A network pharmacology-based exploration of the active compounds and potential drug targets of Si-Jun-Zi decoction in the treatment of cutaneous squamous cell carcinoma

Introduction

Cutaneous squamous cell carcinoma (cSCC) is a kind of skin tumor with an incidence second only to that of basal cell carcinoma (1). It arises from keratinocytes in the epidermis or appendages. Currently, management of cSCC involves surgical treatment, radiation therapy, chemotherapy, and targeted therapy. Surgical treatment is the main method for early cSCC. However, the survival problems faced by cSCC patients include postoperative recurrence, lymph node metastasis, and distant metastasis, all of which require clinical solutions (2,3). Although targeted therapy is the main treatment for these cSCC patients, the prognosis remains poor (4).

Network pharmacology is a new research method combining pharmacology and pharmacodynamics based on a variety of network databases, which can help us understand the interactions among traditional Chinese medicines (TCM), compounds, disease, and targets in a more systematic and comprehensive way.

For a long period of time, TCM has successfully treated a variety of complex diseases through the use of multi-compound decoctions aimed at multiple targets. Through online pharmacology, researchers can not only explore the compounds of TCM formulae, but also understand the interaction between active ingredients and their related targets, which provides a new, highly applicable method for clarifying the mechanism of TCM treatment of diseases.

Si Jun Zi (SJZ) decoction is a classical Chinese medicine formula extracted from Ginseng (Ren Shen), Glycyrrhiza inflata (Gan Cao), Largehead Atractylodes (Bai Zhu), and Indian Bread (Fu Ling) in a ratio of 3:3:3:2. It has been used as a main therapeutic method or complementary therapy for the treatment of various diseases, including cancer, for more than 1,000 years. Given that the function of inhibiting tumor growth, improving tumor cachexia, regulating tumor microenvironment, and regulating body immunity, the SJZ decoction is widely used to facilitate the swift recovery of cancer patients, since disease or chemotherapy may lead to poor physical fitness (5). Nowadays, the research of SJZ Decoction has been reported internationally for the treatment of esophageal squamous cell carcinoma and lung squamous cell carcinoma, but there are no reports or studies on the treatment of cSCC. The main compounds and mechanisms of SJZ decoction in the treatment of tumors have not been clarified. As the main active constituent in ginseng, ginsenosides have been widely used in the treatment of photoaging, hair loss, and trauma, among others (6). In recent years, Licochalcone A, Licochalcone B, and Licochalcone D have been reported to inhibit the growth of lung cancer cells through different pathways (7-11). Licochalcone C, Licochalcone H, and GIP1 can induce apoptosis of human oral squamous cell carcinoma cells (HOSCC) (12). Studies have found that Atractylodes I is the main effective ingredient in alleviating the symptoms of gastric cancer, and it can also improve the occurrence and development of melanoma to a certain extent (13,14). Currently, the main active ingredients of SJZ decoction, which is made up of four Chinese herbs, remain to be confirmed.

Therefore, in this study, we used network pharmacology and bioinformatics analysis to try to construct the action network of SJZ decoction and cSCC, with the aim of solving the following questions: (I) what are the active ingredients of SJZ decoction; (II) which cSCC disease targets are related to the active compounds of SJZ decoction; and (III) what new information can network pharmacology yield in the study of the treatment of cSCC by SJZ Decoction.

Methods

Active ingredients and potential targets of TCMs

We used three databases to identify the chemical ingredients in Ginseng, Glycyrrhiza inflata, Largehead Atractylodes, and Indian Bread: (I) Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; available online: https://old.tcmsp-e.com/tcmsp.php); (II) Traditional Chinese Medicines Integrated Database (TCMID; available online: http://47.100.169.139/tcmid/search/); and (III) TCM Database@Taiwan (http://tcm.cmu.edu.tw/). Specified oral bioavailability (OB) and drug-likeness (DL) properties were treated as filter criteria in the TCMSP^TM, which may allow the various components of the drug to perform the desired activity. Compounds that met both criteria were considered as candidate molecules.

Network analysis and building

Constructing a ‘drug-target-disease-pathway’ network allows us to gather a variety of information regarding active ingredients and their corresponding targets and to better understand the anti-tumor mechanism of active ingredients from another perspective. Firstly, we sorted out the screened active ingredients and corresponding potential targets of the four TCM ingredients. Then, we extracted cSCC disease-related targets from GeneCards online database (https://www.genecards.org/) for sorting and conducted functional enrichment analysis of these common potential targets, including “Gene Ontology (GO)” and “Kyoto Encyclopedia of Genes and Genomes (KEGG)“ analyses. Finally, all data were processed by Cytoscape 3.5.1 software (https://cytoscape.org/) to construct a complex network of “drug-component-target-pathway-disease”.

Plant sample preparation

The SJZ decoction is a mixture of Ginseng, Indian Bread, Largehead Atractylodes, and Glycyrrhiza inflata at a mass ratio of 3:3:3:2. The components were purchased from the Chinese Pharmacy of the Guangdong Second Provincial General Hospital (GD2H; Guangzhou, China). A total of 1,650 g of the Chinese herbal medicine (CHM) formula consisting of Ginseng, Indian Bread, Largehead Atractylodes, and Glycyrrhiza inflata was soaked for 30 minutes and extracted with 100 ℃ water twice. The sample was then cooled and concentrated.

Ultra-high performance liquid chromatography for sample

An ultra-high performance liquid chromatography (UPLC) system (Water, Milford, MA, USA) was used for dealing with the filtered sample solutions, combining with the annotation and classification of mass spectrometry database information were completed using precise characterization Instrument ASTAT-DAP. LC-MS (Thermo, Ultimate 3000 LC, HF) fitted with a C18 column [Zorbax Eclipse C18 (1.8 µm × 2.1 mm × 100 mm)] and the separation conditions were as follows: column temperature =30 ℃; flow rate =0.3 mL/min; mobile phase A=water + 0.1% formic acid, mobile phase B = pure acetonitrile; Injection volume =2 µ; and active autokinetic nozzle =4 ℃.

Statistical analysis

In this study, TCMSP, TCMID, TCM Database@Taiwan, and GeneCards database were used for data acquisition, David online tool and Cytoscape software were used for data analysis and image rendering respectively.

Results

cSCC-related gene pathways and networks

A total of 748 human genes with a high score (≥30.0) associated with cSCC were identified in the GeneCard database, and the encoded proteins were assembled into a set of 94 pathways and 25 networks using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; (https://cn.string-db.org/cgi/input.pl) and Cytoscape software. Cluster 1, which has the highest relevant scores among the networks, is shown in Figure 1A. The top five pathways involved cytokine-cytokine receptor interaction, lipid and atherosclerosis, rheumatoid, arthritis, malaria, and hepatitis B. The GO enrichment and network analysis showed that “T positive regulation of cytokine production”, “response to molecule of bacterial origin”, “leukocyte proliferation, positive regulation of mononuclear cell proliferation”, and “regulation of mononuclear cell proliferation” covered the top 5 biological processes of cSCC target proteins (Figure 1B).

Figure 1 GO enrichment and network analysis of cSCC target genes. (A) Interaction networks between Cluster 1 with the highest score by using MCODE algorithm in Cytoscape software. (B) Top 20 functionally enriched biological processes with corresponding adjusted P values analyzed by cluster Profiler, which are displayed scales indicated the different thresholds of adjusted P values, and the sizes of the dots represented the gene count of each term. GO, Gene Ontology; cSCC, cutaneous squamous cell carcinoma.

Screening of common targets of TCM compound and cSCC

The targets of the four herbs were predicted using the TCMSP database. A total of 136 candidate compounds were screened from 559 chemicals with DL (≥0.18) and OB (≥30%), including 22 in Ginseng, 7 in Largehead Atractylodes, 15 in Indian Bread, and 92 in Glycyrrhiza inflata. Several compounds have been reported to have more than one biological activity in recent studies. A total of 112 different herb targets obtained from 136 candidate compounds (Table 1) were further subjected to the UniProt (https://www.uniprot.org/) for conversing ID name. A total of 54 common candidate targets (Table 2) were selected from 748 different candidate cSCC targets filtered from GeneCards and 112 herb targets (Figure 2). The STRING database was used to construct the protein-protein interaction (PPI) networks (Figure 3).

Figure 2 Venn diagram. Among 748 cSCC targets and 112 traditional Chinese medicine targets, 54 co-targets were screened. cSCC, cutaneous squamous cell carcinoma.

Figure 3 PPI network from 54 co-candidate targets. The PPI network is built through the STRING website. Network nodes represent proteins, each of them represents all the proteins produced by a single, protein-coding gene locus. Edges represent protein-protein associations, which are meant to be specific and meaningful. PPI, protein-protein interaction; STRING, Search Tool for the Retrieval of Interaction Genes/Proteins.

Network pharmacology of SJZ decoction

The 54 targets of drug active compounds in SJZ which are related to cSCC are mainly enriched in “response to steroid hormone”, “response to metal ion”, “regulation of apoptotic signaling pathway”, “response to lipopolysaccharide”, “response to molecule of bacterial origin”, “extrinsic apoptotic signaling pathway”, “cellular response to oxidative stress”, “response to antibiotic”, “reproductive structure development”, and “regulation of DNA-binding transcription factor activity” by GO analysis for biological process. In this study, we reintegrated “TCMs-compounds”, “compounds-targets”, “TCMs-targets”, “targets-pathways”, and “disease-targets” by using Cytoscape software and obtained a “TCMs-compounds-targets-pathways-disease” network (Figure 4).

Figure 4 Network pharmacology of ‘TCMs-compounds-targets-pathways-disease’. Blue dot represents cSCC, green dots represent key pathways related to biological processes obtained by GO analysis, red dots represent the nine kinds of TCMs, pink dots represent active compounds related to those TCMs, and yellow dots represent common targets related to diseases and active compounds of TCMs. TCM, traditional Chinese medicine; GO, Gene Ontology; cSCC, cutaneous squamous cell carcinoma.

Screening and analysis of cluster and hub genes

We selected the module with the highest score from 54 common targets through the MCODE plug-in in Cytoscape. This cluster contains the 25 most correlated targets (Figure 5A). The 25 targets are mainly enriched in “response to steroid hormone”, “response to metal ion”, “myeloid cell differentiation”, “epithelial cell proliferation”, and “regulation of epithelial cell proliferation” by GO analysis for biological process (Figure 5B). The CytoHubba plug-in was used to sort degree value as a standard from high to low, and ten genes with the highest degree value were selected (Figure 6). Through GO analysis of hub genes, we found that “mammary gland alveolus development”, “mammary gland lobule development”, “positive regulation of epithelial cell proliferation”, “epithelial cell proliferation”, and “response to light stimulus” were the most closely related biological processes, in which “positive regulation of epithelial cell proliferation” and “epithelial cell proliferation” and Cluster 1 had the same closest relationship. These two processes included the MYC, ESR1, EGFR, HIF1A, CCND1, VEGFA, and ERBB2 genes. The pathways which had the top five highest relevant scores the candidate genes in Cluster 1 were mainly enriched in: “Kaposi sarcoma-associated herpesvirus infection”, “hepatitis B”, “human cytomegalovirus infection”, “chemical carcinogenesis-receptor activation”, and “lipid and atherosclerosis”. The common target-related main signaling pathways might have included: the JNK/NF-κB signaling pathway, the NF-κB/VEFG signaling pathway, the NF-κB/ICAM-1 signaling pathway, the JNK/AP-1 signaling pathway, the AP-1/ICAM-1 signaling pathway, the AP-1/VEFG signaling pathway, the HIF1A/VEFG signaling pathway, and the Caspase-9/Caspase-3 signaling pathway.

Figure 5 Cluster 1 of interacted proteins in SJZ against cSCC by use of MCODE algorithm. (A) 25 targets in cluster1; (B) GO enriched analysis with biological processes of functionally. SJZ, Si Jun Zi decoction; cSCC, cutaneous squamous cell carcinoma; GO, Geno Ontology.

Figure 6 Hub targets of SJZ. As results, the 10 regulator targets were finally identified, showing CASP3, MYC, ESR1, EGFR, HIF1A, CCND1, VEGFA, IL-6, ERBB2, and FOS. SJZ, Si Jun Zi decoction.

UPLC fingerprints

Through comparison with the “Similarity Evaluation System for Chromatographic Fingerprint of TCMs (Version 2012)”, the fingerprints of chromatograms in SJZ decoction showed 13 common peaks, which were identified as Atractylon, Liquiritin, 25-hydroxyporicoic acid H, Isoliquiritin apioside, ginsenoside Rg1, Ginsenoside Rg3, Ginsenoside Re, Kanzonol H, Ginsenoside Ro, Poricoic acid, Licoricesaponin G2, Glycyrrhizic acid, and Liquiritigenin, respectively (Figure 7). These ingredients may provide important laboratory evidence for antitumor therapy of SJZ.

Figure 7 UHPLC-ESI-MS/MS analysis of SJZ: Total ion chromatograms of water extraction of SJZ decoction by negative mode (A) and positive mode (B). The numbers in the chromatograms showed the constituent peaks. Peak 1 was identified as Atractylon; Peak 2 was identified as Liquiritin; Peak 3 was identified as 25-hydroxyporicoic acid H; Peak 4 was identified as Isoliquiritin apioside; Peak 5 was identified as Ginsenoside Rg1; Peak 6 was identified as Ginsenoside Rg3; Peak 7 was identified as Ginsenoside Re; Peak 8 was identified as Kanzonol H; Peak 9 was identified as Ginsenoside Ro; Peak 10 was identified as Poricoic acid; Peak 11 was identified as Licoricesaponin G2; Peak 12 was identified as Glycyrrhizic acid; Peak 13 was identified as Liquiritigenin. UHPLC-ESI-MS/MS, ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry; SJZ, Si Jun Zi decoction.

Discussion

As a kind of non-melanoma skin tumor, cSCC has a high incidence and metastasis rate. In the treatment of this disease, surgery is suitable for the early, non-metastatic type, but is not the best choice for metastatic or recurrent cSCC (15). Formulae of TCM have been applied and studied by medical and scientific researchers in many countries worldwide for many years, which have been reported to have the characteristics of high absorption, complex ingredients, and multi-target (16). The different combinations and proportions of various CHMs in the prescription make the drugs have different mechanisms and effects, which has also become a research hotspot in clinical treatment.

The SJZ decoction, as a classical prescription, has been used by many patients to improve the weakness caused by malignant tumors, and is an auxiliary treatment method. In recent years, there are a number of reported results show that SJZ decoction or SJZ based Chinese traditional medicine in the bladder cancer mice, lung cancer mice that accept chemotherapy have enhanced the effect of chemotherapy drugs, reduce the effect of chemotherapy drugs toxic side effects through inhibiting tumor growth and can prolong the survival of mice (17,18). In addition to gastric cancer (19-22), it has also been studied at an animal level or cellular level in the clinical treatment of HOSCC and multiple types of lung cancer (23), but we did not find any previous studies related to SJZ in cSCC.

In this study, the constituents of Ginseng, Glycyrrhiza inflata, Largehead Atractylodes, and Indian Bread in SJZ decoction were screened by network pharmacology, and the targets of these candidate constituents were further analyzed and categorized. At the same time, comprehensive comparison and intersection were made between these potential targets above and potential targets related to cSCC, and candidate targets were screened out for functional enrichment analysis. Meanwhile, a network diagram of “drug-component-target-pathway-disease” was made for future reference and use.

The study found that the potential targets of the more prominent active ingredients of SJZ decoction were mainly concentrated in the biological processes: response to steroid hormone and may be most closely related to the pathways: Kaposi sarcoma-associated herpesvirus infection. At the same time, these targets, pathways, and biological processes are related to the occurrence and development of cSCC.

The results of UPLC analysis showed that the top ten active ingredients extracted from SJZ decoction were Atractylon, Liquiritin, 25-hydroxyporicoic acid H, Isoliquiritin apioside, ginsenoside Rg1, Ginsenoside Rg3, Ginsenoside Re, Kanzonol H, Ginsenoside Ro, Poricoic acid, Licoricesaponin G2, Glycyrrhizic acid, and Liquiritigenin. The ingredients come from the four different Chinese herbs.

In short, the ten main active components of SJZ decoction analyzed by UPLC may act on cSCC-related targets through specific pathways, and then play a role in the treatment or adjuvant treatment of cSCC.

Conclusions

This study conducted correlation analysis on the active components of SJZ decoction and the disease target of cSCC through network pharmacology, and constructed a network diagram with software combining TCM-component-target-disease-pathway. The analysis of the effective components of SJZ decoction by UPLC is helpful to provide a reference direction and theoretical basis for omgoing research on the treatment of cSCC by SJZ decoction.

Limitation

Whether SJZ decoction has the effect of preventing and treating cancer and its mechanism of action still need to be further explored.

Acknowledgments

Funding: This work was supported by Project of Administration of Traditional Chinese Medicine of Guangdong Province, China (No. 20221014) and supported by The Science Foundation of Guangdong Second Provincial General Hospital (YQ2019-002).

Footnote

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-1716/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Jones)