NF-κB signaling pathway (Fig. 4F). Differential metabolites were compared between groups, and the differential metabolites were annotated to metabolic pathways via the KEGG database. Differential metabolic pathway enrichment was closely related to glutathione metabolism, arginine, proline metabolism, arachidonic acid metabolism and pentose phosphate pathway. Based on the references, indicators of inflammation are associated with metabolites including triacid, acetic acid, ornithine, and arginine (Zhang et al., 2022). The arachidonic acid (AA) metabolic pathway primarily act on the synthesis of inflammatory mediators and can regulate the production of various inflammatory factors (Lee et al., 2008). Modifying arginine, tryptophan, or purine metabolism has been shown to effectively alleviate excessive inflammatory responses (Xiao et al., 2021). Metabolic pathway analysis showed that the main metabolites and enriched signaling pathways were closely related to inflammation and immune response, and combined with the results of network pharmacological analysis suggested that the Toll receptor signaling pathway and the NF-κB signaling pathway were the key pathways in SFJD for the treatment of influenza viral pneumonia.

Fig. 3.

Fig. 3

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Metabonomic analysis results of mouse lung tissue. (A) Results of PCA (Different colours show clustering relationships between groups). (B) Venn diagram of metabolites between different groups. (C) Volcano map of control vs FM1. (D) Volcano map of FM1 vs SFJD-H.

Fig. 4.

Fig. 4

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Results of differential metabolite analysis of mouse lung tissue. (A) Results of hierarchical clustering heat map. (Each column of graph represents a sample, each row represents a metabolite, and colours in graph indicate magnitude of relative expression of metabolite in that group of samples.) (B–E) Expression statistics of metabolite alpha-D-glucose 1,6-bisphosphate, glutathione, oxiglutatione and contignasterol. (Mean ± SD, n = 6). (F) Bubble diagram of KEGG enrichment pathway. (The horizontal coordinate is rich factor; the vertical coordinate is KEGG pathway. The size of bubbles in graph represents how much of pathway is enriched to metabolic set of compounds, and the colours of bubbles indicate size of P-values for different enrichment significance. ###P < 0.001 vs control group; ***P < 0.001 vs model group; ns indicates that the difference is not statistically significant.

3.4. SFJD had anti-inflammatory and protective effects on lung tissue

To further evaluate the effects of the drugs in influenza virus-infected mice, we assessed the anti-inflammatory effects of the drugs by H&E staining and immunofluorescence staining. In the control group, the lung tissue was structurally normal, with no obvious swelling of the lung tissue and no obvious stasis or inflammatory exudation in the interstitium. Compared with the control group and control + SFJD-H group, the model group had more inflammatory cell infiltration, significant thickening of the alveolar wall with congestion and edema, and abnormal alveolar structure. Compared with the model group, the OP group, SFJD-H group and SFJD-L group showed improved lung histopathology with reduced cellular infiltration and more normal alveolar structure. The protective effect on lung tissue was better in the high dose group than in the low dose group. (Fig. 5A). The extent of macrophage inflammatory infiltration in lung tissue was determined using F4/80 immunofluorescence staining. Inflammatory signals trigger the recruitment of macrophages to the lungs. The fluorescence intensity was significantly enhanced in the FM1 group compared to the control group and control + SFJD-H group (P < 0.001). Macrophage infiltration in the lung tissue of mice was reduced after administration of SFJD and OP treatment (P < 0.01, 0.001), suggesting that SFJD may suppress the inflammatory response by inhibiting macrophage infiltration (Fig. 5B–C).

Fig. 5.

Fig. 5

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Protective and anti-inflammatory effects of SFJD on lung tissue. (A) H&E staining to evaluate lung histopathology, magnification factor: × 200, scale bar = 100 µm. (B) Images of macrophages in lung tissue detected by immunofluorescence staining (F4/80), × 50, scale bar = 20 µm. (C) Expression of F4/80 in lung tissue (Mean ± SD, n = 3). ###P < 0.001 vs control group and control + SFJD-H group; **P < 0.01, ***P < 0.001 vs model group.

3.5. SFJD modulated TLR4-mediated immune signaling pathways for treatment of viral pneumonia

TLR4 pattern recognition receptor plays a role in the antiviral response and activates the intracellular NF-κB signaling pathway via MyD88 and further promotes the secretion of the inflammatory cytokines TNF-α and IL-6. Therefore, Western blot was used to detect the expression of TLR4, MyD88, TRAF6 and p-NF-κB in lung tissues, and ELISA was used to detect the expression of TNF-α and IL-6 in lung tissues. Protein expression of TLR4, MyD88, TRAF6 and p-NF-κB was significantly increased in lung tissues after viral infection compared with control group and control + SFJD-H group (P < 0.05, 0.01). TLR4, MyD88, TRAF6 and p-NF-κB protein expression was reduced in lung tissues after administration of the drugs compared with the FM1 group, and the difference was statistically significant. TLR4, MyD88 and TRAF6 protein expression were reduced in lung tissues of the OP group compared with the FM1 group (P < 0.05). TLR4, MyD88, TRAF6 and p-NF-κB protein expression was significantly lower in lung tissues of the SFJD-H group compared with the FM1 group (P < 0.05). Notably, the results in the SFJD-H group were better than those in the SFJD-L group (Fig. 6A–E). The expression of pro-inflammatory factors in lung tissues was further examined, and the expression of both TNF-α and IL-6 was significantly increased in the model group when compared with the control group and the con + SFJD group, respectively (P < 0.01, 0.001); compared with the model group, each administered group could significantly down-regulate the expression of TNF-α in the lung tissues (P < 0.05, 0.01, 0.001), and the expression of IL-6 in the lung tissues could be statistically down-regulated in the OP group and the SFJD-H group (P < 0.001) (Fig. 6F–G). The above results suggest that SFJD can play a role in the treatment of viral pneumonia by down-regulating the expression of inflammatory factors and regulating the expression of related proteins on the TLR4/MyD88/NF-κB signaling pathway.

Fig. 6.

Fig. 6

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SFJD treated viral pneumonia in mice via the TLR4/MyD88/NF-κB immune signaling pathway. (A) Protein expression strip chart. (B−E) Protein expression of TLR4, MyD88, TRAF6 and p-NF-κB in lung tissues. (F−G) ELISA for TNF-α and IL-6 expression in lung tissue (n = 6). Data were shown as the mean ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001 vs control group and control + SFJD-H group; *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

3.6. SFJD protected mucosal epithelial cells and reduced airway inflammation and modulated TLR4-mediated signalling pathways

Krausgruber et al. utilized cellular phenotyping, transcriptome sequencing, chromatin open profiling, and epigenomics to investigate the interactions between mucosal epithelia and hematopoietic immune cells within specific organs like the lung or intestine (Krausgruber et al., 2020). The study revealed that mucosal epithelial cells play a crucial role in the initial stages of viral replication and infection establishment (Yang & Yan, 2021). These cells, similar to immune cells, express various PRRs that recognize viral PAMPs and activate the NF-κB pathway to carry out their functions (Wang and Proud, 2020, Yang and Yan, 2021). Therefore, in this experiment, we chose the influenza virus FM1-infected mouse model and combined it with the results of pharmacodynamic experiments to observe the morphologic changes in lung epithelial cells at the electron microscopic level after infection and treatment. In the control group, the cell membrane was intact, with a few microvilli (Mv) on the cell surface and no obvious swelling; the structure of tight junctions (TJ) was acceptable, and no widening of the cell gap was observed; the nuclear membrane of the nucleus (N) was structurally intact; the mitochondrion (M) was partially swollen, and in some cases of severe damage, the membrane was bulging, and the matrix was dissolved and became vacuolated (shown by the red arrowheads). In the FM1 group, cell membranes were intact, cells were moderately to severely edematous, with localized rupture of cell membranes (shown by red arrows), extensive lysis of intracellular matrix, and severe swelling of organelles; mitochondria (M) were severely swollen, with rupture and disintegration of membranes. In the SFJD-H group, the cell membrane was intact, the cells were moderately edematous, the intracellular matrix was reduced in electron density and thinning, and the swelling of the organelles was reduced. The nucleus (N) had an intact nuclear membrane structure, no widening of the perinuclear gap, and homogeneous chromatin; the mitochondria (M) were moderately swollen, with most of the membranes intact, and the matrix was extensively lysed and thinned (Fig. 7A). Further co-localization staining of lung epithelial cells and major proteins on the signaling pathway was performed in lung tissue. EpCAM was expressed in a variety of epithelial tissues, and molecule-specific antibodies can distinguish between tissues of epithelial and non-epithelial origin (Wang et al., 2020). Immunofluorescence co-localization results showed that the lung epithelial cells exhibited strong EpCAM protein fluorescence signals, and the protein fluorescence signals of TLR4, MyD88, and NF-κB in the FM1 group were significantly enhanced after viral stimulation compared to control group (P < 0.05, 0.01); the fluorescence signals of the relevant proteins were reduced compared with those of the model group after the administration of SFJD (P < 0.05), which suggested that SFJD protected the lung epithelial cells to a certain degree, and may act via the TLR4/MyD88/NF-κB signaling pathway (Fig. 7B–G).

Fig. 7.

Fig. 7

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SFJD protected mucosal epithelial cells and reduced airway inflammation. (A) Morphology of lung epithelial cells under electron microscope. (the left scale bar = 5 µm, × 3 000; the right scale bar = 2 µm, × 8 000). (B, D, F) Fluorescent expression of TLR4, MyD88 and NF-κB in mouse lung tissue (scale bar = 20 µm, × 100). (C, E, G) Expression of TLR4, MyD88 and NF-κB in mouse lung tissue (n = 3). Data were shown as the mean ± SD. #P < 0.05, ##P < 0.01 vs control group; *P < 0.05 vs model group.

4. Discussion

SFJD is a TCM compound preparation based on the experience formula of famous doctors, which has the efficacy of dispersing wind and clearing heat, detoxifying and soothing the throat and has been clinically applied for many years with precise efficacy. This product, which possesses independent intellectual property rights, demonstrates significant efficacy in the treatment of acute upper respiratory infections. It has been officially recognized as a recommended medicine for the treatment of influenza A (H1N1) by the National Health Commission of the People’s Republic of China. SFJD is widely used in the prevention and treatment of viral infectious diseases, especially emerging infectious diseases (EIDs), in vitro experiments confirmed the involvement of type I interferon and the MAPK/NF-κB signaling pathway in anti-H1N1 infection effect (Tao et al., 2020). Previous studies have shown that SFJD has antiviral and antimicrobial effects and is effective in treating fever caused by viral upper respiratory tract infections (Ji et al., 2020, Xia et al., 2018). Studies have shown that various components of SFJD may ameliorate p21-activated kinase (PAK)-induced upper respiratory tract infections through multiple targets (Li et al., 2017). Several studies have confirmed the ability of SFJD to treat lung tissue damage caused by influenza virus infection, with anti-inflammatory and antiviral effects. In recent years, there has been an increasing focus on what role the respiratory mucosa plays in antiviral and immunological related research. Mucosal tissue is an important barrier to the body’s immune system. Mucous membranes indicate the presence of a large number of microorganisms, and more than 90% of human pathogen infections are associated with mucous membranes. The mucous membranes of the respiratory tract are both the first site of invasion by the influenza virus and an important part of the body’s immune defence (Currier et al., 2016). Influenza viruses entering the host through the oral or nasal cavity are initially countered by the mucus covering the respiratory tract’s epithelium. If the virus successfully crosses the mucosal layer, it must then adhere to and invade the respiratory epithelial cells. Subsequently, the virus can spread to both non-immune and immune cells, such as macrophages and dendritic cells, within the respiratory tract (Manicassamy et al., 2010, Perrone et al., 2008). Studies have shown that respiratory mucosal epithelial cells have their own characteristic pattern recognition receptor expression and distribution, and through PRRs sensing viral PAMPs, they complete the earliest recognition of viruses by the organism, initiate the process of viral infection, and achieve rapid inflammatory response and antiviral effects; at the same time, they recruit, activate, and coordinate the intrinsic immune response (Weitnauer; Mijošek, & Dalpke, 2016). PRRs TLR2 and TLR4 on the membrane of respiratory mucosal epithelial cells were found to rapidly sense PAMPs on the surface of viruses and activate the intracellular NF-κB signaling pathway via MyD88 (Whitsett & Alenghat, 2015). It further promotes the secretion of inflammatory cytokines TNF-α, IL-1 and IL-6 (Mirzaei et al., 2022, Whitsett and Alenghat, 2015).

The antiviral effects of SFJD have been demonstrated in vivo, particularly in treating lung tissue damage caused by influenza virus infection. However, its mechanism of action is still not well studied and further experimental studies are needed. In this study, we firstly used the modelling method of nasal drip infection, and verified the protective effect of SFJD on influenza virus-infected mice by observing the changes in lung histological imaging before and after the administration of the infection, calculating the inhibition rate of the lung index, and detecting the viral load. μCT is an emerging medical imaging technique that allows for non-destructive volumetric imaging of surgical tissue specimens with high spatial resolution (Bompoti et al., 2021, Ivanciuc et al., 2023). μCT has been shown to be a safe and very effective tool for longitudinal visualization of lung tissue. We evaluated the imaging results of the lungs by μCT. The μCT system is uniquely suited for, among other things, the examination of lung tissue in small animals. μCT, a state-of-the-art CT imaging technology, has spatial resolution on the order of micrometres (> 9 μm) and can provide three-dimensional images (Ritman, 2002). Studies have shown that SFJD effectively inhibits lung tissue damage caused by influenza virus infection, reduces viral load in the lungs, prevents inflammatory cell infiltration and overexpression of inflammatory cytokines, and provides protection to the lung tissue. Specifically, we found that SFJD ameliorated lung tissue lesions, decreased the expression of influenza virus nucleic acids in lung tissue, and reduced macrophage infiltration in lung tissue, resulting in significant anti-inflammatory and antiviral protection.

The innate immune system is involved in the earliest stages of host defense against foreign microorganisms and perceives viral PAMPs through PRRs (Kurt-Jones et al., 2000). Conducted airway cells and type II alveolar cells express multiple TLRs, including TLR2 and TLR4 (Whitsett & Alenghat, 2015), the latter can be activated in response to lipopolysaccharides, viruses and inflammatory cytokines (Baltina et al., 2019, Iwasaki and Pillai, 2014, Root-Bernstein, 2021). Combined with the results of the network pharmacology data and metabonomic analysis, it is thought that the mechanism of pneumonia resistance to influenza virus may be related to pathways such as the Toll-like receptor signaling pathway. Consistent with the analysis, the experimental results confirm that SFJD downregulates TLR4-mediated signaling pathway protein expression in lung tissue. Studies have shown that TLR signaling in non-immune cells, including lung epithelial cells, is essential for driving mucosal immune responses (Cleaver et al., 2014, Hammad et al., 2009). PRRs direct epithelial cell-intrinsic defence response. In epithelial cells, signal transduction activates NF-κB mainly through the signal adapter MyD88 and MyD88-dependent mechanisms. The activated NF-κB signaling pathway further promotes the expression of inflammatory factors. Based on this, we further observed the structural changes in the lung epithelium and analysed the co-localisation of lung epithelial cells and proteins such as TLR4 and MyD88 in in vivo experiments. The findings support the notion that TLR4 plays a role in regulating protein secretion of the natural immune system and the survival of respiratory epithelial cells following influenza virus infection. Maintenance of metabolic homeostasis is the basis for normal physiological functioning of the organism, and host metabolism can influence viral infection and disease progression by directly affecting viral replication and modulating the immune response (Ayres, 2020, Codo et al., 2020). The development of metabolomics technology has made it possible to study the relationship between metabolic regulation and viral infection. Viral infections can influence the body’s metabolic responses, while metabolic responses and metabolites can also modulate the host immune response and thus influence viral infections (Jia et al., 2023). In the present study, we analysed the metabolomics results of lung tissues and the differential metabolites were mostly closely related to glutathione metabolism, arginine and proline metabolism, arachidonic acid metabolism and pentose phosphate pathway. Viral infection activates innate immunity and induces a massive inflammatory response, excessive inflammation can cause tissue damage (Boechat, Chora, Morais, & Delgado, 2021). It has been shown that peripheral blood metabolites in COVID-19 patients are closely related to the secretion of inflammatory cytokines (Su et al., 2020). Differential metabolites in the analysed results were mostly associated with inflammatory responses and viral infections. Significantly, there is evidence that glutathione inhibits replication of various viruses at different stages of the viral life cycle, thereby reducing viral load and potentially preventing the massive release of inflammatory cells into the lungs (Checconi et al., 2023). Arginine showed strong association with the related inflammatory cytokines IL-6, IL-1β, IL-12 p70, and IFN-α2, and thus it is hypothesised that important metabolic pathways have a potential role in the regulation of pro-inflammatory cytokine/chemokine secretion (Xiao et al., 2021). The pentose phosphate pathway is one of the key metabolic pathways in living cells that could produce energy and maintain cellular homeostasis. It was shown that the transketolase (TKT) inhibitor benfotiamine (BOT) could inhibit SARS-CoV-2 replication, which was closely related to the non-oxidative pathway of pentose phosphate (Bojkova et al., 2021).

The cells in the respiratory tract are constantly exposed to the external environment, making the lungs a particularly susceptible site for infection. The mucosal immune system, which is the first line of defence of the organism, has an important task in resisting the invasion of pathogenic microorganisms. Lung epithelial cells are important target cells for influenza virus lung infection and key cells for respiratory mucosal immunity. Therefore, in this experiment, transmission electron microscopy was used to observe the morphological changes of lung epithelial cells, and immunofluorescence staining was used to detect the expression of related proteins. The results confirmed that SFJD has a protective effect on lung epithelial cells and may act through the TLR4/MyD88/NF-κB signaling pathway. This provides some basis for exploring whether SFJD can exert antiviral effects based on respiratory mucosal immune mechanisms. This experimental study focuses on exploring and validating the antiviral effects of SFJD based on the immune mechanism of the respiratory mucosa, and it is worth our attention that macrophages also play an important role in antiviral and modulation of the immune response. In the results of this experiment we found that SFJD could provide anti-inflammatory and antiviral protection by reducing macrophage infiltration in lung tissues, but the proliferation and phagocytic activity of primary macrophages before and after administration were not investigated, and the subsequent experiments will take more account of the changes in macrophage function.

Acute respiratory infections caused by influenza viruses pose a significant threat to human health and public health safety (Sempere Borau & Stertz, 2021). Influenza has a wide host range and spreads rapidly. Global influenza prevention and control still faces many challenges. Currently, the main medications used to treat influenza are anti-influenza viral medications, and the most effective way to prevent influenza infections and their complications is to get vaccinated against influenza. However, the therapeutic efficacy of antiviral drugs has declined due to side effects and the emergence of drug-resistant mutants (Ison, 2011, Javanian et al., 2021). Based on the practical experience accumulated by TCM in the prevention and treatment of epidemics, Chinese medicines are able to act directly on viruses, effectively blocking a certain part of the viral growth and reproduction process, so as to achieve the effect of killing viruses or inhibiting their replication, and are also able to promote the immune function of the body to achieve the purpose of suppressing viruses, which play an irreplaceable and important role in the face of sudden-onset infectious diseases. Chinese medicine exerts its anti-influenza activity by modulating the immune response to interfere with viral infection and host response, and is likely to be another therapeutic option for the treatment of influenza virus infection (Dai et al., 2020; Huang et al., 2021). With its reasonable organic combination, SFJD has brought into play the unique advantages of compound Chinese medicine. The chemical basis of SFJD has been investigated using HPLC-Q-TOF-MS/MS, identifying 94 of these compounds, and 46 exogenous compounds associated with SFJDC have been identified in rat plasma, including 27 prototypical absorbent fractions and 19 metabolites (Han et al., 2023). In this study, starting from the PRRs of respiratory mucosal epithelial cells and the inflammatory pathway as the main links, we obtained the potential molecular mechanism of SFJD antiviral on the basis of experimental validation of SFJD efficacy, combined with network pharmacology and metabolomics. SFJD was able to exert its antiviral effects through the TLR4/MyD88/TRAF6/NF-κB signaling pathway. This provides insights into the clinical management of viral pneumonia by SFJD.

CRediT authorship contribution statement

Shan Cao: Investigation, Writing – original draft, Validation. Lei Bao: Data curation. Zihan Geng: Data curation. Yingli Xu: Software. Bo Pang: Data curation. Jingsheng Zhang: Data curation. Qiang Zhu: Project administration. Jun Pan: Project administration. Yaxin Wang: Data curation. Mengping Chen: Data curation. Yu Zhang: Data curation. Yanyan Bao: Conceptualization, Supervision. Jing Sun: Conceptualization, Supervision. Ronghua Zhao: Conceptualization, Supervision. Xiaolan Cui: Funding acquisition, Project administration. Shanshan Guo: Funding acquisition, Project administration.

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.

Acknowledgments

This research was funded by National Natural Science Foundation of China (Nos. 82141206 and 82151210).

Contributor Information

Xiaolan Cui, Email: cuixiaolan2812@126.com.

Shanshan Guo, Email: ssguo@icmm.ac.cn.

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