Abstract

Background

   Yinqin oral liquid (YOL) has curative effect for upper respiratory
   tract infections, especially for chronic pharyngitis (CP). Since the
   traditional Chinese herbal formulae are complicated, the
   pharmacological mechanism of YOL remains unclear. The aim of this work
   was to explore the active ingredients and mechanisms of YOL against CP.

Methods

   First, the profile of putative target of YOL was predicted based on
   structural and functional similarities of all available YOL components,
   which were obtained from the Drug Bank database, to the known drugs
   using TCMSP. The chemical constituents and targets of honeysuckle,
   scutellaria, bupleurum and cicada were searched by TCMSP, CTD,
   GeneCards and other databases were used to query the CP-related genes,
   which were searched by UniProt database. Thereafter, the interactions
   network between compounds and overlapping genes was constructed,
   visualized, and analyzed by Cytoscape software. Finally, pathway
   enrichment analysis of overlapping genes was carried out on Database
   for Annotation, Visualization, and Integrated Discovery (DAVID)
   platform.

Results

   The pathway enrichment analysis showed 55 compounds and 113
   corresponding targets in the compound-target network, and the key
   targets involved PTGS1, ESR2, GSK3β, NCOA2, ESR1. The PPI core network
   contained 30 proteins, including VEGFA, IL6, ESR1, RELA and HIF1A. A
   total of 148 GO items were obtained (p<0.05), 102 entries on biological
   process (BP), 34 entries on biological process (BP) and 12 entries on
   cell composition (CC) were included. A total of 46 signaling pathways
   were obtained by KEGG pathway enrichment screening (p<0.05), involving
   cancer, PI3K-AKT, hepatitis, proteoglycans, p53, HIF-1 signaling
   pathways.

Conclusion

   These results collectively indicate YOL (including the main ingredients
   luteolin and baicalein) as a highly effective therapeutic agent for
   anti-inflammation, through the NF-kB pathway.

   Keywords: Yinqin oral liquid, chronic pharyngitis, network
   pharmacology, NF-kB, multi-target analysis

Introduction

   Chronic pharyngitis (CP) is a very common disease associated with
   chronic inflammation involving pharyngeal mucosa, submucosal and
   lymphatic tissues. Clinically, CP is mainly manifested as pharyngeal
   discomfort (foreign body sensation, burning sensation, irritation,
   etc.), with occasional pharyngeal itch, cough, etc., which belongs to
   the category of “slow throat arthralgia” in the traditional Chinese
   medicine (TCM).[36]1 CP has a high incidence and accounts for 10–12% of
   all pharyngeal diseases, with a long disease course, recurrence and is
   difficult to cure. At present, western medicine uses antibiotics
   supplemented by hormone preparations, such as dexamethasone and
   antiviral drugs, for the treatment of pharyngitis. These treatment
   methods have drawbacks such as narrow therapeutic spectrum, recurrence,
   poor tolerance and major toxic and side effects. Hence, developing more
   effective therapeutic strategies against CP, and reducing the
   occurrence of side effects from the current therapeutic options is of
   great clinical importance.

   TCM is a major component of complementary and alternative medicine.
   Owing to its excellent clinical efficacy, TCM is a research hotspot
   worldwide. Dialectical theory of TCM is used to treat acute and chronic
   pharyngitis and has unique advantages such as minor toxic side effects,
   obvious curative effect and treatment of both symptoms and root
   causes.[37]2–4 The pharmacological effects of TCM herbal formulae play
   an important role in their appropriate application. However, the
   complex nature of herbal formulae has impeded this understanding.
   Numerous chemical ingredients involving multiple potential targets are
   present in an herbal formula. It is not adequate to explain the effects
   produced by a whole herbal formula if we individually consider every
   single ingredient in it. Yinqin oral liquid (YOL) is composed of
   extracts of honeysuckle, scutellaria, bupleurum and cicada. YOL has the
   effects of relieving wind, muscle and clearing heat, which is mainly
   used for cold, fever, aversion to cold, pharyngeal pain and other upper
   respiratory tract infections caused by wind fever and wind-cold.
   Long-term clinical observation has proven its effect in relieving
   pharyngeal pain and treating hand-foot-mouth disease. YOL contains
   several chemical compounds, which regulate diverse targets, and thus
   precisely determine the pharmacological mechanisms involved in its
   therapeutic actions. In addition, there are several challenges in the
   relationships between the herbs and diseases.[38]5,[39]6

   Network pharmacology is based on the theory of system biology. As a new
   subject, it selects specific signal nodes to design multi-target drug
   molecules through the network analysis of system biology. It is
   possible to study both the active components and the potential gene
   targets from TCM because of the establishment of network pharmacology
   and bioinformatics. This study predicted and analyzed the active
   components and target of YOL using network pharmacology, to explore the
   rationality of its formula and the scientific nature of treating CP,
   and further explore the pharmacological mechanisms of YOL on CP.

Materials and Methods

Chemicals and Reagents

   Yinqin Oral Liquid (YOL) was provided by su zhou si yuan natural
   products research and development Co. Ltd. YOL is composed of
   honeysuckle, scutellaria, bupleurum and cicada, decompressed and
   concentrated to 2 g/mL through boiling and alcohol sinking, and stored
   at 4°C for later use. Primary antibodies: COX-2 and iNOS antibodies
   were purchased from Abcam (Cambridge, UK); glyceraldehyde-3-phosphate
   dehydrogenase (GAPDH) antibody was obtained from Millipore (Billerica,
   MA, USA); PI3K, p-AKT, AKT, p-Stats, Stat3 and p-p65 (S536) antibodies
   were purchased from Cell Signaling Technology (Danvers, MA, USA); and
   p65 antibodies was obtained from Santa Cruz (CA, USA). The horseradish
   peroxidase (HRP)-conjugated sheep anti-mouse or anti-rabbit secondary
   antibodies were purchased from Thermo Fisher (Waltham, MA, USA). The
   proteins were visualized using an ECL detection kit (Thermo Fisher).

Cell Culture

   RAW264.7 cells, a mouse macrophage cell line (Cell Bank of Chinese
   Academy of Sciences, Shanghai, China) were cultured using DMEM
   supplemented with FBS (10%) and penicillin-streptomycin (1%). The cells
   were cultured at 37°C in a humidified environment of 5% carbon dioxide.
   The medium was changed every other day and the cells were passaged at a
   dilution of 1:3.

Collection of Drug Molecular Information and Screening of Active Ingredients

   In the TCMSP database ([40]https://tcmsp-e.com/),[41]7 the “Herb name”
   was selected to retrieve the molecular ADME parameter information of
   honeysuckle, scutellaria, bupleurum and cicada slough, and then the
   screen conformed to the oral bioavailability (OB) ADME parameter
   information of honey (Drug-likeness, DL) of ≥0.18 or more active
   ingredients ([42]Table 1). YOL ingredients were supplemented through
   literature review. Potential targets related to active ingredients were
   searched through the TCMSP database and Cytoscape 3.6.1 software.

Table 1.

   55 Active Compounds Predicted by OB and DL Among 4 Herbs in YOL
   Drug Mol ID Molecule Name MW OB% DL
   Honeysuckle MOL001494 Mandenol 308.56 42 0.19
   MOL001495 Ethyl linolenate 306.54 46.1 0.2
   MOL002914 Eriodyctiol (flavanone) 288.27 41.35 0.24
   MOL003006
   (-)-(3R,8S,9R,9aS,10aS)-9-ethenyl-8-(beta-D-glucopyranosyloxy)-2,3,9,9a
   ,10,10a-hexahydro-5-oxo-5H,8H-pyrano[4,3-d]oxazolo[3,2-a]pyridine-3-car
   boxylic acid_qt 281.29 87.47 0.23
   MOL003014 Secologanic dibutylacetal_qt 384.57 53.65 0.29
   MOL002773 Beta-carotene 536.96 37.18 0.58
   MOL003036 ZINC03978781 412.77 43.83 0.76
   MOL003044 Chryseriol 300.28 35.85 0.27
   MOL003095 5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone 358.37
   51.96 0.41
   MOL003111 Centauroside_qt 434.48 55.79 0.5
   MOL003117 Ioniceracetalides B_qt 314.37 61.19 0.19
   MOL003128 Dinethylsecologanoside 434.44 48.46 0.48
   MOL000358 Beta-sitosterol 414.79 36.91 0.75
   MOL000422 Kaempferol 286.25 41.88 0.24
   MOL000449 Stigmasterol 412.77 43.83 0.76
   MOL000006 Luteolin 286.25 36.16 0.25
   MOL000098 Quercetin 302.25 46.43 0.28
   Scutellaria MOL000073 Ent-Epicatechin 290.29 48.96 0.24
   MOL000173 Wogonin 284.28 30.68 0.23
   MOL000228 (2R)-7-hydroxy-5-methoxy-2-phenylchroman-4-one 270.3 55.23
   0.2
   MOL000359 Sitosterol 414.79 36.91 0.75
   MOL000525 Norwogonin 270.25 39.4 0.21
   MOL000552 5,2ʹ-Dihydroxy-6,7,8-trimethoxyflavone 344.34 31.71 0.35
   MOL001458 Coptisine 320.34 30.67 0.86
   MOL001490 Bis[(2S)-2-ethylhexyl] benzene-1,2-dicarboxylate 390.62 43.59
   0.35
   MOL001689 Acacetin 284.28 34.97 0.24
   MOL002714 Baicalein 270.25 33.52 0.21
   MOL002879 Diop 390.62 43.59 0.39
   MOL002897 Epiberberine 336.39 43.09 0.78
   MOL002909 5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone 376.34 33.82 0.45
   MOL002910 Carthamidin 288.27 41.15 0.24
   MOL002913 Dihydrobaicalin_qt 272.27 40.04 0.21
   MOL002914 Eriodyctiol (flavanone) 288.27 41.35 0.24
   MOL002915 Salvigenin 328.34 49.07 0.33
   MOL002917 5,2ʹ,6ʹ-Trihydroxy-7,8-dimethoxyflavone 330.31 45.05 0.33
   MOL002925 5,7,2ʹ,6ʹ-Tetrahydroxyflavone 286.25 37.01 0.24
   MOL002927 Skullcapflavone II 374.37 69.51 0.44
   MOL002928 Oroxylin a 284.28 41.37 0.23
   MOL002932 Panicolin 314.31 76.26 0.29
   MOL002933 5,7,4ʹ-Trihydroxy-8-methoxyflavone 300.28 36.56 0.27
   MOL002934 Neobaicalein 374.37 104.3 0.44
   MOL002937 Dihydrooroxylin A 286.3 66.06 0.23
   MOL008206 Moslosooflavone 298.31 44.09 0.25
   MOL010415 11,13-Eicosadienoic acid, methyl ester 322.59 39.28 0.23
   MOL012266 Rivularin 344.34 37.94 0.37
   MOL000490 Petunidin 317.29 30.05 0.31
   MOL004598 3,5,6,7-tetramethoxy-2-(3,4,5-trimethoxyphenyl)chromone
   432.46 31.97 0.59
   Bupleurum MOL002776 Baicalin 446.39 40.12 0.75
   MOL001645 Kaempferol 308.56 42.1 0.2
   MOL004718 Linoleyl acetate 412.77 42.98 0.76
   MOL004653 α-spinasterol 426.5 46.06 0.66
   MOL004624 Stigmasterol 348.48 47.72 0.53
   MOL004609 Areapillin 360.34 48.96 0.41
   MOL000354 Isorhamnetin 316.28 49.6 0.31
   MOL013187 Cubebin 356.4 57.13 0.64
   [43]Open in a new tab

Known Therapeutic Targets of Chronic Pharyngitis (CP)

   In the drug treatment of CP, the known therapeutic targets were mainly
   obtained from two sources: GeneCards database
   ([44]https://www.genecards.org/) and CTD database
   ([45]http://ctd.mdibl.org/). The data analysis was conducted in 5878
   therapeutic targets for the treatment of CP, after the redundant
   entries were removed. [46]Figure S1 shows the specific information
   about these known therapeutic targets.

Network Construction and Analysis

   To understand the relationship between the herbs and chemical compounds
   that consist of YOL and its putative targets, and the therapeutic
   targets known for CP, Network Visualization was conducted using
   Cytoscape 3.6.1 software,[47]8 and the degree between compounds and
   targets was analyzed.

Protein-Protein Interaction (PPI) Data

   PPI core network (PPICN) is used to study the relationship between
   chemical compounds and disease-associated protein molecules based on
   biochemistry, signal transduction and genetic networks.[48]9 The
   differing ID types of the proteins were converted to UniProt IDs. In
   order to further understand how YOL and CP interact at the protein
   level, the selected targets in this study were uploaded on the online
   Venn diagram ([49]http://bioinfogp.cnb.csic.es/tools/venny/index.html).
   Moreover, the genes at the intersection of the active compound and CP
   were selected and uploaded on STRING 10.5 ([50]https://string-db.org)
   to obtain the PPI relationship.

Pathway Enrichment Analysis

   The data was imported in the format Gene Symbol into David 6.8 database
   ([51]https://david.ncifcrf.gov/). Molecular function (CC), biological
   process (BP), and cellular function (MF) were selected, respectively. A
   pathway enrichment analysis was prospectively performed using
   biological process enrichment GO analysis and KEGG pathway analysis
   ([52]http://www.genome.jp/kegg/) to clarify the pathways involving
   putative CP targets, and visualized by online mapping website Omicshare
   Tools ([53]http://www.omicshare.com/tools/index.php/).

Statistical Analysis

   Each experiment was repeated at least in triplicate and data were shown
   as mean ± SD. Statistical difference in GO and KEGG analysis was
   performed using hypergeometric and Fisher's exact tests. P<0.05 was
   considered statistically significant.

Results

Screening and Analysis of Active Compounds

   First, all the compounds related to four TCMs were retrieved by TCMSP.
   Second, according to the screening conditions of OB ≥30% and DL
   ≥0.18,[54]10 23 active constituents of honeysuckle (17 predicted
   relevant targets), 36 of baicalensis (30 predicted relevant targets),
   17 of bupleurum (13 predicted relevant targets), and no suitable
   compounds of cicada slough were obtained. Among them, baicalensis and
   honeysuckle contained two identical ingredients, while bupleurum and
   honeysuckle contained three identical components. Finally, a total of
   55 active components were obtained for the data analysis after removing
   redundant entries ([55]Table 1).

Network Analysis of “Active Component-Target” Interaction in YOL

   The compound-target network contained 175 nodes including 55 compound
   nodes and 113 target nodes, of which two had no corresponding target
   and 577 edges. As shown in [56]Figure 1, blue represents compounds in
   bupleurum, red represents compounds in scutellaria, purple represents
   compounds in honeysuckle, green represents drug targets, each edge
   indicates the interaction between compounds and their targets, and two
   of the 55 compounds were excluded from the network construction. The
   degree value of a node represents the amount of connected routes in the
   network, and the larger the shape, the higher the degree value. The
   network in line with its topological properties showed that the nodes
   with higher degree of screening were analyzed. These nodes that connect
   compounds or targets act as hubs in the whole network and may predict
   key compounds or targets. The top five compounds ranked by degree were
   MOL000098-quercetin, MOL000422-Kaempferol, MOL000449-stigmasterol,
   MOL000358-quebrachol and MOL000006-luteolin, which could interact with
   154, 68, 39, 32 and 28 target proteins, respectively. PTGS1, NCOA2, AR,
   PRSS1 and PPARG were the top five targets with the highest degree,
   which could interact with 42, 38, 29, 28 and 17 compounds,
   respectively.

Figure 1.

   [57]Figure 1
   [58]Open in a new tab

   Compounds-target network diagram. Blue represents compounds in
   bupleurum, Red represents compounds in scutellaria, Purple represents
   compounds in honeysuckle, Green represents drug targets. The size
   represents the degree value. The larger the shape, the larger the
   degree value.

The Construction of Key Protein Network of YOL and CP

   Through the internationally recognized CTD and GeneCards disease
   databases, 5150 and 1247 pharyngitis-related targets were obtained,
   respectively. Through the Venny online tool, 30 targets of honeysuckle,
   scutellaria, bupleuri and pharyngitis were obtained, including VEGFA,
   IL6, ESR1, RELA, HIF1A, etc. ([59]Figure 2A). Finally, STRING online
   tool was used to construct the PPI network interaction map of drug and
   disease. It contained 30 nodes representing proteins and 48 edges
   representing the interaction between proteins. The thicker the line,
   the higher is the correlation degree. Through the protein interaction
   network, we could further explore the therapeutic target and mechanism
   of YOL for CP ([60]Figure 2B).

Figure 2.

   [61]Figure 2
   [62]Open in a new tab

   (A) Venn diagram of compounds-target network diagram; (B) Core network
   diagram of protein interaction between YOL and CP.

Pharmacological Mechanisms of YOL Acting on CP

   A total of 30 common targets were obtained by DAVID online tool and 148
   GO items were obtained (p < 0.05) by performing the functional
   enrichment analysis. There were 102 entries on biological process (BP),
   34 entries on Molecular Function (MF), and 12 entries on cell
   composition (CC) ([63]Figure 3A). A total of 46 signaling pathways were
   obtained by KEGG pathway enrichment screening (p<0.05), involving
   cancer, PI3K-AKT, hepatitis, proteoglycans, p53, HIF-1 signaling
   pathways, etc. ([64]Figure 3B and [65]Table S1).

Figure 3.

   [66]Figure 3
   [67]Open in a new tab

   (A) GO analysis function annotation diagram. Biological process (BP),
   molecular function (MF), and cell composition (CC). (B) Enriched KEGG
   pathways of YOL selected targets for CP.

Experimental Verification of Predicted Results

   In order to experimentally demonstrate the predictions about the
   molecular mechanism of YOL, we chose two ingredients for testing
   ([68]Figure 4), based on their composition score, the group to which
   they belong, and their herbal origin to examine how ingredients from
   different herbs affect the same proteins. The effects of these
   compounds on the expression levels of related proteins were analyzed by
   Western blot. Macrophages secrete several inflammatory cytokines, which
   play significant roles in inflammation. Signal transduction and
   transcriptional activator 3 (STAT3), nuclear factor kappa B (NF-kB),
   and phosphatidylinositol 3-kinase (PI3K)/Akt pathways are critical
   inflammatory pathways. LPS can significantly increase the production of
   many proinflammatory factors, including inducible NO synthase (iNOS)
   and cyclooxygenase-2 (COX-2) in macrophages. After pretreatment of
   macrophages with YOL, luteolin and baicalein, the protein expression
   levels of iNOS and COX-2 induced by LPS were significantly inhibited in
   a dose-dependent manner ([69]Figure 4A). The related protein changes
   were consistent with iNOS and COX-2 inflammation protein expression
   ([70]Figure 4B–[71]D).

Figure 4.

   [72]Figure 4
   [73]Open in a new tab

   The YOL inhibits PI3K/p-AKT/p-Stats pathway in RAW 264.7 cells.
   Representative Western blot images show the relative expressions of
   iNOS, COX-2 (A), p-p65, p65 (B), PI3K, p-AKT, AKT (C), p-Stats, Stat3
   (D) in the groups. The protein levels (normalized) of iNOS, COX2 (E),
   and p-p65, p65, PI3K, p-AKT, AKT, p-Stats, Stat3 (F) in cells. All the
   values are presented as mean ± SD. ^#P < 0.05 and ^##P < 0.01 vs
   control group. *P < 0.05 and **P < 0.01 vs LPS group.

Discussion

   Many diseases, including cancer and chronic inflammation, are known to
   be regulated by multiple signaling pathways. TCMs are considered as
   multi-component and multi-target therapeutic drugs, which is in
   accordance with the methodologies of network pharmacology. The holistic
   view of TCM considers the human body as a complex biological network
   system, which is connected with network pharmacology. By combining the
   network pharmacology with the holistic view of TCM, the
   disease-target-drug network is obtained. The network shows that
   different components of TCM may act on one target, and different
   targets may act on the same pathway. It is also possible that the same
   component of TCM acts on different targets, and the same target acts on
   different pathways. The multi-component, multi-target and multi-pathway
   of TCM can be clearly visualized through network pharmacology, which
   resolves many difficulties encountered in the study of TCM.[74]13–15 CP
   is the diffuse inflammation of pharyngeal mucosa, submucosa and lymph
   tissues. Clinically, CP is mainly manifested as pharyngeal discomfort
   (foreign body sensation, burning sensation, stimulation sensation,
   etc.), pharyngeal itching, cough, etc. Pharyngitis belongs to the
   category of “Fenghou Bi” in TCM. Pharyngitis pertains to the category
   of “wind throat arthralgia” in Chinese medicine, and is often caused by
   evil heat entering the body, and heat in the lungs and stomach. YOL is
   an oral TCM preparation, which is further optimized on the basis of
   Jinchan oral liquid developed by Children’s Hospital of Suzhou
   University.[75]5,[76]6 However, there is a lack of research on its
   active ingredients and its mechanism of action.

   In this study, 30 potential targets, 102 biological processes, 12
   molecular functions, 34 cell components, and 46 KEGG pathways were
   obtained. The active ingredients in YOL can treat CP through multiple
   targets and pathways. To elucidate the biological pathways that may be
   affected by YOL, a significantly overexpressed KEGG pathway was
   identified. The results showed VEGFA, IL6, ESR1 and RELA as the key
   targets and the involvement of PI3K-Akt, p53 and HIF-1 signaling
   pathways. TCM aims to restore a patient’s health through the use of a
   TCM prescription, which is usually composed of two or more TCMs in
   optimal proportions.

   YOL, for example, consists of four TCMs. As expected, most of the
   selected active ingredients are directly or indirectly associated with
   inflammation. In a previous study, neochlorogenic acid was demonstrated
   to inhibit LPS-activated inflammatory responses through up-regulation
   of Nrf2/HO-1 and AMPK pathways.[77]16 The decoction extract from six
   natural herbs, which contain honeysuckle, exhibits anti-inflammatory
   and immunomodulatory effects by targeting NF-κB/IL-6/STAT3
   signaling.[78]17 Luteolin is abundant in honeysuckle, which alleviates
   CP by inhibiting the NF-κB pathway and anti-inflammatory polarization
   of M1 macrophages.[79]11 Protective effects of baicalein on liver
   injury in mice induced by multi-microbial septicemia were found to be
   based on inhibiting inflammation.[80]12 Network pharmacological studies
   have shown that the main active components of honeysuckle may play a
   role through inflammation-related proteins such as HSP90AA1, HSP90AB1,
   ESR1, PTGS2, TERT and PPARG, and especially heat shock protein HSP90A
   (HSP90AA1).[81]18 Flavonoids such as baicalin and wogonin alone and in
   combination have anti-inflammatory activity in Scutellaria.[82]19
   Network pharmacological studies have also shown that alkaloids such as
   coptisine and epiphone in Scutellaria and components such as
   dihydrokaempferol, and rographolide flavonoids also show
   anti-inflammatory activity. MAPK14, TNFRSF1A, EGFR and SELE are the
   primary anti-inflammatory components of Scutellaria. The
   anti-inflammatory active components in Scutellaria can directly affect
   MAPK14 and EGFR to produce anti-inflammatory effects, and can also
   indirectly affect other targets to exert anti-inflammatory effects. As
   one of the four p38 MAPKs, MAPK14 plays a vital role in the cellular
   cascade induced by extracellular stimuli such as proinflammatory
   cytokines or physical stimuli.[83]20 Studies predicted that the
   pharmacodynamic basis of Bupleurum is mainly saponins, flavonoids,
   volatile oils, fatty acids and other components. Flavonoids mainly act
   on PI3K-Akt, NF-kB and other pathways, participate in regulating
   inflammatory factors, estrogen signal transduction and other
   physiological processes. Bupleurum-Scutellaria drug pair, first used in
   small bupleurum decoction in Treatise on Febrile Diseases, is an
   important part of Bupleurum prescription. Bupleurum is bitter, flat and
   clear, and can disperse the stagnation of bile fire. Scutellaria has a
   bitter and cold taste, and can clear internal heat. Both of them must
   be used together to regulate the liver and gallbladder, and clear the
   dampness and heat of internal accumulation, which are commonly used to
   treat fever, pharyngitis, and respiratory diseases.[84]21 Cicada slough
   can evacuate wind and heat, and clear the throat. Clinically, it is
   common in classical prescriptions such as Sheng jiang San and
   Pharyngitis San. However, the active components of Cicada slough did
   not satisfy the prediction requirements in this research.

   YOL is composed of honeysuckle, scutellaria, bupleurum and cicada
   slough. According to TCM theory, bupleurum is bitter and flat, enters
   the liver and gallbladder meridian, penetrates and clears Shaoyang, and
   can drain the stagnation of gas. Scutellaria is bitter and cold, and
   clears the heat of Shaoyang. The actions of Bupleurum facilitate the
   clear discharge of Scutellaria, the two are compatible, and achieve the
   goal of reconciliation and Shaoyang. Honeysuckle cools through the
   surface, has heat-clearing and detoxifying effects, and also has the
   effect of fragrant obscenity. Cicada slough is light floating, clears
   the coke gas, shows honeysuckle compatibility, understanding the table
   evil, and is combined with the “treatment of the coke such as feather.”
   The combination of the four drugs, Xuan and descent together, clear the
   evil, thereby clearing heat and detoxification, warming the evil
   without hiding, scattered outside and recovered. Therefore, the
   treatment of CP with YOL can control the pharyngitis, regulate the
   balance of Yin and Yang, strengthen the foundation and improve the
   immunity of the body.

Conclusion

   This study used the network pharmacology platform to probe the YOL
   treatment of CP by multi-target analysis. The results showed that YOL
   could treat CP by acting on related targets, which were consistent with
   the reported literature. In addition, we have verified that YOL and its
   two monomer compounds, Luteolin and Baicalein inhibited activation of
   the NF-kB, Stat3 and PI3K-Akt pathways. Although the potential
   advantages of the “network target, multi-component” strategy of network
   pharmacology are obvious, the content of TCM ingredients is usually
   ignored in the research of network pharmacology of Chinese medicine,
   and the influence of content and concentration on the efficacy cannot
   be ignored. The predicted targets, which have not been discussed, may
   provide clues for further research on the mechanism of YOL.

Funding Statement

   This work was supported by the Third Batch of Suzhou High-tech District
   Health Talents Project SGXWS2020 (to Dr. Ji) and Suzhou Science and
   Technology Plan Project, (SYSD2019171, SYS2020076).

Disclosure

   The authors report no conflicts of interest in this work.

References