Potential Mechanisms of Yanghe Decoction in the Treatment of Soft Tissue Sarcoma and Arteriosclerosis Obliterans Based on Network Pharmacology

Abstract Objective  The objective of this study was to investigate potential mechanisms of Yanghe Decoction ( , YHD) in treating soft tissue sarcoma (STS) and arteriosclerosis obliterans (ASO) based on the use of network pharmacology. Methods  Candidate compounds and potential targets were identified through the TCM Systems Pharmacology database and a comprehensive literature search. Related targets of STS and ASO were collected in the GeneCards database, DisGeNET database, and Drugbank database. Furthermore, The STRING 11.0 database was used to determine protein–protein interaction (PPI) networks; common targets were obtained and imported into Cytoscape 3.7.2. Then, a PPI network comprising common targets was drawn, and network topology analysis was performed to screen for key shared targets. Gene ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of key shared targets were performed by using Metascape software. Subsequently, a compound–target–pathway network was constructed via Cytoscape 3.7.2. Results  The following signaling pathways were found to be associated with the mechanisms of YHD in treating STS and ASO: AGE-RAGE signaling pathway, IL-17 signaling pathway; HIF-1 signaling pathway, TNF signaling pathway, interactions between cytokines and cytokine receptors, Th17 cell differentiation, and NOD-like receptor signaling pathway. Among the compounds and targets involved in these pathways, quercetin, luteolin, and kaempferol were found to be core compounds, and TNF, IL-6, and MAPK1 were found to be core targets. Conclusion  Taken together, our findings elucidated that potential mechanisms of YHD in treating STS and ASO involved cellular proliferation/differentiation, angiogenesis, inflammation, immune responses, oxidative stress, and other related signaling pathways.


Introduction
Soft tissue sarcoma (STS) is a malignant solid tumor derived from fat, fascia, muscle, fibers, lymph, and blood vessels. The incidence rate of STS in China is approximately 2.38/100,000 individuals per year, and this rate continues to increase. 1 Arteriosclerosis obliterans (ASO) involves chronic occlusion of peripheral arteries caused by long-term atherosclerosis, which mostly occurs in the lower extremities. The prevalence of ASO in the elderly is particularly high. Due to the increase in the proportion of the aging population in China, the incidence of ASO is also increasing each year, and it has become an urgent public health and medical problem in China. 2 Traditional Chinese medicine (TCM) posits that STS belongs to the categories of "sarcoma" and "stone gangrene." The occurrence of STS is related to a lack of vital qi and attack by cold pathogen, which leads to qi stagnation, blood stasis, and phlegm resistance. Yanghe Decoction ( YHD), used as a treatment in STS patients, has been shown to significantly improve clinical symptoms and extend survival time. 3,4 ASO belongs to the category of "necrosis" in TCM and is mostly the result of yang deficiency in spleen and kidney, with coldness and wetness as external manifestations, which leads to qi and blood stagnation, and obstruction of channels. YHD has the function of warming yang and tonifying blood, dispersing cold and dredging stagnation. Therefore, it is commonly used in the treatment of ASO. 5,6 Although the above findings suggest that YHD may be efficacious in treating both STS and ASO, its underlying mechanisms in this therapeutic process remain unknown. Unfortunately, traditional pharmacological research methods alone are not sufficient to fully elucidate the mechanisms of action of YHD. In contrast, network pharmacology is a powerful tool for investigating the mechanisms of action of TCM compounds. In particular, network pharmacology assesses multilevel relationships of compounds, targets, and pathways to provide insights into the mechanisms of action of TCM compounds. 7,8 In the present study, we have used network pharmacology to investigate the mechanisms of action of YHD in treating both STS and ASO, which may provide useful findings for further experimental research and clinical applications.

Screening of Chemical Components of YHD
We used the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database and analysis platform 9 and an extensive literature search to identify candidate compounds and targets of the following six key components in YHD: Shudihuang (Rehmannia glutinosa, SD), Rougui (Cinnamomum cassia Presl, RG), Mahuang (Ephedra sinica Stapf, MH), Jiezi (Semen sinapis, JZ), Sheng jiang (Zingiber officinale Rosc, SJ), and Gancao (Glycyrrhiza uralensis Fisch, GC). Lujiao Jiao (Colla Corni Cervi) was excluded since it was not suitable for our present pharma-cological study. According to the absorption, distribution, metabolism, and excretion parameters in the TCMSP database, Chinese medicinal compounds with oral bioavailability !30% and with a drug-like-index (DL) !0.18 were selected as candidate compounds.

Collection and Treatment of Targets in YHD
We obtained potential targets of the candidate compounds through the TCMSP database, uploaded them into the Uni-Prot database (https://www.uniprot.org/), limited the species to "human," and obtained the genetic information corresponding to each protein target. Then, we imported these data into Cytoscape 3.7.2 to build a compound-targetpathway network diagram.

Acquisition of Common Targets of YHD in the Treatment of STS and ASO
We used "soft tissue sarcoma" and "arteriosclerosis obliterans" as search terms in the GeneCards database (https:// www. genecards.org), DisGeNET database (https://www.disgenet.org/), and Drugbank database (https: //www.drugbank.ca) to collect disease targets related to STS and ASO. If there were too many disease targets in the database, we defined targets with scores greater than the median score as potential targets. Then, we merged potential targets obtained from each database and removed any duplicates. Subsequently, we determined cross targets, and the cross targets were displayed via a Venn diagram through Omic-Share (https://omicshare.com/index.php).

Screening of Key Shared Targets
We entered the obtained common targets into the STRING 11.0 database (https://string-db.org/). The organism category was set to Homo sapiens. We obtained protein-protein interaction networks of the common targets via a combined score >0.4 as the screening criterion. Then, we imported the screened information into Cytoscape 3.7.2 to plot the protein-protein interaction network between the common targets and performed network topology analysis. Key shared targets were selected according to values greater than the median.

GO Function Enrichment and KEGG Signal Pathway Enrichment Analysis
We uploaded the key common targets to Metascape (https:// metascape.org/) and set the parameter to H species to obtain the gene ontology (GO) function enrichment analysis results and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis results. GO functional enrichment analysis includes biological process (BP), cell composition (CC), and molecular function (MF). According to the value of log (10)p, we used the bioinformatics online mapping tool to visualize various top-ranked GO items and filtered out the top 20 signaling pathways according to theirs. OmicShare was used to visualize the results of enrichment analysis of the top 20 KEGG pathways. Finally, we imported these data into Cytoscape 3.7.2 to construct a compoundtarget-pathway interaction network.

Screening of Chemical Components of YHD
We identified a total of 76 chemical constituents of SD, 103 chemical constituents of RG, 363 chemical constituents of MH, 37 chemical constituents of JZ, 265 chemical constituents of SJ, and 280 chemical constituents of GC. A total of 129 candidate chemical constituents were obtained after screening, including two chemical constituents of SD, three chemical constituents of RG, 23 chemical constituents of MH, three chemical constituents of JZ, five chemical constituents of SJ, and 92 chemical constituents of GC. After deleting duplicates, a total of 118 chemical components were obtained (►Table 1).

Collection and Treatment of Targets in YHD
We obtained 28 targets of SD, 153 targets of RG, 209 targets of MH, 17 targets of JZ, 49 targets of SJ, and 213 targets of GC. After deleting duplicates, a total of 233 drug targets were obtained, which were imported into Cytoscape 3.7.2 to construct a drug-compound-target interaction network (►Fig. 1).

Acquisition of Common YHD Targets in the Treatment of STS and ASO
We obtained 1,615 STS disease targets and 223 ASO disease targets. The above two groups of disease targets were intersected with 233 compound targets in YHD (►Fig.

GO and KEGG Analyses
A total of 745 BPs were obtained and were mainly related to cell migration, cell apoptosis, cytokine metabolism, and cell response to biological stimuli. A total of 12 CCs were obtained and involved membrane rafts, membrane microdomains, membrane domains, and the extracellular matrix. A total of 14 MFs were obtained and involved cytokine activity, receptor ligand activity, growth factor activity, and chemokine receptor binding. The top-ranked items included cytokine-mediated signaling pathways, positive regulation of cell migration, positive regulation of cell motility, positive regulation of cellular-component movement, positive regulation of locomotion, and other matters (►Fig. 4). KEGG pathway enrichment analysis yielded 65 signaling pathways, among which the top 20 pathways included the AGE-RAGE signaling pathway (related to diabetic complications), IL-17 signaling pathway, and HIF-1 signaling pathway and other matters (►Table 2). The results of KEGG pathway enrichment analysis of the top 20 KEGG pathways were visualized using Omic-Share (►Fig. 5) and were then imported into Cytoscape 3.7.2 to build a compound-target-pathways interaction network (►Fig. 6).

Discussion
YHD is derived from the Waike Zhengzhi Quansheng Ji. 10 SD can nourish yin and blood. RG and SJ can warm the yang and dissolve the cold. MH can open the sweat pores of the skin and release pathogens. JZ can dredge collaterals and resolve phlegm. GC can nourish qi and detoxify and harmonize medicines. STS is a type of malignant solid tumor. The formation of STS is due to an imbalance of yin and yang. Yang deficiency leads to cold coagulation, phlegm obstruction, blood stasis, and eventually leads to tumors. Cold dampness pathogen is the superficial reason of ASO, while yang deficiency of spleen and kidney is the root cause. Most of the patients are middle aged and elderly people, who often have the syndromes of deficiency, blood stasis, and phlegm. It can be seen that the above two diseases have yang deficiency, phlegm dampness, and blood stasis in varying degrees. YHD can warm yang to regenerate qi and blood and remove cold to dredge collaterals and disperse accumulation. Therefore, it theoretically explains the reason why YHD can treat STS and ASO.
In the present study, we used network pharmacology to identify signaling pathways involved in the effects of YHD in treating STS and ASO, which included the following: the AGE-RAGE signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, interactions of cytokines and cytokine receptors, Th17 cell differentiation, and NOD-like receptor pathway. The topological properties were analyzed based on the degree values and corresponding intermediary values in the two networks of drug-compound-targets and compound-target-pathways. The most important compounds were determined to be quercetin, luteolin, and kaempferol. Additionally, the most critical targets were found to be TNF, IL-6, and MAPK1. To corroborate these findings, we performed an extensive literature search and further explored the mechanisms of YHD in the treatment of STS and ASO.
Quercetin 11 and luteolin 12 exhibit proteasome inhibitory activity and have significant effects in overcoming multidrug resistances of various tumors such as sarcomas. Quercetin 12 and kaempferol 13 can inhibit tumor invasion and metastasis by inhibiting the activity of MMP-9 in human fibrosarcoma HT1080 cells. Quercetin can induce apoptosis of human liposarcoma SW 872 cells by down-regulating Bcl-2, cleaving PARP, and activating caspase-3, Bax, and Bak. 14 Luteolin can down-regulate β-catenin expression, inhibit Wnt signaling, and reduce the formation of fibromatosis, sarcoma, and Chinese Medicine and Natural Products © 2022. The Author(s)    15 Quercetin, 16 luteolin, 17 and kaempferol 18 all have anticoagulant, antithrombotic, antiplateletaggregation, and defibrillating effects. Quercetin can also exert antiarterial effects by inhibiting the expression of SDF-1 and CXCR4 in the sera of APOE mice, regulating blood lipid levels in atherosclerotic rats and interfering with the activities of key proteins in the PI3K/Akt/NF-κb pathway. 19,20 In addition, luteolin also can reduce atherosclerosis by reducing inflammation in APOE mice. 21 TNF is a cytokine that can directly kill tumor cells and exerts antitumor effects by activating the immune system. Ubiquitin-specific protease 20 can inhibit smooth-muscle cell inflammation caused by TNF overexpression by deubiquitinating β and relieving atherosclerosis. 22 IL-6 is a pleiotropic cytokine expressed by immune cells and various tumor cells. IL-6 induces inflammation, promotes cancer cell proliferation, and inhibits apoptosis, thereby promoting chemotherapy resistance. Studies have shown that IL-6 promotes the progression of Ewing's sarcoma by increasing resistance to apoptosis and promoting metastasis under cellular stress. 23 Inflammation is a major factor leading to atherosclerosis. IL-6 is an upstream inflammatory cytokine that plays a central role in downstream inflammatory responses leading to atherosclerosis. 24 MAPK1 is a member of the MAP kinase family and is also known as ERK2. MAPK1/ ERK2 is involved in processes such as cellular proliferation, differentiation, and transcriptional regulation. Most patients with angiosarcoma have obvious genetic changes related to the MAPK signaling pathway, which activates the MAPK pathway and increases tumor cell proliferation. In the occurrence and development of alveolar rhabdomyosarcoma, HGF/MET signaling (mainly through ERK2 signaling)  3.06987E-27  ICAM1, IL-1β, IL-6, CXCL8, MMP2, NOS3,  SERPINE1, MAPK1, CCL2, STAT3, TNF,  VCAM1, VEGFA   hsa05418  Fluid shear stress and  atherosclerosis   3.1339E-20  HMOX1, ICAM1, IFNG, IL-1β, MMP2, MMP9 The AGE-RAGE signaling pathway not only causes oxidative stress, inflammation, thrombosis, and fibrosis in a variety of cells but also activates a variety of signal transduction pathways related to cellular proliferation and apoptosis. Furthermore, the AGE-RAGE signaling pathway plays an important role in the occurrence, development, and metastasis of tumors. 29,30 The IL-17 signaling pathway not only participates in autoimmune diseases and chronic inflammatory diseases but also participates in tumor cell survival, angiogenesis, chemokine production, tissue remodeling, and immune modification of the tumor microenvironment, thereby affecting the occurrence and development of STS and ASO. [31][32][33][34] The HIF-1 signaling pathway participates in the regulation of angiogenesis, cellular metabolism, and autophagy, as well as in the occurrence or development of malignant tumors and inflammatory responses. Some studies have shown that the HIF-1 signaling pathway affects cellular metabolism, differentiation, angiogenesis, proliferation, and metastasis and this pathway is related to the prognosis of patients with STS and chondrosarcoma. 35,36 The HIF-1 signaling pathway can also cause endothelial cell dysfunction, angiogenesis, and inflammation by up-regulating VEGF, NO, ROS and PDGF. Moreover, these responses play a role in the development of ASO. 37 Cytokines are small Fig. 1 Network of drugs-compounds-targets related to YHD in the treatment of STS/ASO. Notes: The regulatory network was constructed by 357 nodes (six Chinese herbs, 118 candidate compound nodes, and 233 target nodes) and 1,769 edges; among the 118 candidate compounds, eight compounds had no corresponding targets found in the database; purplish-red represents Chinese herbs, blue represents compound, yellow represents target,and the size of node is directly proportional to the Degree value of node.   polypeptides or glycoproteins that are synthesized and secreted by a variety of cells and include interleukins, interferons, chemokines, growth factors, the tumor necrosis factor superfamily and colony-stimulating factors. Interactions between cytokines and cytokine receptors can regulate the growth and differentiation of cells, regulate immune responses, participate in inflammatory responses, repair damaged tissues, and have regulatory effects on STS and ASO. [38][39][40][41] In addition, TNF signaling pathway, Th17 cell differentiation, and NOD-like receptor signaling pathway are all signal pathways related to immunity and inflammation, which can activate NF-κB, MAPK, and endoplasmic reticulum emergency pathway and promote the release of inflammatory factors such as IL-6 and mediate inflammatory response, which is closely related to tumors, inflammatory diseases, and autoimmune diseases. 42,43 Conclusions In summary, this study reveals that the mechanism of YHD in the treatment of STS and ASO mainly involves cell proliferation, differentiation, angiogenesis, inflammation, immune response, oxidative stress, and other related signal pathways, which is consistent with the current research on the mechanism of STS and ASO. To some extent, it is proved that the results predicted by the network pharmacology method are reliable, but further experimental verification is still needed. This study can not only guide the experimental research in