Abstract Background and Aim Platycodon grandiflorum (PG) has been widely used for treating chronic bronchitis (CB). However, the material basis and underlying mechanism of action of PG against CB have not yet been elucidated. Methods To analyze the ingredients in PG, ultraperformance liquid chromatography-quadrupole-time-of-flight tandem mass (UPLC-Q-TOF-MS/MS) technology was performed. Subsequently, using data mining and network pharmacology methodology, combined with Discovery Studio 2016 (DS), Cytoscape v3.7.1, and other software, active ingredients, drug-disease targets, and key pathways of PG in the treatment of CB were evaluated. Finally, the reliability of the core targets was evaluated using molecular docking technology and in vitro studies. Results A total of 36 compounds were identified in PG. According to the basic properties of the compounds, 10 major active ingredients, including platycodin D, were obtained. Based on the data mining approach, the Traditional Chinese Medicine Systems Pharmacology Database, and the Analysis Platform (TCMSP), GeneCards, and other databases were used to obtain targets related to the active ingredients of PG and CB. Network analysis was performed on 144 overlapping gene symbols, and twenty core targets, including interleukin-6 (IL-6) and tumor necrosis factor (TNF), which indicated that the potential signaling pathway that was most relevant to the treatment of CB was the IL-17 signaling pathway. Conclusion In this study, ingredient analysis, network pharmacology analysis, and experiment verification were combined, and revealed that PG can be used to treat CB by reducing inflammation. Our findings provide novel insight into the mechanism of action of Chinese medicine. Furthermore, our data are of value for the research and development of novel drugs and the application thereof. Keywords: chemical ingredient, chronic bronchitis, experiment verification, mechanism of action, network pharmacology, Platycodon grandiflorum Introduction Chronic bronchitis (CB) is chronic, nonspecific inflammation of the trachea, bronchial mucosa, and surrounding tissues, which may be due to infection or noninfection (allergies, oxidative stress) ([41]Malesker et al., 2020). CB is one of the common clinical diseases and has a high incidence in the middle-aged and elderly population. According to statistics, the prevalence of CB in China is about 4.0%, among which 10% to 15% is accounted for by elderly patients, and the incidence is increasing ([42]Luo et al., 2019). The main clinical manifestations of patients are cough, phlegm, or wheezing ([43]Zhang, 2019). The pathological changes mainly involve damage of the epithelium of the central airways. Infiltration of inflammatory cells and hypertrophy of smooth muscle cells further leads to increased mucus secretion, decreased immune function of the epithelium, and ultimately leads to airway remodeling ([44]You, 2014). In Chinese medicine, CB can be divided into six types: phlegm dampness lung type, external cold cohesion type, phlegm heat closed lung type, lung and spleen qi deficiency type, lung and kidney weakness type, and spleen and kidney yang deficiency type. Only when symptomatic treatment is performed, the recurrence of CB can be relieved from the root cause ([45]Weng et al., 2014; [46]Zhu, 2017; [47]Bai and Li, 2019). At present, Western medicine and Chinese medicine have been shown to be successful in the treatment of CB ([48]Sun et al., 2015; [49]Zhu, 2017). Treatment involving Western medicine mainly uses antiinfective, antiallergic, relieves bronchial smooth muscle spasm and other antitussive, phlegm-reducing drugs to treat CB, including ambroxol hydrochloride ([50]Sun et al., 2015), budesonide ([51]Wesseling et al., 1991), and levofloxacin ([52]Blasi et al., 2013). Short-term use of Western medicine may temporarily relieve symptoms, but due to the long course of the disease, long-term use has several shortcomings, including toxic side effects, patient intolerance, and high costs. In addition, patients with CB are often frail and other systems are affected, therefore, they are often forced to discontinue treatment because of the toxic side effects of certain drugs ([53]You, 2014; [54]Jin, 2019). In view of the shortcomings of Western medicine, it is of utmost importance to develop drugs that can safely and effectively treat CB. For the treatment of CB, traditional Chinese medicine (TCM) is mainly used to clear the lungs and phlegm, spleen and kidney function, and has effectively relieved symptoms, including cough and phlegm. Long-term use of TCM can effectively enhance human immunity and reduce the frequency of attacks of CB, including Shegan Mahuang Decoction ([55]Bai and Li, 2019), Zhi Chou San ([56]Bai and Li, 2019), and Tianxing Kechuan Patches ([57]Fan et al., 2012). Compared with Western medicine, TCM has the characteristics of a multiingredient and multitarget action. In addition, it can perform overall regulation and multitarget intervention on CB ([58]Zhao and Li, 2018). Platycodon grandiflorum (PG) is the dried root of campanulaceae, and PG extracted from platycodon has been shown to have a good effect on ventilating lung and eliminating phlegm. According to the TCM theory, PG mainly acts on the lung and its related structures. It has a cough-relieving effect and has been used to treat CB with good curative effect ([59]Chen et al., 2010; [60]Chen et al., 2013; [61]He et al., 2013). PG contains triterpenoid saponins, flavonoids, phenolic acids, polyacetylene, and sterols ([62]Deng et al., 2020; [63]Ji et al., 2020). Among them, platycodin D is one of the main active ingredients ([64]Deng et al., 2020). Multiingredients are both independent and have connections, and may lead to cross-links between targets. Therefore, for the treatment of CB, PG should act as a multiingredient and multitarget in synergy to exert drug effects, which means that its mechanism of action is complex. Thus, a method that establishes the relationship between ingredients, targets, and diseases is warranted for exploring the underlying mechanism of action by which PG treats CB. Network pharmacology is a theory based on systems biology, and emphasizes the multichannel regulation of signaling pathways, which coincides with the characteristics of multiingredient-multitargets of TCM ([65]Zhang et al., 2019; [66]Li W. J. et al., 2020; [67]Ou et al., 2020). Network pharmacology integrates TCM, active ingredients of TCM, TCM targets, disease targets, constructs drug-ingredients-gene symbols-disease four-dimensional graphs, and comprehensively analyzes common targets of Chinese medicine ingredients on diseases, and thoroughly analyzes target genes, proteins, and signal pathways, to identify possible mechanism of Chinese medicine treatment to treat diseases ([68]Park et al., 2018; [69]Yang et al., 2019; [70]Ye et al., 2020). Therefore, the network pharmacology approach is a tool that is sufficient to identify the mechanism underlying the treatment of CB by PG. PG is mainly produced in the provinces of Northeast, North China, East China, Central China, and Guangdong. There are differences in PG ingredients from different areas. Shandong is one of the genuine producing areas of PG, and the PG produced there has long roots, few bifurcations, and a high content of active ingredients ([71]Zhu et al., 2013). However, the ingredients of PG from Shandong have not yet been systematically analyzed and identified. Therefore, in this study, ingredient analysis of PG produced in Shandong was conducted, and based on the relevant principles and methods of network pharmacology, drug-ingredients-gene symbols-disease (D-I-G-D) network was constructed to explore the potential molecular mechanism for treating CB. Subsequently, the reliability of the core targets was verified by molecular docking verification and in vitro studies ([72] Figure 1 ). Our findings will provide a theoretical basis for the clinical application of PG and the development of novel drugs. Figure 1. [73]Figure 1 [74]Open in a new tab A comprehensive strategy diagram the chemical ingredients analysis, targets prediction, network calculation and experimental validations for investigation the mechanism of action of Platycodon grandiflorum (PG) on chronic bronchitis (CB). Materials and Methods Chemicals and Herb Materials Methanol, acetonitrile, and formic acid for high performance liquid chromatography (HPLC) were purchased from ACS (Washington D.C., MD, USA). Methanol for herb extraction was purchased from Xilong Scientific Co., Ltd. (Guangdong, China). Ultrapure water was obtained from a Milli-QB system (Bedford, MA, USA). PG pieces were purchased from Jiangxi Jiangzhong Herbal Pieces Co., Ltd. (Jiangxi, China; batch number: 181024). The original PG medicinal material was purchased from Yiyuan, Shandong province, and was identified as the dried root of PG (Jacq.) A. DC. Campanulaceae by Professor Fu Xiaomei. PG decoction pieces were processed by Jiangxi Jiangzhong TCM Decoction Co., Ltd. according to the processing method of the Chinese Pharmacopeia 2015 edition. Next, dried PG pieces were crushed into powder (40 mesh) and stored in the laboratory of the Jiangxi University of TCM. As reference standards, 13 pure compounds were used (purity≥98%). Among these compounds, chlorogenic acid (2), caffeic acid (3), ferulic acid (7), lobetyolin (8), luteolin (15), kaempferol (16), apigenin (17), deapio-platycodin D (19), and platycodin D (21) were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Sichuan, China). Lobetyol (4), rutin (5), 3-O-β-D-glucopyranosyl platycodigenin (30), and linoleic acid (36) were purchased from Sichuan Vicky Biotechnology Co., Ltd. (Sichuan, China). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were purchased from Huamei Biotechnology (Wuhan, China). The minimum detectable dose of mouse TNF-α, IL6, and IL-1β is typically less than 3.9 pg/ml, 0.39 pg/ml, and 7.8 pg/ml, respectively. Intra-assay and inter-assay precision of these three ELISA kits is less than 8% and 10%. Ultra-Performance Liquid Chromatography-Quadrupole-Time-of-Flight Tandem Mass Analysis Preparation of Standard and Sample Solutions Ten milligrams of each reference compound (luteolin; chlorogenic acid; caffeic acid; ferulic acid; lobetyolin; kaempferol; apigenin; lobetyol; deapio-platycodin D; linoleic acid; 3-O-β-D-glucopyranosyl platycodigenin; rutin; platycodin D) was weighed, transferred to 10-ml volumetric flasks, methanol was added to reach the volumetric mark, shaken well, and used as a stock solution. Then, the appropriate amount of stock solution was added to a 5-ml volumetric flask, and methanol was added to reach the volumetric mark. The solutions were filtered through 0.22-μm microporous membranes to obtain the standard solutions. PG powder (2.0 g) was accurately weighed and placed in a round bottom flask with 50 ml 50% methanol, mixed well, soaked for 0.5 h at room temperature, and treated ultrasonically for 30 min using an ultrasonic cleaning instrument (Jiangsu, China). The extract solution was centrifuged at 14,000 rpm for 15 min at room temperature, then filtered through a 0.22-μm microporous membrane before qualitative analysis. Ultra-Performance Liquid Chromatography-Quadrupole-Time-of-Flight Tandem Mass Conditions Chemical analysis was conducted on a connected system of UPLC (Nexera X2 LC-30A, Shimadzu Corp., Japan)-hybrid triple quadruple time-of-flight mass spectrometer (Triple TOF™ 5600^+, AB Sciex, Forster City, CA, USA) with an electrospray ionization source (ESI). Acquity UPLC BEH C[18] column (2.1×100 mm×1.7µm) was used to perform chromatographic separation with a flow rate of 0.25 ml/min at 40°C. A linear gradient program with a mobile phase system including solvent A (100% acetonitrile, v/v) and solvent B (0.01% formic acid in water, v/v) was described in detail: solvent A (5%~23%) for 10 min, (23%~25%) for 6 min, (25%) for 4 min, (25%~29%) for 3 min, (29%~95%) for 7 min, (95%~5%) for 2.1 min, isocratic eluted at 5% for 2.9 min. The instrumental settings of Q-TOF-MS/MS were as follows: ion source gas 1 (GSI) and gas 2 (GS2) were both set to 50 psi, curtain gas (CUR) was set to 40 psi, ion spray voltage floating (ISVF) was set to 5500 V in the positive mode while 4500 V was set in the negative mode, ion source temperature (TEM) was 500°C, collision energy (CE) was 60 V, collision energy spread (CES) was 15 V, declustering potential (DP) was 100 V, and nitrogen was used as a nebulizer and auxiliary gas. Samples were analyzed in both positive and negative ionization modes with a scanning mas-to-charge (m/z) range from 100 to 1,250. Data were collected in information-dependent acquisition (IDA) mode and analyzed by PeakView^®1.2 software (AB Sciex, Foster City, CA, USA). Ingredients Identification Analysis The chemical ingredients of PG were collected from existing databases, including SciFinder ([75]https://scifinder.cas.org/), the Traditional Chinese Medicine Systems Pharmacology Database, and the Analysis Platform (TCMSP, [76]http://lsp.nwu.edu.cn/tcmsp.php) database. Then, a PG ingredients database was established, which contained basic information, such as ingredient name and molecular formula. A total of 161 known ingredients in PG were collected, and specific information is presented in [77]Schedule 1 . MS data was imported into PeakView^® 1.2 for ingredient analysis. Chemical identifications were based on reference standards, chromatographic elution behaviors, chemical ingredient, mass fragment patterns, and mass spectral library (Natural Products HR-MS/MS Spectral Library, Version 1.0, AB Sciex, Forster City, CA, USA). Collecting Related Targets for Active Ingredients in PG In this study, four databases were searched to identify the target of active ingredients in PG. Databases searched included Swiss Target Prediction ([78]http://www.swisstargetprediction.ch/) ([79]Gfeller et al., 2014), Pubchem ([80]https://pubchem.ncbi.nlm.nih.gov/) ([81]Liu et al., 2012), TCMSP ([82]Ru et al., 2014), and Pharmmapper ([83]http://www.lilab-ecust.cn/pharmmapper/) ([84]Liu et al., 2010). Targets were converted into gene symbols by Uniprot ([85]http://www.uniprot.org/), and gene symbols were combined. CB-Associated Targets Collection In this study, “Chronic bronchitis” was used as a keyword to search for relevant CB targets in DisGenet ([86]http://www.disgenet.org/) ([87]Li B. T. et al., 2019; [88]Su et al., 2019) and GeneCards databases ([89]https://www.genecards.org/) ([90]Zhang et al., 2018; [91]Yang et al., 2019). Protein-Protein Interaction Network Construction To obtain overlapping targets, VENNY 2.1 ([92]http://www.liuxiaoyuyuan.cn/) software was used to cross PG-related targets with CB-related targets. Overlapping targets of Chinese medicine-disease were added into STRING11.0 ([93]https://string-db.org/) ([94]Snel et al., 2000; [95]Yang et al., 2019), and the screening condition used was “Homo sapiens”, the minimum interaction score was 0.4, and the results were saved. The resulting file was imported into Cytoscape v3.7.1 software, and the plugin CentiScape was used to calculate the degree centrality (DC). The core target of the protein-protein interaction (PPI) network was filtered ([96]Li Z. et al., 2020). D-I-G-D Network Construction Related files were established of “drug-core ingredients,” “core ingredients-core targets,” and “disease-core targets,” and files were imported into Cytoscape v3.7.1 to build a “drug-ingredients-gene symbols-disease” network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis Metascape ([97]http://www.metascape.org/) is a gene annotation tool that integrates multiple authoritative data sources such as gene ontology (GO), Kyoto Encyclopedia of Genes and Genome (KEGG), UniProt, and DrugBank. It not only completes pathway enrichment and bioprocess annotation, but also performs gene-related protein network analysis and drug analysis, and is committed to providing comprehensive and detailed information on each gene ([98]Zhou et al., 2019). On the premise of retaining the advantages of DAVID, Metascape has perfectly made up for its vacancy, and data are frequently updated, which guarantees the timeliness and credibility of the data. Gene symbols of the core targets were imported into Metascape, and “Homo sapiens” was selected for enrichment analysis to further explain the role of the core targets in gene function and signaling pathways. Computational Validation of Ingredients-Target Interactions In this study, we aimed to ascertain the interaction between active ingredients and their protein targets, and explore their binding modes. Hence, we selected seven core ingredients and twenty core targets for verification of molecular docking. The PDB format of core ingredients was obtained from the Uniprot database, and the X-ray crystal structures were downloaded from the RCSB database ([99]https://www.rcsb.org/). The molecular docking function of Discovery Studio 2016 (DS) was used for ingredient-target molecular docking in the LibDock module. Experimental Verification In Vitro Cell Culture RAW264.7 cells were obtained from the Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Solarbio, Beijing, China), supplemented with 10% fetal bovine serum (FBS, Zhejiang, China). Cells were cultured at 37°C and 5% CO[2]. Cell Viability Assay RAW264.7 cells in the logarithmic phase were seeded at 1×10^4 cells/well in 96-well plates. After incubation for 24 h, RAW264.7 cells were exposed to luteolin (0, 15, 20, 40, 45, 60, 80, and 100 μM). After treatment for 24 h, 20 μl of Cell Counting Kit (CCK-8) assay solution (Solarbio, Beijing, China) was added to each well, and cells were incubated for 4 h at 37°C and 5% CO[2]. The absorbance at 450 nm was measured by a microplate reader (FLUOstar Omega, LABTECH, Offenburg, Germany). Cell survival was calculated as: absorbance/absorbance of control ×100%. TNF-α, IL-6, and IL-1β Expression RAW264.7 cells (5×10^3 cells/well in 96-well plates) were incubated with lipopolysaccharide (LPS; 1 μg/ml) for 24 h, then treated with luteolin (20, 40, or 60 μM) for 24 h. Supernatants were harvested and levels of IL-6, TNF-α, and IL-1β were determined by ELISA. Real-Time Quantitative Polymerase Chain Reaction Total RNA was extracted using Trizol reagent (Beijing, China), then treated with RNase-free DNase (Promega, Beijing, China), and reverse transcribed with oligo-DT (Beijing, China) using MMLV (TOYOBO, Shanghai, China) reverse transcriptase according to the instructions of the reverse transcription kit. Reverse transcription reaction conditions were as follows: 30°C for 10 min, 42°C for 60 min, 99°C for 5 min, and 4°C for 5 min. The reaction was carried out in a PCR machine (Applied Biosystems, Foster City, CA, USA). Primers were commissioned by Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China) and primers sequences were as follows: * IL-1β: (Forward primer) 5’-TGCCACCTTTTGACAGTGATG-3’ * (Reverse primer) 5’-AAGGTCCACGGGAAAGACAC-3’ * IL-6: (Forward primer) 5’-ACAAGTCCGGAGAGGAGACT-3’ * (Reverse primer) 5’-TGTGACTCCAGCTTATCTCTTGG-3’ * TNF: (Forward primer) 5’-ACCCTCACACTCACAAACCA-3’ * (Reverse primer) 5’-ACCCTGAGCCATAATCCCCT-3’ * GAPDH: (Forward primer) 5’-GGGGTCCCAGCTTAGGTTC A-3’ * (Reverse primer) 5’-TTCCCATTCTCGGCCTTGAC-3’ Real-time quantitative polymerase chain reaction (qRT-PCR) was performed by SYBR™ Green Master Mix (Vazyme, Beijing, China) in an QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, USA). The PCR cycling profile was as follows: one cycle at 95°C for 3 min and 72°C for 10 min, 35 cycles at 94°C, 60°C, and 72°C for 30 s. Fluorescence signals were detected using the QuantStudio 6 Flex system. Gene-expression data were normalized to that of the endogenous control GAPDH. The 2^-△△Ct method was used as the basis for relative gene expression. Statistical Analyses Data were analyzed by SPSS 25.0 (SPSS Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation (SD). All experiments were performed in triplicate. Data were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) testing. p<0.05 was considered statistically significant. Results Identification of the Chemical Constituents in PG by Ultraperformance Liquid Chromatography-Quadrupole-Time-of-Flight Tandem Mass Ultraperformance liquid chromatography-quadrupole-time-of-flight tandem mass (UPLC-Q-TOF-MS/MS) is a high-throughput analytical technology that has rapidly developed in the past decade, and is widely used in the fields of environmental science, medicine, drug research, and others ([100]Ren et al., 2020). Typical total ion chromatograms (TICs) of nonvolatile ingredients extracted by PG are presented in [101]Figure 2 . Although TICs are complex, most of the chromatographic peaks are well separated. A total of 36 chemical constituents were identified in the 50% aqueous methanol extract of PG based on the reference standards, chromatographic elution behaviors, chemical ingredient, mass fragment patterns as well as mass spectral library with the high resolution UPLC-Q-TOF MS/MS system, including triterpene saponins, flavonoids, phenolic acids, and polyacetylene. Among them, 13 compounds (2, 3, 4, 5, 7, 8, 15, 16, 17, 19, 21, 30, and 36) were identified by comparing with the reference standards. By comparing references ([102]Guo, 2007;