Network Pharmacology Analysis of the Mechanisms of Compound Herba Sarcandrae (Fufang Zhongjiefeng) Aerosol in Chronic Pharyngitis Treatment

Introduction

Chronic pharyngitis (CP) is a diffusive inflammation of the pharyngeal mucosa, submucosa, and pharyngeal lymphoid tissues, with a high incidence that accounts for approximately 10–20% of throat diseases.^1,2 According to the epidemiological data, at least 20% of the adult population suffers from chronic pharyngitis worldwide.³ The symptoms of CP are stubborn, and the disease is difficult to cure and causes great inconvenience to the patient. At present, well-known international medical treatments are lozenges, laser and cryotherapy, and antibiotics. However, the disease is likely to break out repeatedly. In recent years, the abuse of many antibiotic drugs leads to the increased the risk of antibiotic resistance and the risk of adverse reactions⁴ as well as a significant decrease of the curative effects. Group A β-hemolytic Streptococcus (GAS)-induced pharyngitis is often considered as a trigger of CP, and in 56–97% of CP cases, GAS pharyngitis precedes lesion formation.⁵

CP recurs easily, often has a prolonged disease course, and is difficult to treat.⁶ The causes of CP can be infectious (bacterial or viral), non-infectious (excessive alcohol use, smoking, voice abuse or misuse),⁷ or a recurring acute pharyngitis that is not being treated properly.^7–11 Among the bacterial CP, GAS bacterium is the most common pathogen associated with the disease.^7,12,13 Compound Herba Sarcandrae aerosol, also known as the Fufang Zhongjiefeng (FFZJF) aerosol, has been shown to be clinically effective in treating CP and it has been approved by Guizhou Medical Products Administration for human use. A previous study reported that FFZJF has anti-inflammatory and anti-bacterial properties with small side effects, in vitro and in animals,¹⁴ long-term use, but its exact mechanism remains unclear. Traditional Chinese medicine (TCM) has a long history of viewing an individual or patient as a system with different statuses and has accumulated numerous herbal formulae. The holistic philosophy of TCM shares much with the key ideas of emerging network pharmacology and network biology, and meets the requirements of overcoming complex diseases.^15,16 Network pharmacology is a technique used to systematically evaluate the pharmacological effects of drugs with multiple components and multiple targets by establishing links between targets, drugs, and diseases based on the principles of systems biology, which shares a similar holistic philosophy to traditional Chinese medicine (TCM).^15,16 Therefore, network pharmacology is an important tool to analyze the effective components and therapeutic target of TCM.¹⁷

In this study, we used a network pharmacology approach to determine the effective ingredients, key targets, and related signaling pathways of FFZJF for the treatment of CP. A rat bacterial CP model⁵ was then established to evaluate the pharmacodynamic effects of FFZJF and to verify the molecular targets and signaling pathways predicted by network pharmacology. The results of this study will aid further investigation of the underlying mechanism of FFZJF in treating chronic pharyngitis.

Materials and Methods

Experimental Animal, Drugs and Reagents

Sixty specific-pathogen-free Sprague Dawley rats, including 30 females and 30 males, weighing 210–230 g, were purchased from Qinglongshan Experimental Animal Farm (Nanjing, Jiangsu Province, China) [License number: SCXK (Su) 2017–0001]. FFZJF was prepared using Zhongjiefeng (Herba Sarcandrae), Maidong (Ophiopogonis Radix), Honghua (Carthami Flos), Yuxingcao (Houttuyniae Herba), and Jiegeng (Platycodonis Radix) at a 4:3:2:1:1 ratio. The authenticity of herbs was validated by Deputy Chief Pharmacist Xianmei Xu (Guiyang College of Traditional Chinese Medicine Second Affiliated Hospital). The FFZJF aerosol was prepared by the hospital laboratory (Qian Yao Z20140036, equivalent to 2 g raw herbs per mL aerosol preparation). Ampicillin Sodium for Injection (AS) (Guo Yao H13020426) was purchased from North China Pharmaceutical (Shijiazhuang City, Hebei Province, China). Group A β-hemolytic Streptococcus (GAS, Cat# FSCC225016) was purchased from Guangzhou Huankai Bio-Technology.SYBR^® Premix Ex Taq (RR820A), PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A), RNAiso Plus (9108), all purchased from Takara Japan; DEPC (D105557, Aladdin Biochemistry in Shanghai); TLR-4 Antibody (GR163524-6, British Abcam Corporation), MyD88 Antibody (GR142247-2,British Abcam Corporation), NF-κB P65 Antibody (00057397, American Proteintech Corporation); TLR-4, MyD88, NF-κB P65 PCR primers (Synthesis of Nanjing Kingsley Biotechnology Co., Ltd). TNF-α, IL1β, and IL6 ELISA Kit (CSB-E11987r, CSB-E08055r, CSB-E04640r, Cusabio).

Identification of FFZJF Active Compounds and Putative Compound Targets

The active compounds in FFZJF were collected from the TCM Systems Pharmacology Database and Analysis Platform (TCMSP;https://old.tcmsp-e.com/tcmsp.php)¹⁸ and Chemistry Database (http://www.organchem.csdb.cn/scdb/default.htm?nCount=13303008).¹⁹ The absorption, distribution, metabolism, and excretion (ADME) profiles of these compounds were obtained via literature searches and used to facilitate the screening of effective ingredients.²⁰ The pharmacokinetics and pharmacodynamics criteria included oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, and half-life (HL) ≥ 4 h(The main screening conditions of drug components were certain OB and DL levels. If one component does not meet one of the two conditions, it is still retained if it has good DL and HL levels or activities).^21,22 The corresponding targets of the putative compounds were searched in the TCMSP and the Swiss Target Prediction database (http://www.swisstargetprediction.ch/).^15,23 To expand the potential targets, the molecular similarity match tool, the Simplified Molecular Input Line Entry System (SMILES) in PubChem (https://pubchem.ncbi.nlm.nih.gov/#query=Nevadensia),²³ was used. These two groups of targets were entered into the UniProt database with the organism defined as “Homo sapiens” and were considered FFZJF potential targets. UniProt (https://www.uniprot.org),²³ is used for finding gene names and UniProt numbers of the putative target genes.²⁴

Identification of Chronic Pharyngitis-Associated Targets

Potential target genes of CP were collected from Online Mendelian Inheritance in Man (OMIM) (https://omim.org/),²⁵ DrugBank Online (https://www.drugbank.ca/),²⁶ DisGeNT database (https://www.disgenet.org/),²⁶ and Genetic Association Database (GAD) (https://geneticassociationdb.nih.gov/)²⁵ with the keyword of “chronic pharyngitis”. The overlapping and repeat targets were excluded. UniProt (https://www.uniprot.org) was used for unifying gene names and UniProt numbers.

Network Construction and Pathway Enrichment

After the putative targets of the FFZJF active compounds and the known therapeutic targets of CP were identified as described above, the protein-protein interaction (PPI) analysis was performed using the Search Tool for the Retrieval of Interacting Genes (String, Version 10.5, https://string-db.org/) database.²⁷ A Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/)²⁵ was then produced to identify the intersection nodes of FFZJF and chronic pharyngitis.

The FFZJF–active compound–CP target network and herbs-active ingredient network were then constructed and visualized using Cytoscape software (version 3.6.1).²⁷ Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to predict gene functions using Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8 (https://david.ncifcrf.gov/).²⁷ The GO describes three distinct aspects gene functions in terms of biological process, cellular component, and molecular function. P-value < 0.05 was set as the cut-off criteria for significantly enriched KEGG pathways. The top key pathways related to FFZJF treatment of CP were selected based on the count values.

Animal Model and Drug Treatment

Experimental procedures used in this study followed the procedures described previously.^28,29 All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC)(Ethical number:1709051). After one-week acclimation, the 60 rats were randomly divided into two groups. The first cohort (n = 10) received no drug intervention and were used as blank control group. The other 50 rats received pharyngeal inoculation of 10¹² CFU/mL GAS to establish a CP model. After inoculation, the bacteria were allowed to colonize for 15 days. The general health conditions of the animals were monitored daily. Five rats died due to the improper operations of a laboratory staff during the creation of the animal model. On Day 16, 3 rats were randomly selected and euthanized, and the pharyngeal mucosal tissues were collected for histopathological examination. The establishment of the GAS CP rat model was confirmed according to the symptoms, signs, and histopathological examination. The rats with confirmed CP were randomly divided into the control model group, ampicillin sodium group (AS), FFZJF high-dose (FFZJF-H), FFZJF medium-dose (FFZJF-M), and FFZJF low-dose (FFZJF-L).

According to the literature,³⁰ the rats in the FFZJF-L, FFZJF-M, and FFZJF-H group received 2.33, 4.66, and 9.32 g/kg FFZJF in 20 mL saline nebulization, respectively. The rats in the control and treated CP model groups received an equal volume of saline nebulization once daily for 14 consecutive days as a control treatment. The rats in the AS group received a daily intraperitoneal injection of ampicillin sodium at a dose of 0.93 g/kg for 14 days. At 24 h after the last treatment, the animals were euthanized and the serum and pharyngeal mucosal tissues were collected for further analysis. Half of each tissue sample was fixed in 4% paraformaldehyde, and the other half was frozen and stored at −80°C.

Measurement of the Serum Content of TNF-α, IL1β, and IL6 Protein

24 hours after the final medication, several steps are taken: 1) Anesthesia with 10% chloral hydrate solution mL/100 g intraperitoneal injection; 2) taking blood from the abdominal aorta; 3) after 2 h at room temperature, 3000 r/min, 4°C Centrifugation (radius 13.5 cm) for 10 min; 4) take the supernatant and keep it in −80°C Refrigerator; 5) serum TNF-α, IL1β, IL6 levels in rats were measured by ELISA method. The operation was carried out strictly according to the instructions of the kit. TNF-α, IL1β, IL6 levels in serum were calculated.³¹

Measurements of mRNA Expression Levels of TLR-4, MyD88, and NF-κB P65 in Pharyngeal Mucosa Tissue by Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

The mRNA levels of TLR-4, MyD88, and NF-κB P65 in the pharyngeal mucosal tissue were determined using a qRT-PCR. In brief, total RNA was extracted using the Trizol reagent and cDNA was prepared. SYBR^® Premix Ex Taq, PrimeScript™ RT reagent Kit with gDNA Eraser, and RNAiso Plus were purchased from Takara (Kusatsu, Shiga, Japan). qRT-PCR was performed in a 20 µL reaction volume using the following conditions: pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 1 min. StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for qRT-PCR. The primers were synthesized by GenScript Biotechnology (Nanjing, Jiangsu Province, China) and their sequences are shown in Table 1. The expression levels of TLR-4, MyD88, and NF-κB P65 mRNAs were normalized to GAPDH and calculated using the 2^−ΔΔCt method.

Table 1 List of Primer Sequences Used for RT-PCR Analysis in This Study

Histological Examination of Pharyngeal Mucosal Tissue and Immunohistochemical (IHC) Staining

Serial sections (4 µm) were obtained from paraformaldehyde-fixed, paraffin-embedded rat pharyngeal mucosal tissues for histological analysis and IHC. The pharyngeal mucosal tissue morphology and anatomy were examined and compared using hematoxylin and eosin (HE) staining.³²

For IHC, after antigen retrieval, the endogenous peroxidase activity was quenched, and the nonspecific antibody binding was blocked. The tissue sections were then incubated at 4°C overnight with specific primary antibodies (at 1:200 dilution), followed by incubation with the corresponding secondary antibodies (at 1:200 dilution) at 4°C for 50 min. Mouse anti-TLR-4 (lot GR163524-6) and rabbit anti-MyD88 (lot GR142247-2) antibodies were purchased from Abcam (Shanghai City, China), and the rabbit anti-NF-κB P65 (lot 00057397) antibody was from Proteintech (Wuhan City, Hubei Province, China). Following the secondary antibody incubation and washing, the sections were developed with 3, 3’, 4, 4’-biphenyltetramine tetrahydrochloride (DAB) and counterstained with hematoxylin. The positive staining was identified as brown color granules. The expression patterns and subcellular localization of the TLR-4, MyD88, and NF-κB P65 protein were examined. A Nikon H550S microscope (Nikon, Tokyo, Japan) was used to observe the staining. The relative expression levels of TLR-4, MyD88, and NF-κB P65 proteins were quantitated using Integrated Optical Density (IOD) measured in Image-Pro plus 6.0 software and compared between the experimental groups.

Statistical Analysis

Statistical analyses were performed using SPSS software version 22.0 (Armonk city, New York state, USA). Unless otherwise noted, all data are presented as mean ± standard error of mean (SEM). The data were first subjected to the Shapiro–Wilk test for normality. Normally distributed data were analyzed using one-way analysis of variance (ANOVA), and the Kruskal–Wallis test was used if the data did not pass the normality test. If the results from the ANOVA tests were statistically significant, the pair-wise comparisons were then performed. For pair-wise comparisons, the Fisher’s least significant difference (LSD) tests were used. Comparisons with p values < 0.05 were considered statistically significant.

Results

Active Compounds Identified in FFZJF and Their Putative Targets

Database screening identified 189, 161, 200, 220, and 162 compounds in the five FFZJF herbal extracts, namely Zhongjiefeng, Maidong, Honghua, Yuxingcao, and Jiegeng, respectively. Using OB ≥ 30%, DL ≥ 0.18, and half-life ≥ 4 h as the cut-off criteria, we identified 45 compounds from FFZJF, of which 7 from Zhong Jie Feng (15.6%), 10 from Mai Dong (22.2%), 15 from Hong hua, 6 from Yu Xing cao (33.3%), and 7 from Jiegeng (15.6%). According to relevant literature,^21,22 the components with good retention activity but not satisfying the screening conditions were retained (Table 2). The network diagram of active components of Chinese herbal medicine was established (Figure 1), Pink nodes represent herbs, Purple nodes represent key active ingredients after network analysis, and Green nodes represent other active ingredients. In the network diagram, the effective compounds were analyzed and arranged in descending order. The first four active compounds were quercetin, Spinasterol, Stigmasterol, kaempferol (Table 3). In addition, a total of 704 putative protein targets of these compounds were identified using the TCMSP and the Swiss Target Prediction databases.

Table 2 The Active Compounds from the 5 Herbs in FFZJF

Table 3 Information on Four Key Active Ingredients

Figure 1 The network construction for herbs-active ingredient.

Prediction of FFZJF Molecular Targets for the Treatment of CP

The CP-related target genes were screened from OMIM (https://omim.org/), DrugBank Online (https://www.drugbank.ca/), DisGeNT (https://www.disgenet.org/), and GAD (https://geneticassociationdb.nih.gov/). Using the keyword “chronic pharyngitis” to search the databases, 43 associated genes were identified. From the comparative analysis between the 43 CP-related and 704 putative FFZJF targets, 11 were predicted as FFZJF targets for the treatment of CP (Figure 2).

Figure 2 Venn diagram of FFZJF drug targets and CP-disease proteins.

The network relationship between FFZJF, the active compounds, and CP targets are shown in Figure 3, which shows the interactions of targets containing 384 nodes and 2,252 edges (connecting lines) with an average node degree of 11.73. The node size represented the degree value of the target. The shapes indicate nodes and the grey lines indicate interaction edges. The triangles and green colour indicate drugs. Diamond shapes and yellow colour indicate compounds. Circles and red colour indicate targets. According to the node degree, Honghua and Maidong were identified as the principle herbs. The top 10 predicted compounds and targets are shown in Table 4.

Table 4 Topology Features of FFZJF-Active Compounds–CP Network

Figure 3 (A) Gene ontology analysis of key target genes. (B) Topology features of FFZJF-active compounds–CP network.

Analysis of PPI

The FFZJF-compounds-CP PPI network included 11 nodes and 37 edges (Figure 4).The larger the node and the darker the color was, the larger the degree value was. The larger the degree value was, the more important role the part played in the PPI. The average node degree was 6.73. Eight hub proteins, which were identified based on the node degree > 6.73, ranked from the highest to the lowest degrees were IL6, PTGS2, TLR-4, TNF, ELANE, CXCR4, TLR9, and IL1B (Table 5). These targets are likely to be key targets for FFZJF treatment of CP, and TNF-α, IL1β, IL6, TLR-4 targets are selected for subsequent experimental verification.

Table 5 Information on the Top-11 Genes in PPI Network

Figure 4 (A) PPI network of CP targets of FFZJF. (B) Hub genes of the PPI network.

GO Enrichment and KEGG Pathway Analysis of the 11 Putative FFZJF Targets

The 11 common targets shared by FFZJF active compound and CP disease-related proteins were further characterized for known functions using DAVID pathway analysis. GO enrichment was analyzed from the perspective of molecular function, biological process, and cellular component. Analyses using the GO database showed that the main enrichment of genes in the BP category was “inflammatory response”, “positive regulation of NF-kappaB import into nucleus”, “positive regulation of nitric oxide biosynthetic process”, “positive regulation of interleukin-6 production”, and “positive regulation of gene expression”. The main MF categories were “heme binding”, “cytokine activity”, “enzyme binding”, “interleukin-1 receptor binding”, “cytokine binding”, “steroid hydroxylase activity”, and “oxygen binding”.The main CC categories were “cytoplasm”, “cell surface”, “extracellular region”, “extracellular space”,and “endoplasmic reticulum membrane” (Figure 5).

Figure 5 Gene ontology analysis of key target genes.

In addition, KEGG pathways of the target genes were analyzed and the top 15 of the 27 enriched pathways are shown in Figure 6. The 27 enriched pathways were found to be associated with viral infections, cancer, inflammation, and immunity. Among these pathways, the classic signaling pathways, including the toll-like receptor signaling pathway, TNF signaling pathway, NF-κB signaling pathway, interactions between cytokine-cytokine receptors, NOD-like receptor signaling pathway, and HIF-1 signaling pathway were among the top 15 enriched with the most genes. In particular, the top-ranked Toll-like receptor signaling pathway, TNF signaling pathway, and NF-κB signaling pathway were enriched with genes such as IL6, TNF, TLR-4, and TLR9, these targets were tested and validated.

Figure 6 Top 15 pathways by KEGG analysis.

General Health Conditions of the Animals in the Treatment Groups

Four days after GAS inoculation, the animals started to show some symptoms, such as scratching in the mouth area, drinking more frequently, lethargy, fluffed coat, and oral discharge (occasionally bloody). Some animals died during the CP model establishment. By day 16, oral discharge decreased gradually. However, the rats exhibited weight loss (p<0.05) (Figure 7A), became less active, and consumed less food. Oral visual examination with a headlight showed chronic congestion in pharyngeal mucosal tissue, indicated by a dark red-purple colored, dry, dull, and swollen mucosa with some yellow discharge. After FFZJF or AS treatment, the overall health conditions of the animals markedly improved, as evidenced by the lustrous coat and increased food consumption, the weight increased (p<0.05), but did not return to the weight of the rats in the blank group (p<0.05) (Figure 7B). Such improvement was particularly significant in the FFZJF-H and AS treatment groups. Moreover, oral examination showed overall normal pharyngeal mucosal tissue in the FFZJF-H and AS groups. In the FFZJF-L and FFZJF-M groups, the pharyngeal mucosal tissue showed a relatively normal color and morphology, except for some mild chronic congestion. The rats in the control group were healthy and had no anomalies throughout the study. The rats in the treated model group had CP symptoms and signs, which remained unrelieved throughout the study.

Figure 7 Changes of body weight of rats during modeling and administration.

Serum Levels of TNF-α, IL1β, and IL6

The treated model group had significantly elevated serum levels of TNF-α, IL1β, and IL6 compared to the non-CP control group (p < 0.01), which indicated the successful establishment of the CP model in vivo. Ampicillin sodium treatment significantly reduced the serum levels of TNF-α, IL1β, and IL6 as compared to the CP model group (p < 0.01). The animals treated with different doses of FFZJF showed decreased serum levels of TNF-α, IL1β, and IL6 at different degrees (p < 0.01), with FFZJF-H showing the greatest effect The curative effect of FFZJF-L group was the most obvious. Compared to the AS group, there was no significant difference in serum TNF-α, IL1β, and IL6 in FFZJF-H group (p > 0.05) (Table 6, Figure 8A–C).

Table 6 The Effect of FFZJF Aerosol on the Rat’s Serum Levels of TNF-α, IL1β, and IL6 (Mean ± SEM, n = 8)

Figure 8 The effect of FFZJF aerosol on the rat’s serum levels of TNF-α, IL1β, and IL6 (Mean ± SEM, n = 8). (A) TNF-α; (B) IL1; (C) IL6. Notes: As compared to treated non-CP control group: *p < 0.05, **p < 0.01; as compared to treated CP model group: #p < 0.05, ##p < 0.01; as compared to treated Ampicillin sodium group: ΔP<0.05.

Histopathological Examination

In the non-CP control group, the pharyngeal mucosal tissue exhibited normal microscopic anatomy. In the CP model group, submucosal edema with extensive inflammatory cell infiltration was observed, which also confirmed the successful establishment of CP model. The inflammatory cell infiltration reduced significantly in the AS group. In FFZJF groups, the alleviation of inflammatory cell infiltration increased as FFZJF dose increased, with the most significant reduction in the FFZJF-H group (Figure 9).

Figure 9 Histopathological examinations of the pharyngeal mucosal tissue of the rats from each group (HE, 200×). (A) Control group; (B) Model group; (C) FFZJF-L group; (D) FFZJF-M group; (E) FFZJF-H group; (F) AS group. It can be seen that the morphology of the pharyngeal tissues in the control group remain intact based on microscopy. In the model group, the mucous membrane appears necrotic, the submucosal layer shows swelling (edema), and many inflammatory cells are present. The inflammation is significantly reduced in the FFZJF treatment groups. As the dose of FFZJF increases, the condition of inflammation improved.

The Expression of TLR-4, MyD88, and NF-κB P65

The mRNA expression levels of TLR-4, MyD88, and NF-κB P65 in pharyngeal mucosal tissues were measured using qRT-PCR and compared between the experimental groups. The CP model group had significantly increased mRNA levels of TLR-4, MyD88, and NF-κB P65 in pharyngeal mucosa as compared with the no-CP control group (p < 0.01). Compared to the CP model group, the FFZJF-L group had a moderately reduced NF-κB P65 mRNA level (p < 0.05). The FFZJF-M group had a significant reduction in TLR-4 (p < 0.05), MyD88 (p < 0.01), and NF-κB P65 (p < 0.01) mRNA expression level, while the FFZJF-H and AS groups showed the greatest reduction in the expression of all three genes (p < 0.01) in their pharyngeal mucosal tissues.Compared with the AS group, the mRNA levels of TLR-4, MyD88 and NF-κB P65 in the FFZJF-H group were not statistically significant (p>0.05) (Table 7,Figure 10A–C).

Table 7 The Effect of FFZJF Aerosol on the mRNA Levels of TLR-4, MyD88, and NF-κB P65 in the Rat’s Pharyngeal Mucosal Tissues (Mean ± SEM, n = 8)

Figure 10 The effect of FFZJF aerosol on the mRNA levels of TLR-4, MyD88, and NF-κB P65 in the rat’s pharyngeal mucosal tissues (Mean ± SEM, n = 8). (A) TLR-4; (B) MyD88; (C) NF-κB P65.Note: As compared to treated non-CP control group: *p < 0.05, **p < 0.01; as compared to treated CP model group: #p < 0.05, ##p < 0.01; as compared to treated Ampicillin sodium group: ΔP<0.05.

The protein expression levels of TLR-4, MyD88, and NF-κB P65 in pharyngeal mucosal tissues were assessed using IHC and compared across the experimental groups. The subcellular location and tissue distribution of each protein were first evaluated. Positive TLR-4 staining was mainly associated with the cell membrane and cytosol of the lymphocytes, but the staining was relatively weak in other tissue types (Figure 11). MyD88 positive staining was mainly associated with the cytosol of all tissue and cell types (Figure 12). NF-κB P65 positive staining was mainly associated with the cytosol and nucleus in all tissue and cell types (Figure 13). Consistent with the mRNA expression data, the treated model group had elevated TLR-4, MyD88, and NF-κB P65 protein expression in the pharyngeal mucosal tissue compared to the non-CP controls (p < 0.01), which could be reduced effectively by AS (p < 0.01) or by FFZJF treatment at all dosages tested (p < 0.01), moreover, the effects of FFZJF on decreasing TLR-4,MyD88,and NF-κB P65 expression levels are dose-dependent. The higher the FFZJF concentration level is, the more obvious the treatment effects are. Compared to the AS group, the protein expressions of TLR-4, MyD88, and NF-κB P65 in FFZJF-L and FFZJF-M groups were significantly different (P < 0.05), and the protein expressions of TLR-4 in FFZJF-H group were significantly different (P< 0.05), but there was no statistical significance observed in the expression levels of MyD88 and NF-κB P65 (P > 0.05) (Table 8, Figure 14).

Table 8 The Effect of FFZJF Aerosol on the TLR-4, MyD88, and NF-κB P65 Protein Expression Levels in the Rat’s Pharyngeal Mucosal Tissues (Mean ± SEM, n = 8)

Figure 11 The effect of FFZJF aerosol on the TLR-4 protein expression in rat’s pharyngeal mucosal tissues by immunohistochemistry (400×). TLR-4 is stained brown. (A) Control group; (B) Model group; (C) FFZJF-L group; (D) FFZJF-M group; (E) FFZJF-H group; (F) AS group.

Figure 12 The effect of FFZJF aerosol on the MyD88 protein expression in rat’s pharyngeal mucosal tissues by immunohistochemistry (400×). MyD88 is stained brown. (A) Control group; (B) Model group; (C) FFZJF-L group; (D) FFZJF-M group; (E) FFZJF-H group; (F) AS group.

Figure 13 The effect of FFZJF aerosol on the NF-κB P65 protein expression in rat’s pharyngeal mucosal tissues by immunohistochemistry (400×). NF-κB P65 is stained brown. (A) Control group; (B) Model group; (C) FFZJF-L group; (D) FFZJF-M group; (E) FFZJF-H group; (F) AS group.

Figure 14 The effect of FFZJF aerosol on the TLR-4, MyD88 and NF-κB P65 protein expression in rat’s pharyngeal mucosal tissues by immunohistochemistry. (A) TLR-4; (B) MyD88; (C) NF-κB P65.Note: As compared to treated non-CP control group: *p < 0.05, **p < 0.01; as compared to treated CP model group: #p < 0.05, ##p < 0.01; as compared to treated Ampicillin sodium group: ΔP<0.05.

Discussion

Chronic pharyngitis is a diffuse inflammation of the mucosa, submucosa, and lymphoid tissue of the pharynx. It has high prevalence, long duration of disease, easy recurrence, and a variety of symptoms. The main symptoms are sore throat, dry pharynx, itchy pharynx, foreign body sensation in pharynx, hyperemia and edema of pharyngeal mucosa, and hyperplasia of lymphoid follicles. The long-term use of antibiotics to treat bacterial chronic pharyngitis is likely to produce drug resistance and other side effects.^7–11 In recent years, traditional Chinese medicine has achieved good results in the treatment of CP. FFZJF is a hospital preparation of Guizhou University of Traditional Chinese Medicine (Guizhou Medicines Z20140036). Prof. Zhang Yanping has optimized the prescription and dosage form of FFZJF according to the experience of Dr. Li Shengyue, a famous traditional Chinese medicine scientist. It has been used in clinic for more than ten years and has achieved satisfactory results. The prescription is composed of five components: Zhongjiefeng, Maidong, Honghua, Yuxingcao, and Jiegeng. It has the functions of clearing heat and detoxifying, reducing swelling and relieving pain, activating blood circulation, and promoting pharynx. It also has the characteristics of low price, rich drug source, convenient use form (atomizer), and small side effects, especially reducing the use of antibiotics. We also confirm that FFZJF single drug is nearly non-toxic. Among them, Honghua and Yuxingcao are well-known plants that are widely used as herbs as well as food.^33–37 FFZJF has shown to be effective in terms of treating CP.¹¹ More importantly, the application of FFZJF avoids the overuse of antibiotics in treating this chronic condition. However, the mechanism of FFZJF has rarely been investigated.^7–11 The network pharmacology is consistent with the holistic view of traditional Chinese medicine, such as the syndrome differentiation and treatment.^15,16 Therefore, this study based on network pharmacology unveils the mechanism of FFZJF treatment of CP.

In this study, we used a network pharmacology approach combined with experimental validation to investigate the mechanisms of FFZJF for the treatment of CP. Our network pharmacology study identified 51 active compounds found in FFZJF. FFZJF-active compound-target CP network showed that the main active compounds included stigmasterol, 6-hydroxykaempferol, quercetin, kaempferol, and quercetagetin. Quercetin has good curative effects on pharyngitis, and kaempferol and quercetagetin have anti-inflammatory effects.^38,39 Meanwhile, the network suggests that the components Honghua, Maidong, Jiegeng, Zhongjiefeng play a key role in the treatment of CP, which was confirmed by previous research.¹¹ Zhongjiefeng, bitter, is the commonly used medicine for the treatment of throat diseases; Maidong, slightly bitter and slightly cold, can moisturize lung and clear throat; Honghua can activate blood circulation and disperse blood stasis to relieve pain; Jiegeng can promote blood circulation and promote pharynx, detumescence, and pain relief. Modern pharmacological studies have shown that Zhongjiefeng and Maidong can reduce TNF-α and IL6 levels and inhibit the activation of transcription factor NF-κB.⁴⁰ The extraction of Yuxincao can downregulate the production of NO, TNF-α and interleukin in cells and inhibit the NF-κBp65 subunit of nuclear translocation.⁴¹ The active component of hydroxysafflor yellow A blocks IκBα phosphorylation and degradation and prevents nuclear translocation.⁴² Platycodin-D can significantly inhibit IL1β induced NF-κB activation.⁴³ Our previous study suggested that the treatment of gastroesophageal reflux pharyngitis symptoms and signs was improved by compound FFZJF aerosol and was better than that of some other widely-used medicines.⁴⁴ Compound FFZJF aerosol can reduce the number of white blood cells, neutrophils and lymphocytes in plasma, and the level of serum IL-1. The lowest inhibitory concentration for streptococcus haemolyticus B in vitro was 105 mg/mL, suggesting that compound FFZJF aerosol may be used to treat pharyngitis by anti-inflammation and anti-bacterial,¹⁴ but the mechanism remains to be further studied. The PPI network revealed the core genes IL6, PTGS2, TLR-4, and TNF potentially targeted by FFZJF. Pathway analysis showed that these genes are involved in the regulation of classic pathways, which included regulation of cytokine secretion, inflammatory response, MyD88-dependent toll-like receptor signaling pathway, regulation of estradiol secretion, positive regulation of smooth muscle cell proliferation, negative regulation of growth of symbionts in the host, toll-like receptor signaling pathway, TNF signaling pathway, NF-κB signaling pathway, interactions between cytokine-cytokine receptors, NOD-like receptor signaling pathway. The results suggest that FFZJF may regulate the expression of toll-like receptor signaling pathway, TNF signaling pathway, NF-κB signaling pathway, and other signaling pathways along with IL6, PTGS2, TLR-4, and TNF genes to achieve the purpose of treating CP.

Studies have shown that a large number of cytokines are secreted in patients with pharyngitis, including TNF-α, IL-1β, IL6, etc. TNF-α is mainly produced by activated macrophages, NK cells, and T lymphocytes. IL-1β and IL6 are cytokines secreted mainly by T and B lymphocytes and monocytes macrophages, which can reflect the degree of injury of inflammatory response. Cytokines in the body can interact with each other. For example, the release of IL-1β and TNF-α can encourage the occurrence of each other and cause severe reactions such as temperature rise.46 We confirm that FFZJF can down-regulate the expression of TNF-α, IL1,β and IL6 in serum of rats with chronic pharyngitis, and achieve the effect of treating chronic pharyngitis. NLRP3 inflammasome play a pathogenic or disease-resistant role in the development of many diseases, with up-regulated expression in patients with pharyngitis.47,48 NLRP3/ASC/Caspase-1 axis induces NLRP3 inflammasome by regulating the maturation and secretion of inflammatory factors such as IL-1βand IL-18, and activation of NLRP3 inflammasome was important to the body’s resistance to invasion of exogenous pathogenic microorganisms. However, the excessive activation of NLRP3 inflammasome resulted in excessive innate immune defense against viral or bacterium with inflammatory cell apoptosis, tissue damage, and aggravated development of inflammatory diseases.49 In this study, network pharmacological analysis shows that FFZJF can achieve the effect of CP treatment by regulating NOD-like receptor signaling pathway, but more details about the underlying mechanism need to be further studied.

TLR-4 is a member of the toll-like receptor family. Upon pathogen recognition, TLR-4 is activated via a MyD88 (Myeloid Differentiation Primary Response Gene 88)-dependent pathway. Activated TLR-4 in turns activates NF-κB signaling pathway¹⁷ that eventually leads to the production and release of inflammatory cytokines (eg, TNF-α, IL1, and IL6).^24,45,46 MyD88 is an adaptor protein that plays a central role in TLR-4 signaling pathways.⁴⁷ TLR-4 recognizes pathogen-associated molecular patterns (PAMPs) and dimerizes with MyD88. MyD88 signaling involves the sequential activation of IL1 receptor-associated kinases (IRAKs), adaptor molecule TNF receptor-associated factor 6 (TRAF6), and NF-κB.⁴⁸ NF-κB activation induces the mRNA expression of inflammatory cytokines, such as TNF-α, IL1β, and IL6,^49,50 which in turn will results in more NF-κB activation. Such positive feedback mechanisms elicit an inflammatory reaction cascade. With respect to TLR-4/MyD88/NF-κB as a classical inflammatory response signaling pathway, NF-κB activation is a central link. Its activation promotes the release of inflammatory factors, which in turn promote the activation of NF-κB and form a positive feedback. Some studies have shown that inhibiting the expression of TLR-4 can reduce the expression of NF-κB, IL6 and improve the CP pathological changes.^51–53 The etiology of CP is well-studied, while its mechanism is less studied.⁵⁴ This study uses network pharmacology to analyze the key components, targets and signaling pathway, which lays a foundation for the study of CP pathogen and the mechanism of FFZJF treatment. In this study, the serum levels of TNF-α, IL1β, and IL6 in the CP model group were significantly higher than those in the non-CP control group. FFZJF treatment significantly reduced the serum levels of these cytokines. Histopathological examination confirmed that the inflammation was alleviated as the dose of FFZJF increased, especially in the FFZJF-H group. These results indicated that FFZJF is effective in treating CP. The significant increases of both mRNA and protein levels of TLR-4, MyD88, and NF-κB P65 in the pharyngeal mucosal tissues of the model group demonstrated that cytokines, TLR-4, MyD88, and NF-κB P65 are involved in the pathogenesis of CP. We postulate that certain substances released by GAS and pharyngeal mucosal cells are recognized by TLR-4, which leads to TLR-4 MyD88, and the eventual NF-κB activation. As a result, the production and secretion of proinflammatory cytokines (eg, TNF-α, IL1β, and IL6, etc.) increase, which in turn promotes chronic inflammation, tissue repair, or tissue damages in the pharynx. Therefore, activation of the TLR-4/MyD88/NF-κB signaling pathway likely plays a critical role in the tissue damage observed in bacterial CP. Compared with the model group, the FFZJF-M, FFZJF-H, and AS groups had significantly decreased mRNA expression of TLR-4, MyD88, and NF-κB P65 in the pharyngeal mucosal tissues of CP animals. In all FFZJF treatment groups, the protein expression levels of TLR-4, MyD88, and NF-κB P65 decreased significantly. These results demonstrated that FFZJF treatment could effectively downregulate the overexpressed TLR-4, MyD88, and NF-κB P65 at both mRNA and protein levels.

Conclusion

In summary, this network pharmacology study revealed potential mechanisms of multi-component, multi-target, and multi-pathway effects of FFZJF in the treatment of CP. Animal experiments further validated that FFZJF treatment could not only reduce the tissue expression of TLR-4, MyD88, and NF-κB P65 at both mRNA and protein levels, but it also reduced the serum levels of TNF-α, IL1β, and IL6 in treated animals. These results suggest that the treatment effect of FFZJF in CP could be mediated by the reduction of cytokine levels via downregulation of TLR-4, MyD88, and NF-κB expression. Therefore, the intervening TLR-4/MyD88/NF-κB signaling pathway might be one of the mechanisms of FFZJF in treating CP. Our study provides a novel direction for deciphering the molecular mechanisms of FFZJF as an efficacious treatment strategy for CP. Since the pathogenesis of CP is less studied, according to the previous study, the FFZJF can be treated by anti-inflammatory characteristics CP and combined with the literature to select TLR-4/MyD88/NF-κB signaling pathway as the mechanism of the phenomenon, the scope is small. Therefore, other inflammatory factors can be selected for further study.Retrieve FFZJF components in the database, some component data are still insufficient, follow up can be retrieved to ensure the accuracy of the data.