Molecular mechanism of the anti-inflammatory effects of Sophorae Flavescentis Aiton identified by network pharmacology

Inflammation, a protective response against infection and injury, involves a variety of biological processes. Sophorae Flavescentis (Kushen) is a promising Traditional Chinese Medicine (TCM) for treating inflammation, but the pharmacological mechanism of Kushen’s anti-inflammatory effect has not been fully elucidated. The bioactive compounds, predicted targets, and inflammation-related targets of Kushen were obtained from open source databases. The “Component-Target” network and protein–protein interaction (PPI) network were constructed, and hub genes were screened out by topological analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on genes in the PPI network. Furthermore, nitric oxide (NO) production analysis, RT-PCR, and western blot were performed to detect the mRNA and protein expression of hub genes in LPS-induced RAW264.7 cells. An immunofluorescence assay found that NF-κB p65 is translocated. A total of 24 bioactive compounds, 465 predicted targets, and 433 inflammation-related targets were identified and used to construct “Component-Targets” and PPI networks. Then, the five hub genes with the highest values-IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2)- were screened out. Enrichment analysis results suggested mainly involved in the NF-κB signaling pathway. Moreover, experiments were performed to verify the predicted results. Kushen may mediate inflammation mainly through the IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2), and the NF-κB signaling pathways. This finding will provide clinical guidance for further research on the use of Kushen to treat inflammation.


Effects of Kushen on cell viability and NO production.
Previous studies have reported that the main components of Kushen, such as Matrine 15 , Oxymatrine 16 , Sophoridine 17 , Kushenol B 18 , own excellent antiinflammatory effects. In this study, LPS-induced RAW264.7 cells inflammatory model was used cell to investigate the mechanism underlying Kushen's anti-inflammatory effect on macrophagocytes. After an incubation period, the effects of Kushen extract on the viability of RAW264.7 cells were detected by CellTriter-Lumi ™ Plus.
A viability assay showed that Kushen extract does not inhibit cell proliferation at any concentrations up to 10 μg/ mL (Fig. 5A). In addition, the anti-inflammatory effects of Kushen extract on NO production in LPS-treated cells were detected by Griess reagent. As shown in Fig. 5B, the Kushen extract significantly inhibited NO production. Furthermore, laser microscopy showed that Kushen extract is a stronger inhibitor of intracellular NO production than LPS stimulation alone (Fig. 6).

Suppression of the mRNA and protein expression of the hub genes by Kushen extract.
To investigate the effects of Kushen extract on the predicted hub genes by network pharmacology, the mRNA levels of IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) were measured by quantitative real-time PCR, whereas the protein levels of IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) were measured using western blot analysis. As shown in Fig. 7, the mRNA expression of IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) was significantly increased after LPS stimulation (0.2 μg/mL). Moreover, the mRNA expression of these genes was significantly inhibited by all concentrations of Kushen extract in a concentration-dependent manner. In addition, the protein expression of IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) in cells treated with Kushen extract was significantly inhibited compared to their expression in the LPS group alone (0.2 μg/mL) group (Fig. 8). Collectively, our study suggests that Kushen extract mainly treats inflammation by inhibiting these genes.
Translocation of the NF-κB p65 subunit. As shown in Table 4, the NF-κB signaling pathway is the key signaling pathway underlying the anti-inflammatory action of Kushen extract. The translocation of the NF-κB p65 subunit was determined by immunofluorescence. As shown in Fig. 9, after stimulation by LPS, p65 (red) was translocated from the cytoplasm to the nucleus; this clearly attenuated by Kushen extract (10 μg/mL), suggesting that the Kushen extract inhibited NF-κB activation.

Discussion
Inflammation is a protective response against infection and injury 19 , which is closely linked to various chronic or malignant diseases, such as type II diabetes 20 , atherosclerosis 21 , and cancer 22 . Inflammation is a complex process involving multiple genes and signaling pathways 23 , and TCM is considered to have anti-inflammatory potential due to its effects via multiple-compounds, multiple-targets, and multiple pathways involved 24 . Kushen, wellknown for its efficacy in clearing body heat, is a TCM mostly used to treat various syndrome that are caused by inflammation 25 or infection 26 . Therefore, Kushen and its preparation, such as Kushen Lotion, Kushen Injection, are clinically used as adjuncts to treat inflammation-related diseases 27 . Through data mining and analysis, network pharmacology systematically interprets the overall relationship between drugs and targets, which perfectly fits TCM's strategy of disease management through multiple ingredients, targets, and pathways 28 . Unlike previous studies, our study was the first to fully elucidate the anti-inflammatory mechanism of Kushen extract via network pharmacology methods and experimental validation, laying the foundation for further clinical research.
In this study, we retrieved 19 bioactive compounds of Kushen from the TCMSP, ETCM, and SymMap databases and five bioactive compounds with noteworthy pharmacological effects from literatures and found 465 predicted targets and used them to construct a "Component-Target" network. In the network, important compounds such as isosophocarpine (degree 218), quercetin (degree 207), dehydromiltirone (degree 166), and luteolin (degree 129) have a high degree value and are associated with many targets. Isosophocarpine, a tetracyclic quinolizidine alkaloid, shows anti-cancer effects on different types of cancer by attenuating inflammation 29 . Quercetin is a flavonoid with antioxidant, antiviral, and antibacterial effects, and is widely distributed in various plants and food. Lin et al. 30 found that quercetin suppresses inflammation by countering the Azoxymethane/ Dextran sodium sulfate (AOM/DSS)-induced carcinogenesis progression. In addition, Yue et al. have found that the dehydromiltirone is an anti-inflammatory compound that initiates p38 and the NF-κB signaling pathway in LPS-induced Kupffer cells 31 . Luteolin is a flavonoid commonly found in plants, such as celery, green pepper, honeysuckle, and chamomile. Previous studies have suggested that luteolin can reduce inflammation via inhibiting inflammation via activating the Nrf2/ARE, NF-κB, and MAPK signaling pathways 32 . These findings suggested that Kushen extract exerts its anti-inflammatory effects through multiple components and multiple targets.
A PPI network was constructed with 81 nodes and 1088 interaction edges to elucidate further the mechanisms of Kushen's anti-inflammatory response. Then, the hub genes, namely IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) were screened out based on the topological properties' analysis. LPS-induced RAW264.7 cells constitute a typical inflammation model, and therefore the present study used them to investigate the antiinflammatory effects of Kushen extract. A NO production assay showed that any concentration of Kushen extract inhibits the production of NO in a concentration-dependent manner, and intracellular NO production assay intuitively showed that Kushen extract shows maximum anti-inflammatory effect at 10 μg/mL. Moreover, RT-PCR and western blot further verified that the mRNA and protein levels of IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2) differ significantly between the LPS-induced and Kushen extract groups, suggesting that Kushen mainly exerts the anti-inflammatory effects via these predicted hub genes. IL-6, IL-1β, and TNF-α are crucial pro-inflammatory cytokines that coordinate a variety of inflammatory and immunomodulatory pathways that have broad effects on cells of the immune system 31,33 . VEGFA, a pleiotropic cytokine, has been considered as the   www.nature.com/scientificreports/ indispensable part of angiogenesis, which mainly leads to cancer-related inflammation 34 . PTGS2 (COX-2), an enzyme induced by pro-inflammatory cytokines, releases prostaglandin E2 (PGE2) and promotes the synthesis of prostaglandins stimulating cancer cell proliferation, development, and metastasis; thus, it serves as a therapeutic target for anti-inflammatory drugs.  www.nature.com/scientificreports/ In, addition, the KEGG enrichment analysis of targets in the PPI network showed that Kushen's anti-inflammatory effect is mainly enriched in the NF-κB signaling pathway and the PI3K-Akt signaling pathway. The PI3K-Akt/NF-κB signaling pathways play different roles in normal physiological responses and inflammatory processes 31,35 , including promoting cell proliferation, survival and differentiation. After activation of PI3K-Akt pathway, Akt enhances the phosphorylation and lowers the phosphorylation of the NF-κB inhibitor protein IκB kinase. PI3K/Akt/NF-κB signaling pathway server as the intersection of T and B cell inflammatory signaling pathway, resulting in increased expression of its maker proteins IKK-α, IκB-α, NF-κB p65, PI3K, p-AKT, p-NF-κB p65 in inflammatory model 36 . Among these, p65/RelA and p50 take important part in the NF-κB signaling pathway; p65 degrades when NF-κB signaling shuts down 37 . The immunofluorescence results also verified that Kushen extract (10 μg/mL) attenuates p65 from the cytoplasm to the nucleus in LPS-induced RAW264.7 cells, suggesting that the Kushen plays a crucial role in modulating inflammation via the NF-κB signaling pathway.   www.nature.com/scientificreports/ In conclusion, a network pharmacology approach was developed to elucidate the underlying molecular mechanism of anti-inflammatory effects of Kushen on inflammation. A total of 24 bioactive compounds, 465 Kushen-related targets, and 433 inflammation-related targets were obtained from open source databases. Furthermore, five hub genes were screened out based on a topological property analysis of the PPI network: IL-6, IL-1β, VEGFA, TNF-α, and PTGS2 (COX-2). Then, an experimental in vitro validation was performed to confirm the mRNA and protein expression of these hub genes and for enrichment analysis. Considering the complexity of the inflammatory process and TCM predicted targets, further research and clinical trials are necessary to confirm our findings.  Preparation of Kushen extract. Kushen herb was purchased from the Tongren Pharmaceutical Co., Ltd.
(BEIJING, CHINA). Kushen (50 g) was soaked in 1 L of cold water for 30 min and then boiled for 30 min; this procedure was repeated three times. The combined extracts were concentrated to 1 g/mL (crude herbal dose)  Forward TGT TAC CAA CTG GGA CGA CA   Reverse  GGG GTG TTG AAG GTC TCA AA   COX-2  Forward TGA GTA CCG CAA ACG CTT CTC   Reverse  TGG ACG AGG TTT TCC ACC AG   TNF-α  Forward TAG CCA GGA GGG AGA ACA GA   Reverse  TTT TCT GGA GGG AGA TGT  www.nature.com/scientificreports/ in a vacuum rotary evaporator. Then, the extract (1 mL) was filtered through microporous membrane before quantification.

Construction of a bioactive component-target network.
To intuitively understand the mechanisms of Kushen extract treatment on anti-inflammation, a "Component-Target" network was constructed via Cytoscape 3.7.0 based on the bioactive components and predicted targets. In this network, the green rhombus node, blue round node, and edges indicate a bioactive component, a predicted target, and the interaction between the bioactive compounds and targets, respectively. The plug-in "Cytohubba" was applied to calculate the "degree" value of the node, which suggests the number of edges between the nodes in the network.
Screening for potential targets of inflammation. The keywords "inflammation, " "anti-inflammatory, " or "antiinflammation" were used to search disease-related genes on the following databases:

Protein-protein interaction network construction and hub genes analysis.
To further elucidate the potential mechanism underlying Kushen's anti-inflammatory effect, a website was used to find overlapping inflammatory-related and predicted targets of Kushen. These overlapping targets were used to construct a protein-protein interaction (PPI) network on the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https ://strin g-db.org/) with the "Homo sapiens" setting. Cytoscape 3.7.0 was used to visualize PPI network, and the plug-in "Network Analysis" was performed to visualize the topological properties of each node in the network. To further elucidate the mechanism by which Kushen treats inflammation, the hub genes were screened out based on the topological properties of nodes in the PPI network. The plug-in "cyto-Hubba" was applied to calculate the value of degree in the PPI network, and the five genes with the highest values of degree were selected as anti-inflammatory hub genes for Kushen. Further, information on the target type (protein class) of the hub genes was taken from the DisGeNET database (https ://www.disge net.org).
Gene ontology and KEGG pathway enrichment analyses. Gene ontology (GO) enrichment analysis is a bioinformatics tool for predicting gene function, while the Kyoto Encyclopedia of Genes and Genomes (KEGG, https ://www.kegg.jp/) is a database for identifying the systematic functions and biological relevance of targets 42 . In order to analyze the biological pathways of genes in the PPI network, the "clusterProfiler" package (https ://bioco nduct or.org/packa ges/relea se/bioc/html/clust erPro filer .html) in R (version: 3.6.3) 43 was applied to analyze GO enrichment and KEGG pathway enrichment (adjusted to P. < 0.05).
Cell culture. RAW264 Nitrite assay. RAW264.7 cells were cultured in 96-well plates at 1 × 10 4 cells/well and treated as described above. NO production in the supernatant of the medium was measured by the Griess assay according to the manufacturer's instructions and absorbance at 540 nm (OD 540 ) was measured with a multifunctional microplate reader. In addition, DAF-FM was applied to qualitatively detect the concentration of NO. The cells were cultured and treated as described above. According to the instructions for the DAF-FM DA Kit, the images were generated with a Laser microscope (495 nm/515 nm).
Total mRNA extraction and RT-PCR. Total  License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.