Material basis and pharmacodynamic mechanism of YangshenDingzhi granules in the intervention of viral pneumonia: Based on serum pharmacochemistry and network pharmacology

Abstract

Background

YangshenDingzhi granules (YSDZ) are clinically effective in preventing and treating COVID‐19. The present study elucidates the underlying mechanism of YSDZ intervention in viral pneumonia by employing serum pharmacochemistry and network pharmacology.

Methods

The chemical constituents of YSDZ in the blood were examined using ultra‐performance liquid chromatography‐quadrupole/orbitrap high‐resolution mass spectrometry (UPLC‐Q‐Exactive Orbitrap MS). Potential protein targets were obtained from the SwissTargetPrediction database, and the target genes associated with viral pneumonia were identified using GeneCards, DisGeNET, and Online Mendelian Inheritance in Man (OMIM) databases. The intersection of blood component‐related targets and disease‐related targets was determined using Venny 2.1. Protein–protein interaction networks were constructed using the STRING database. The Metascape database was employed to perform enrichment analyses of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways for the targets, while the Cytoscape 3.9.1 software was utilized to construct drug‐component‐disease‐target‐pathway networks. Further, in vitro and in vivo experiments were performed to establish the therapeutic effectiveness of YSDZ against viral pneumonia.

Results

Fifteen compounds and 124 targets linked to viral pneumonia were detected in serum. Among these, MAPK1, MAPK3, AKT1, EGFR, and TNF play significant roles. In vitro tests revealed that the medicated serum suppressed the replication of H1N1, RSV, and SARS‐CoV‐2 replicon. Further, in vivo testing analysis shows that YSDZ decreases the viral load in the lungs of mice infected with RSV and H1N1.

Conclusion

The chemical constituents of YSDZ in the blood may elicit therapeutic effects against viral pneumonia by targeting multiple proteins and pathways.

We used UPLC‐Q‐Exactive Orbitrap‐MS and network pharmacology to investigate the pharmacodynamic mechanism that enables the blood components of YangshenDingzhi granules to intervene in viral pneumonia. In vitro and in vivo experiments verified that the drug inhibited H1N1, RSV, and SARS‐CoV‐2 Replicon viruses through the synergistic effects of multiple components, targets, and pathways.

1. INTRODUCTION

Viral pneumonia, a rapid‐onset respiratory infection, predominantly strikes in winter and spring and can spread widely or appear as outbreaks in densely populated areas. It is caused by a diverse group of viruses, including influenza A virus (IAV), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). It can also result from co‐infection with multiple viruses or bacterial pathogens. ¹ Traditional Chinese medicine (TCM) has classified it as an ‘epidemic disease’, that is, a contagious and widespread disease, a definition similar to the ‘acute infectious disease’ classification in modern medicine. “The five epidemics can be transmitted to each other, regardless of whether they are adults or children, and the symptoms are the same,” according to Huangdi Neijing (Inner Canon of Yellow Emperor). ² Currently, conventional available antiviral drugs like acyclovir, adenosine, and interferon are susceptible to drug resistance and may cause various side effects. In contrast, TCM has shown certain advantages in preventing and treating viral pneumonia due to its multi‐component and multi‐target nature. ³ Hence, there is a pressing need for research in TCM approaches for the management and prevention of viral pneumonia.

YangshenDingzhi granules (YangshenDingzhi Keli, YSDZ) are a traditional proprietary herbal formula developed by Professor Zhaojun Yan at the Hospital of Shandong University of Traditional Chinese Medicine (SDUTCM). The formulation is based on a modification of ‘Renshu Powder’ from the classical text “Introduction to Medicine”. ⁴ YSDZ has been recognized as a TCM compound, and is composed of Rehmanniae Radix Praeparata (Shudihuang, SDH), Polygala tenuifolia Willd (Zhiyuanzhi, ZYZ), Poria cocos (Schw.) Wolf (Fushen, FS), Saposhnikovia divaricata (Trucz.) Schischk (Fangfeng, FF), Cyrtomium fortunei J. Sm. (Mianmaguanzhong, MMGZ), Talinum paniculatum (Jacq.) Gaertn. (Hongshen, HS), Chrysanthemum indicum L. (Yejuhua, YJH), and Aurantii fructus (Fuchaozhiqiao, FCZQ). ⁵ The last two decades of clinical application and research have shown the potential efficacy of YSDZ in treating several mental health conditions, such as anxiety, fear, and insomnia. It has also demonstrated efficacy in preventing and treating respiratory diseases due to its heat‐clearing, detoxifying, phlegm‐dissolving, and cough‐relieving properties. ⁶ , ⁷ , ⁸ The formulation utilizes specific herbs to replenish and promote the circulation of qi (vital energy) and blood, stabilize the spirit, expel wind, relieve external heat, dissolve phlegm, and tranquilize the spirit. YSDZ has demonstrated significant clinical efficacy in preventing and treating SARS‐CoV‐2 infection and was utilized in Tanzania's epidemic in 2020, marking a significant milestone in the international application of TCM against COVID‐19. Later, it was extensively used in the Square Cabin Hospital in Shanghai in 2022, making a substantial contribution to the care of COVID‐19 patients. Overall, it has been recognized as an effective TCM compound for strengthening the body, harmonizing form and spirit, and addressing disease symptoms and underlying causes.

Serum pharmacochemical investigation of TCM formulae is an efficient approach for identifying and analyzing components migrating in the blood after oral administration. This method enables rapid screening to determine the pharmacodynamic material basis of the TCM formula and explains the compound compatibility rule. TCM has several complex components, but only the components in the blood can be transported to the target site as active ingredients, using blood as the carrier, to produce therapeutic effects. ⁹ , ¹⁰ Network pharmacology is employed to investigate the mechanisms and network of disease and drug‐related targets by integrating systems biology, genetics, high‐throughput omics data analysis, and network database retrieval. ¹¹ , ¹² The combination of serum pharmacochemistry and network pharmacology allows the precise screening of active drug ingredients that directly affect the human body, revealing their complex pharmacological mechanisms of action. ¹³ , ¹⁴ In this study, ultra‐performance liquid chromatography‐quadrupole/orbitrap high‐resolution mass spectrometry (UPLC‐Q‐Exactive Orbitrap MS) was utilized along with in vitro cellular experiments and in vivo animal experiments to perform a comprehensive analysis of the blood‐entry components of YSDZ. This approach aimed to explore the mechanism of YSDZ intervention in viral pneumonia by integrating network pharmacology and to validate its antiviral efficacy by employing in vitro and in vivo experiments. This study provides detailed data describing the material basis of YSDZ in the treatment of viral pneumonia and its molecular mechanism of antiviral therapy.

2. METHODS

2.1. Instruments and reagents

Cutting‐edge laboratory equipment, including the Ultimate 3000 ultra‐performance liquid chromatography, the a mass spectrometry workstation Xcalibur3.0, low‐temperature high‐speed centrifuge and vortex mixer, and HPLC grade formic acid and acetonitrile were acquired from Thermo Fisher Scientific (Waltham, MA, USA). An electronic analytical balance with a precision of one ten‐thousandth was obtained from Sartorius in Gottingen, Germany. Methanol of analytical purity was supplied by Tedia (Fairfield, OH, USA), and YSDZ was received from the Affiliated Hospital of SDUTCM (Jinan, China) (Batch No.: Luyao Preparation [Emergency] No. Z2020003).

2.2. Animals and housing

Forty 8‐week‐old male Sprague–Dawley (SD) rats, and eighteen 5‐week‐old and eighteen 4‐week‐old female Balb/c mice were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd (SCXK2021‐0006, Beijing, China). All the animal experiments were approved by the Animal Experiment Ethics Committee of Shandong University of Traditional Chinese Medicine.

2.3. Serum pharmacokinetic analysis of TCM

2.3.1. Preparation of YSDZ

YSDZ granules were dissolved in a methanol–water (1:1) solution using ultrasonication (300 W power, 50 kHz frequency) for 5 min. The solution was mixed with control solution to obtain a concentration of approximately 0.125 mg/mL. The resulting solution was filtered through a 0.22 μm microporous membrane.

2.3.2. Animal administration and serum sample collection

In the animal administration phase, 8‐week‐old rats were randomly allocated to the mock group (n = 15) and the YSDZ group (n = 25). Rats in the YSDZ group were orally administered 300 mg/kg/d of YSDZ for 14 days, based on the clinically applied human equivalent dose recommended by Professor Yan Zhaojun of the Affiliated Hospital of SDUTCM. Meanwhile, the mock group received an equivalent volume of saline. Blood samples were centrifuged at 3000 r/min at 4°C for 15 min to obtain serum samples, which were then subjected to inactivation at 56°C for 30 min, transferred to a 1.5 mL Eppendorf tube, and stored at −80°C.

2.3.3. Pretreatment of serum samples

Serum samples (1 mL) obtained from YSDZ and the control group were mixed with 4 mL acetonitrile and immediately vortexed for 3 min. Then, the mixture was centrifuged at 13 000 r/min at 4°C for 15 min. Subsequently, the supernatant was dried using nitrogen at room temperature. The resulting residue was reconstituted with 100 μL of methanol–water (1:1) solution, followed by centrifugation at 14 000 r/min at 4°C for 15 min. Lastly, the supernatant was extracted to obtain the test solution for UPLC‐Q‐Extractive Orbitrap MS analysis.

2.3.4. UPLC‐Q‐Exactive Orbitrap MS detection conditions

The liquid chromatography with tandem mass spectrometry (LC–MS/MS) data was obtained using the UHPLC Ultimate 3000 instrument coupled with a Q‐Exactive Orbitrap‐MS spectrometer (Thermo Fisher Scientific). Chromatographic analysis was performed on a Halo‐C₁₈ column (2.1 × 100 mm, 1.8 μm). The mobile phase consisted of 0.1% formic acid for solvent A and acetonitrile for solvent B. The gradient elution program was set as follows: 0–5 min, 5% B; 5–15 min, 5%–13% B; 15–25 min, 13%–25% B; 25–35 min, 25%–45% B; 35–40 min, 45%–60% B; and 40–45 min, 60%–95% B. The column temperature was maintained at 40°C, and the flow rate was fixed at 0.3 mL/min, while the injection volume was set at 3 μL. The mass spectrometer was equipped with a heated electrospray ionization (HESI) source using the following parameters: sheath gas pressure, 45 arb; auxiliary gas pressure, 10 arb; spray voltage, 3.00 kV; capillary temperature, 350°C; heater temperature, 350°C.

2.4. Network pharmacology analysis

2.4.1. Identification of targets for blood‐entry components

Chemical structures of the blood‐entry components were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and optimized using ChemDraw 20.0 software. The target proteins were predicted by using the SwissTargetPrediction platform (http://www.swisstargetprediction.ch/). ¹⁵ All the information related to target proteins was collected from the UniProt database (https://www.uniprot.org).

2.4.2. Identification of disease targets

The disease‐specific targets for ‘virus pneumonia’ were identified by GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org/), and OMIM (https://omim.org/) databases. ¹⁶ , ¹⁷ , ¹⁸ Duplicate entries were removed by data integration, and the nomenclature of the target proteins was standardized using the UniProt database.

2.4.3. Protein–protein interaction network analysis

The Venny 2.1 online platform (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was employed to identify the common targets between blood components and viral pneumonia. The protein–protein interaction (PPI) network was constructed on the basis of molecular interaction information from the STRING database (https://string‐db.org/), which was further visualized by Cytoscape 3.9.1 software. The primary targets were screened out based on the degree value, and nodes with higher degree values are considered to be key targets for potential blood component treatment of viral pneumonia. ¹⁹

2.4.4. Signaling pathways and functional enrichment analysis

The common targets were imported into the Metascape database (https://metascape.org/) to perform enrichment analyses for Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. ²⁰ The study was focused on identifying biological functions and pathways associated with the antiviral pneumonia target genes of YSDZ blood‐entry components. The GO and KEGG enrichment analysis results were visualized using the microbiology platform (https://www.bioinformatics.com.cn).

2.4.5. Building a ‘drug‐component‐disease‐target‐pathway’ network

To visualize the relationship between drug components, disease targets, and pathways for YSDZ intervention in viral pneumonia, a network was constructed using Cytoscape 3.9.1 software. This network integrated the identified compounds, common targets, and pathways.

2.5. In vitro antiviral activity assay for YSDZ

2.5.1. In vitro evaluation of the antiviral activity of YSDZ against H1N1

Madin‐Darby Canine Kidney (MDCK) cells were seeded at a density of 10⁵ cells/mL in 96‐well plates and incubated at 37°C with 5% CO₂ for 12 h. Subsequently, the cells were infected with an IAV A/PR/8/34 (H1N1) viral solution at a concentration of 100 median tissue culture infectious doses (TCID₅₀). Next, the cells were treated with YSDZ drug serum after diluting six mass concentrations at a 2‐fold ratio. After the 48‐h incubation period, the cells were subjected to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining, and the optical density (OD) value (λ = 490 nm) of each group was measured using an enzyme marker. Thereafter, the percentage viral inhibition and percentage cell viability were calculated based on the obtained OD values.

2.5.2. In vitro evaluation of the antiviral activity of YSDZ against RSV

Human larynx epidermoid carcinoma (Hep‐2) cells were incubated in 96‐well plates for 12 h. Later, the cells were infected with RSV virus solution at a concentration of 100 TCID₅₀. All the other experimental conditions were kept the same as those described above for H1N1.

2.5.3. In vitro evaluation of the antiviral activity of YSDZ against SARS‐CoV‐2 replicon

Human embryonic kidney (293T) cells were incubated in 96‐well plates for 12 h. Then, the cells were infected with SARS‐CoV‐2 Replicon virus solution (1 RLU/cell). After 36 h of incubation, viral replication was assessed by luciferase expression, and the percentage of viral inhibition was calculated. The rest of the experimental conditions were the same as reported above for H1N1.

2.6. In vivo antiviral activity assay for YSDZ

2.6.1. In vivo evaluation of the antiviral activity of YSDZ against H1N1

A total of 18 female Balb/c mice (5 weeks old) were randomly divided into mock, vehicle, and YSDZ groups, with six mice in each group. The mice in the vehicle and YSDZ group were inoculated with 30 μL of H1N1 viral solution (0.8 LD₅₀) per nasal drop, while the mock group received saline nasal drops. After infection, the YSDZ group was orally administered a 0.2 mL dose of YSDZ twice a day for 5 days, equal to 4 times the human equivalent dose of 15.6 g/kg/d, whereas the mock and vehicle groups received an equal volume of saline. One hour after the administration of the last dose, the lung tissue of the mice was dissected and extracted. Subsequently, 30 mg of lung tissue was weighed and tested for viral load using the quantitative polymerase chain reaction (qPCR).

2.6.2. In vivo evaluation of the antiviral activity of YSDZ against RSV

The Balb/c mice were randomly divided into mock, vehicle, and YSDZ groups of six each. The mice in the vehicle and YSDZ group were inoculated with 100 μL of RSV viral solution (3.125 × 10⁴ TCID₅₀) through continuous nasal drops on the first and second day of administration. The mock group received saline nasal drops. Following infection, the YSDZ group was orally administered the same YSDZ dose as for H1N1‐infected mice for 3 days, while the mock and vehicle groups were given an equal volume of saline. One hour after the final dose, lung tissue was obtained as described in the previous section, and viral titer TCID₅₀ was detected using cytopathic effect (CPE) analysis.

2.7. Data analysis

All data were shown as mean ± standard deviation (SD), and the difference between experimental samples was assessed by the univariate t test using GraphPad Prism 9.5.1 software (GraphPad Software, San Diego, CA, USA).

3. RESULTS

3.1. Identification of blood‐entry constituents

Figures 1 and 2 depict the comprehensive ion flow charts of the serum chemistry of the treated and untreated serum samples in positive and negative ion modes. A total of 15 supplementary constituents, including 12 standard components and 3 metabolites, from rat serum were identified using Compound Discoverer 3.1 software (Thermo Fisher Scientific) in conjunction with MS/MS fragments and pertinent literature (Table 1).

FIGURE 1.

Total ion flow diagram of control and YSDZ‐medicated serum in positive ion mode. (A) Control serum, (B) Medicated serum.

FIGURE 2.

Total ion flow diagram of control and YSDZ‐medicated serum in negative ion mode. (A) Control serum, (B) Medicated serum. YSDZ blood‐entry components Viral pneumonia.

TABLE 1.

Identification of YSDZ blood‐entry components.

No.	t R (min)	Adduction	Measured mass (m/z)	Mass error (ppm)	Molecule formula	MS/MS fragments (m/z)	Name	Source
1	2.29	M + H	165.0548	1.2080	C9H8O3	147.0442, 119.0495	Deglycosylation product of catalpol	SDH
2	3.21	M + H	268.1020	0.5950	C10H13O4N5	136.062	Adenosine	FF, FCZQ
3	3.4	M + H	127.0393	2.1990	C6H6O3	109.0288, 100.0762	5‐Hydroxymethylfurfural	FCZQ
4	10.4	M + H	163.0393	1.7750	C9H6O3	135.0445, 117.0707, 107.0859	Umbelliferone	FF, FCZQ
5	11.47	M − H	153.0179	−9.1620	C7H6O4	135.0073,109.0279	Protocatechuic acid	SDH
6	11.49	M + H	377.1461	5.0290	C16H24O10	243.0874	Methylation product of catalpol	SDH
7	13.39	M + H	321.0971	0.9050	C16H16O7	303.0863, 273.0398	Divaricatacid	FF
8	11.56	M − H	183.1016	−5.7220	C10H16O3	139.075	Rehmapicrogenin	SDH
9	17.47	M + H	247.0961	−1.4390	C14H14O4	229.0861, 175.0392, 147.0443	Nodakenetin	FF
10	18	M + H	307.1167	−3.0760	C16H18O6	259.0598, 235.0614	Cimifugin	FF
11	20.73	M − H	433.1137	−0.6470	C21H22O10	257.0815, 175.0234	Naringenin‐7‐O‐β‐D‐glucoside	YJH
12	25.4	M + H	261.1122	0.4000	C15H16O4	243.1018,189.0548,159.0442, 131.0494	Meranzin	FCZQ
13	38.42	M + H	359.1125	−0.1100	C19H18O7	344.0898	5‐Hydroxy‐3′,4′,6,7‐Tetramethoxyflavone	FCZQ
14	42.92	M − H	237.0765	−1.5470	C12H14O5	223.0275,193.0862, 51.0784	3,4,5‐Trimethoxycinnamic acid	ZYZ
15	43.57	M − H	783.4981	10.3310	C42H72O14	391.2854	Ginsenoside Rf	HS

Abbreviations: FCZQ, Fuchaozhiqiao; FF, Fushen; HS, Hongshen; SDH, Shudihuang; YJH, Yejuhua; ZYZ, Zhiyuanzhi.

3.2. Screening of targets associated with blood‐entry components and disease‐related targets

Potential targets for 12 prototype compounds were identified utilizing the SwissTargetPrediction database. Based on a probability threshold greater than zero, 724 potential targets were initially determined, which reduced to 442 targets after merging and removing duplicates. A total of 5610 disease‐related targets for ‘viral pneumonia’ were obtained from GeneCards, DisGeNET, and OMIM databases. Subsequently, the GeneCards database filtered 1490 disease‐related targets after applying two screenings with a relevance score greater than or equal to the median value of 3.579. Then, the Venny 2.1 software was employed to determine the intersection of the 442 compound‐related targets with the 1490 disease‐related targets, resulting in 124 overlapping targets (Figure 3).

FIGURE 3.

Intersection‐targets of YSDZ blood‐entry components and viral pneumonia.

3.3. Analysis of PPI network

The identified intersection‐target genes were utilized in the STRING database to generate the PPI network (Figure 4), which comprises 124 nodes and 2068 edges. Subsequently, the constructed network was visualized and analyzed using the Cytoscape 3.9.1 software. Then, topology analysis was carried out using the CytoNcA plugin, and the median mean degree value was determined to be 28. A filtering process based on a median of greater than 2‐fold degree values resulted in a core set of 19 targets (Figure 5).

FIGURE 4.

PPI network of potential targets.

FIGURE 5.

Core targets network.

3.4. GO functions and KEGG pathway enrichment analysis

The GO functions and KEGG pathway enrichment analyses were performed on 124 intersection targets using the Metascape database. GO functional analysis was employed to characterize the function of gene targets encompassing Biological Process (BP), Cell Component (CC), and Molecular Function (MF). A total of 1660, 104, and 149 entries were obtained for BP, CC, and MF, respectively (p value <0.01). The top 10 enriched pathways in each of the three sections related to viral pneumonia were selected for the histogram (Figure 6). BP is mainly involved in protein phosphorylation and positive regulation of response to external stimulus. CC is primarily concerned with the vesicle lumen, membrane raft, and receptor complex. MF is mainly engaged with protein tyrosine kinase activity.

FIGURE 6.

GO enrichment analysis of the intersection‐targets.

Furthermore, 124 intersection targets were analyzed to assess the enrichment of KEGG signaling pathways. Similarly, 179 signaling pathways were screened with a p value <0.01. Then, a total of 52 pathways were examined to elucidate the mechanisms underlying viral pneumonia, which mainly involved coronavirus disease (COVID‐19), PI3K‐Akt signaling pathway, MAPK signaling pathway, C‐type lectin receptor signaling pathway, and T cell receptor signaling pathway. The visualization of the top 20 enriched KEGG signaling pathways is depicted in Figure 7. These 20 fundamental pathways can be classified into three modules: virus‐related, inflammation‐related, and immunomodulation‐related, as summarized in Table 2.

FIGURE 7.

Enrichment analysis of KEGG pathways for intersection‐targets.

TABLE 2.

Pathways linked to viral pneumonia.

Classifications	No.	Pathways	p‐values
Virus‐related	hsa05171	Coronavirus disease—COVID‐19	2.31488E−25
	hsa05163	Human cytomegalovirus infection	3.75698E−24
	hsa05169	Epstein–Barr virus infection	3.9227E−22
	hsa05164	Influenza A	5.607E−19
	hsa05168	Herpes simplex virus 1 infection	3.65744E−10
	hsa04061	Viral protein interaction with cytokine and cytokine receptor	0.000779006
Inflammation‐related	hsa04151	PI3K‐Akt signaling pathway	6.52818E−34
	hsa04010	MAPK signaling pathway	2.1412E−24
	hsa04066	HIF‐1 signaling pathway	1.99544E−22
	hsa04630	JAK–STAT signaling pathway	9.89651E−21
	hsa04668	TNF signaling pathway	2.15885E−20
	hsa04014	Ras signaling pathway	2.39261E−19
	hsa04012	ErbB signaling pathway	5.29761E−19
Immunomodulation‐related	hsa04625	C‐type lectin receptor signaling pathway	1.46896E−24
	hsa04210	Apoptosis	2.42585E−22
	hsa04660	T cell receptor signaling pathway	2.2797E−19
	hsa04659	Th17 cell differentiation	4.12795E−19
	hsa04657	IL‐17 signaling pathway	2.35655E−18
	hsa04620	Toll‐like receptor signaling pathway	1.03919E−17
	hsa04520	Adherens junction	9.54893E−17

3.5. Establishment of the ‘drug‐component‐disease‐target‐pathway’ network

A network was constructed using the compounds associated with the intersection‐targets and KEGG outcomes (Figure 8). Network topology analysis revealed that five active ingredients, i.e. nodakenetin, 5‐hydroxy‐3′,4′,6,7‐tetramethoxyflavone, cimifugin, adenosine, and divaricatacid, have degree values greater than the median (Table 3). Furthermore, 56 targets were found with degree values above the median. When combined with the PPI core target network, these data indicated that the targets MAPK1, MAPK3, AKT1, EGFR, and TNF might play a vital role in the therapeutic potential of YSDZ in the prevention and treatment of viral pneumonia (Table 4).

FIGURE 8.

The network of ‘drug‐component‐disease‐target‐pathway’. The network comprised 161 nodes (5 drug nodes, 11 compound nodes, 1 disease node, 124 target nodes, 20 pathway nodes) and 593 edges. The size of the nodes indicated the target's degree value, signifying its importance within the network. The green circles in the diagram correspond to various drugs (FCZQ, Fuchaozhiqiao; FF, Fushen; HS, Hongshen; SDH, Shudihuang; YJH, Yejuhua). The hexagons in the diagram represent active ingredients, with the two yellow colors in the middle left representing the common components of FCZQ and FF. The blue octagon in the diagram represents a disease, while the blue diamond represents intersection‐targets, and the red triangles represent pathways.

TABLE 3.

Topological analysis of blood‐entry components.

No.	Components	Degree	Betweenness centrality	Closeness centrality
1	Nodakenetin	39	0.175666	0.425532
2	5‐Hydroxy‐3′,4′,6,7‐Tetramethoxyflavone	37	0.188137	0.418848
3	Cimifugin	32	0.119343	0.40404
4	Adenosine	24	0.103006	0.368664
5	Divaricatacid	23	0.05844	0.37037
6	Umbelliferone	20	0.049161	0.365297
7	Rehmapicrogenin	18	0.107595	0.358744
8	Meranzin	13	0.024547	0.350877
9	Protocatechuic acid	6	0.01795	0.3125
10	Naringenin‐7‐O‐β‐D‐glucoside	6	0.015331	0.305344
11	Ginsenoside Rf	6	0.014064	0.283688

TABLE 4.

Top 10 targets with topological analysis of degree values.

No.	Targets	Degree	Betweenness centrality	Closeness centrality
1	MAPK1	19	0.037586	0.415584
2	MAPK3	18	0.046684	0.415584
3	AKT1	17	0.027532	0.405063
4	EGFR	14	0.04355	0.445682
5	TNF	14	0.029427	0.383693
6	JUN	12	0.007108	0.37296
7	CASP3	10	0.007664	0.37123
8	MTOR	9	0.006361	0.359551
9	IL2	8	0.009482	0.336842
10	SRC	7	0.011696	0.380048

3.6. Efficacy of YSDZ against viral pneumonia

3.6.1. In vitro antiviral activity of YSDZ against H1N1, RSV and SARS‐CoV‐2 replicating serum

In vitro experiments revealed that the YSDZ‐treated serum exhibits inhibitory effects against H1N1, RSV, and SARS‐CoV‐2 Replicon. Within its effective concentration range, the treated serum displayed a decrease in inhibitory activity with increased dilution. Specifically, the inhibition rate of H1N1 exhibited a more pronounced dose–response relation. Furthermore, the treated serum showed inhibition of RSV at high concentrations (1:2) and also demonstrated a more evident dose‐dependent inhibition of SARS‐CoV‐2 Replicon at higher concentrations (1:4 and 1:2) (Figure 9).

FIGURE 9.

In vitro antiviral activity of YSDZ‐medicated serum against pneumonia. (A), H1N1. (B), RSV. (C), SARS‐CoV‐2 Replicon.

3.6.2. In vivo efficacy of YSDZ against viral pneumonia

The animal study was performed to establish the effectiveness of YSDZ in the treatment of viral pneumonia. The YSDZ‐treated 5‐week‐old H1N1‐infected Balb/c mice group showed a reduction in the viral load in the lungs compared to the vehicle group on the fifth day, indicating an inhibitory effect of YSDZ on H1N1. Similarly, the YSDZ‐treated 4‐week‐old RSV‐infected Balb/c mice group exhibited a decrease in virus titer in the lungs compared to the vehicle group on the fifth day, suggesting the inhibitory effect of YSDZ against RSV also (Figure 10).

FIGURE 10.

Effect of YSDZ on viral load in the lungs of H1N1‐infected mice and viral titer in the lungs of RSV‐infected mice. (A), H1N1, (B), RSV.

4. DISCUSSION

In the current investigation, network pharmacology revealed that certain components of YSDZ such as adenosine, 5‐hydroxymethylfurfural (5‐hmf), umbelliferone, protocatechuic acid (PCA), divaricatacid, rehmapicrogenin, nodakenetin, cimifugin, naringenin‐7‐o‐β‐d‐glucoside, meranzin, 5‐hydroxy‐3′,4′,6,7‐tetramethoxyflavone, and ginsenoside Rf, may potentially impact viral pneumonia by influencing the molecular network associated with key target genes such as MAPK1, MAPK3, AKT1, EGFR, and TNF. Additionally, these components may also modulate signaling pathways related to viral infection, inflammation, and immune modulation, including coronavirus disease (COVID‐19), human cytomegalovirus infection, Epstein–Barr virus infection, influenza A, the PI3K‐Akt signaling pathway, the MAPK signaling pathway, the HIF‐1 signaling pathway, the C‐type lectin receptor signaling pathway, apoptosis, and the T cell receptor signaling pathway.

Among the components, adenosine and its derivatives are significant regulators of metabolic processes in animal cells, influencing various metabolic functions through purinergic signaling pathway and exhibiting antiviral, anticancer, and immunomodulatory properties. ²¹ Nucleoside analogs can have therapeutic antiviral effects by inhibiting viral nucleic acid synthesis. For example, Ridecivir, a novel nucleoside analog antiviral, has been found to inhibit coronavirus RNA synthesis. ²² Research has demonstrated that 5‐HMF treatment up‐regulated the expression of β‐interferon (IFN‐β) and IFN‐stimulated chemokine genes in RAW 264.7 cells and primary peritoneal macrophages. It also enhanced innate antiviral immunity in mice, resulting in elevated serum IFN‐β levels and decreased morbidity and viral load after infection with vesicular stomatitis virus (VSV). It has also been shown that 5‐HMF establishes a novel immunomodulatory mechanism against viral infections by enhancing IFN‐JAK/STAT signaling and increasing the production of type I interferon (IFN) through retinoic acid‐inducible gene I (RIG‐I). ²³ PCA, a natural phenolic acid, has many beneficial properties, including antioxidant, anti‐inflammatory, neuroprotective, antibacterial, antiviral, anticancer, anti‐osteoporosis, analgesic, antiaging, metabolism regulation, hepatoprotective, and protective effects on the renal, digestive and reproductive systems. ²⁴ PCA administration to H1N1‐infected mice prevented weight loss and mortality and reduced lung index, viral titer, immune cell infectivity, and cytokine levels. In addition, it also inhibited H1N1 transmission by suppressing H1N1‐induced TLR4/NF‐κB activation. This suggests that PCA may have the potential to treat IAV. ²⁵ Another study revealed that Rehmapicrogenin exhibited potent anti‐inflammatory effects on inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), and interleukin‐6 (IL‐6) in a model of inflammation induced by lipopolysaccharide (LPS)‐induced RAW264.7. ²⁶ Meranzin has been shown to potentially reduce the inflammatory response induced by LPS and enhance the secretion of inflammatory cytokines TNF‐α, IL‐4, and IL‐6 in macrophages. ²⁷ Ginsenoside Rf has been demonstrated to significantly decrease nitric oxide (NO) levels and IL‐6 in macrophages treated with LPS. ²⁸ According to Lim's research, umbelliferone and nodakenetin, coumarin derivatives, exhibited potent anti‐inflammatory effects in both in vitro and in vivo experiments conducted in mice. ²⁹ Cimifugin possesses various pharmacological effects, including antipyretic and analgesic properties, anti‐inflammatory, antioxidant, anti‐allergic, antitumor, and anti‐psoriasis activities. ³⁰ , ³¹ Cimifugin has been found to exert anti‐inflammatory effects by inhibiting the activation of the MAPK/NF‐κB signaling pathway and down‐regulating the expression of iNOS and COX‐2 in LPS‐induced macrophage inflammation models, thereby suppressing the release of inflammatory factors such as NO. ³²

The MAPK pathway is a central hub for several signal transduction pathways involved in cell proliferation, stress response, inflammation, differentiation, functional coordination, cellular transformation, and apoptosis. MAPK1 and MAPK3 are the principal subfamilies in this pathway. In terms of an inflammatory response, MAPK1 transduces extracellular signals from the cell membrane to the nucleus via the NOD‐like pathway, resulting in the release of inflammatory mediators, such as TNF‐α and IL‐1β, through the NF‐κB signaling pathway, thereby exacerbating cellular damage. ³³ , ³⁴ Inhibition of the MAPK3/MAPK1 signaling pathway reduces the expression of inflammatory factors, including TNF‐α and IL‐1β, induced by lung inflammation, suggesting the possibility of potential therapeutic implications for various inflammatory conditions. ³⁵ AKT1 is involved in the regulation of cell proliferation and growth. Its overexpression has been found to enhance viral protein synthesis, while its suppression reduces viral RNA expression and impedes viral capsid formation. Research has demonstrated that the inhibition of AKT1 can decrease the viral load in human hepatocellular carcinoma (Huh7) cells infected with SARS‐CoV‐2. ³⁶ Additionally, AKT1 plays a role in modulating the body's innate immunity, which affects the immune response and macrophage activation properties. ³⁷ Recent studies have shown that EGFR, a receptor tyrosine kinase for epidermal growth factor cell proliferation and signaling, and a member of the ErbB receptor family, is involved in multiple viral infections, including SARS‐CoV‐2, and is upregulated after SARS‐CoV‐2 infection. Various respiratory viruses can utilize EGFR‐related signaling pathways by activating EGFR to invade host cells and suppress the immune response. ³⁸

The enrichment analysis results indicated that the activation of signaling pathways such as MAPK, JAK‐STAT, TNF, and IL‐17 collectively influenced the expression of downstream inflammatory factors, including IL‐6, IL‐4, IL‐1β, COX2, and TNF‐α. These factors are closely related to cellular inflammatory responses and immune regulation. ³⁹ Viral infections are commonly accompanied by oxidative stress, resulting in exaggerated immune response, release of inflammatory cytokines, increased lipogenesis, and altered endothelial and mitochondrial function. Furthermore, the PI3K/Akt signaling pathway could serve as a promising antiviral target by mitigating the detrimental effects of oxidative stress and reducing disease severity through various host cell survival mechanisms. ⁴⁰ C‐type lectin receptors are crucial as pattern recognition receptors within the innate immune system. In viral infections, these receptors indirectly protect the host by reducing excessive inflammatory responses through the immunological receptors of dendritic cells (DCs). In HIV infection, DCs identify and engulf the virus via their expressed C‐type lectin receptors, resulting in viral replication within DCs and subsequent transmission to CD4+ T cells. Activation of C‐type lectin receptors also hinders essential cellular functions, including DC maturation, intracellular Toll‐like receptor pathway signaling, retinoic‐acid‐inducible gene I‐like receptor activation, and cell autophagy. In addition, it has been noted that the S protein of SARS‐CoV‐2 can attach to the C‐type lectin receptor on the surface of DCs, facilitating viral invasion and exacerbating the body's inflammatory response, this promotes the onset of an ‘inflammatory factor storm’. ⁴¹ , ⁴² Studies have indicated that inflammatory marker levels are elevated in viral pneumonia, and YSDZ reduces the expression of TNF‐α, and IL‐1β inflammatory factors. ⁶ , ⁴³ Thus, YSDZ could play a major role in the treatment of viral pneumonia by regulating the inflammatory response.

5. CONCLUSIONS

In this study, in vitro experiments demonstrated that YSDZ exhibited exceptional broad‐spectrum antiviral activities, while in vivo analysis established that YSDZ was effective in treating viral pneumonia. Serum pharmacochemistry and network pharmacology are utilized to identify the active compounds in YSDZ and examine the enrichment analyses of GO and KEGG pathways. Preliminarily findings revealed that YSDZ intervenes in viral pneumonia through multi‐component, multi‐target, and multi‐pathway characteristics. Nonetheless, this research presents certain constraints, and further verification of the active compounds and pathways predicted in network pharmacology is required. Thus, future research should concentrate on the monomeric compounds of YSDZ to elucidate the aforementioned limitations.

AUTHOR CONTRIBUTIONS

Qinghua Cui and Kejian Li conceived and designed the research. Huirong Xu conducted experiments and wrote the manuscript. Meiyue Dong and Chengcheng Zhang designed the experimental procedures and analyzed the data. Zinuo Chen and Guangyu Tian analyzed the data. Ruikun Du provided reagents.

FUNDING INFORMATION

This study was supported by Key R & D Project in Shandong Province, China (Grant number: 2020CXGC010505) and Qingdao Science and Technology Demonstration Program for the Benefit of the People, Shandong Province, China (Grant number: 23‐7‐8‐smjk‐3‐nsh).

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest to declare.

ETHICS STATEMENT

The Ethical Review Board at Experimental Center of Shandong University of Traditional Chinese Medicine approved the animal experiments (no. 201803512).

Xu H, Dong M, Du R, et al. Material basis and pharmacodynamic mechanism of YangshenDingzhi granules in the intervention of viral pneumonia: Based on serum pharmacochemistry and network pharmacology. Anim Models Exp Med. 2024;7:259‐274. doi: 10.1002/ame2.12440

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this published article.