Mechanism of Tripterygium Wilfordii for the Treatment of Idiopathic Membranous Nephropathy Based on Network Pharmacology

Tripterygium wilfordii has been widely used for idiopathic membranous nephropathy (IMN), while the pharmacological mechanisms are still unclear. This study is aimed at revealing potential therapeutic targets and pharmacological mechanism of tripterygium wilfordii for the treatment of IMN based on network pharmacology. Active components of tripterygium wilfordii were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. IMN-associated target genes were collected from GeneCards database, DisGeNET database, and OMIMI database. VENNY 2.1 was used to identify the overlapping genes between active compounds of tripterygium wilfordii and IMN target genes. Using STRING database and Cytoscape 3.7.2 software to analyze interactions among overlapping genes. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the targets were analyzed using the Rx64 4.0.2 software, colorspace, stringi, DOSE, clusterProler, and enrichplot packages. A total of 153 compounds-related genes, 1485 IMN-related genes were obtained, and 77 overlapping genes between them were identied. The protein–protein interactions network indicated that the targets AKT1, TNF, VEGFA, TP53, PTGS2, CXCL8, MAPK8, STAT3, JUN, and CASP3 play an important role in the treatment effect of tripterygium wilfordii for IMN. The enrichment analysis showed that the main pathways of targets were AGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway. This study reveals potential mechanisms of multi-component and multi-target of tripterygium wilfordii for the treatment of IMN based on network pharmacologic approach, and provide a scientic basis for further experimental studies.


Introduction
Idiopathic membranous glomerulonephritis (IMN) is an immune-mediated primary glomerular disease that usually manifested as nephrotic syndrome [1]. Although 30% of patients undergo spontaneous remission, persistent proteinuria are occur in 30%-40% of patients, and even develop end-stage renal disease [2][3]. Although the optional treatment options for IMN include rituximab, glucocorticoids plus cyclophosphamide and glucocorticoids plus calcineurin inhibitors, there are some adverse events. The adverse events of rituximab include infusion reaction, opportunistic infections, allergic reaction and hypogammaglobulinemia, in addition, have some defects including slower effect and expensive.
Cyclophosphamide is associated with a variety of side effects, such as gonad injury, malignancy, bone marrow suppression and hemorrhagic cystitis. The patients who use calcineurin inhibitors have the risk of renal insu ciency, and a high rate of relapse after drug withdrawal.
Tripterygium wilfordii, as a well-known traditional Chinese medicine, has been widely used for the treatment of various autoimmune diseases, such as nephritic syndrome, in ammatory bowel disease, systemic lupus, erythematosus, and rheumatoid arthritis [4]. Clinical trials have demonstrated that Tripterygium wilfordii was effective in the treatment of IMN [5][6]. However, we know little about the molecular mechanisms of the effect of tripterygium wilfordii on IMN.
The incorporation of traditional Chinese medicine into clinical therapy via network pharmacology can provide insights into the possible mechanism and enhance the speci city and effectiveness of the treatment scheme [7]. In our study, we explored the possible molecular mechanism based on network pharmacology.

Materials And Methods
Identi cation and screening of Active Compounds and Targets for Tripterygium Wilfordii and Therapeutic

Construction of Drug-disease Target Protein-Protein Interactions (PPI) Network
The overlapping genes between compounds of tripterygium wilfordii and IMN target genes were imported into STRING [11] (https://string-db.org/cgi/input.pl) to construct and visualize the PPI network. And Cytoscape 3.7.2 software [12] was used to further analyze and visualize the PPI network. The node size and color re ected the number of combined targets (degree), and the larger node and the redder color indicates the higher degree value. The thickness of the side indicates the value of the combine score, and the thicker side indicates the higher combine score.
Gene Ontology (GO) and Kyoto Encyclopedia of Gene, Genomes (KEGG) Pathway Enrichment Analysis of Tripterygium Wilfordii for IMN and the Drug-Compound-Target-Signaling Pathway Network The GO enrichment analysis and KEGG pathway enrichment of the targets were analyzed using the Rx64 4.0.2 software, colorspace, stringi, DOSE, clusterPro ler, and enrichplot packages [13][14], and the screening conditions were adjusted P value < 0.05 and q < 0.05, and the result of GO enrichment analysis included 3 different levels: biological processe (BP), molecular function (MF), and cellular component (CC) [15]. We used Cytoscape 3.7.2 to construct and visualize the drug-compound-target-signaling pathway network.

Identi cation and screening of Active Compounds and Targets for Tripterygium Wilfordii and Therapeutic Targets for IMN
A total of 42 active compounds of tripterygium wilfordii were obtained by searching the TCMSP database, and using ADME parameters such as OB, DL, Hdon and Hacc (Additional le 1). After nding the target data from TCMSP database, and deleting the repeated items, 153 targets were nally obtained.
Then, we used UniprotKB database normalized the protein targets collected in TCMSP database. A total of 1485 therapeutic targets of IMN were acquired from GeneCards, DisGeNET and OMIMI databases after removing duplicates.

Drug-Disease Target PPI Network
The result of Venn diagram showed that 77 overlapping genes were identi ed between 153 genes related to tripterygium wilfordii and the 1485 genes related to IMN (Fig. 1, Table 1). To predicate the relationship between the overlapping genes, we uploaded the overlapping genes to the STRING database. A PPI network was built, with 77 nodes and 1009 edges (Fig. 2). The average node degree of was 26.20, and the average local clustering coe cient was 0.69. The result of PPI network indicated that there was a complex relationship between these genes. The results were used for further analysis through Cytoscape software, and the network was constructed as Fig. 3. The top ten targets AKT1, TNF, VEGFA, TP53, PTGS2, CXCL8, MAPK8, STAT3, JUN, and CASP3 have higher degree in this process, which explained their signi cance in the network.
GO Enrichment Analysis of Tripterygium Wilfordii for the Treatment of IMN.
A total of 1782 BP terms, 119 MF, and 25 CC terms were enriched for the 77 target genes. We selected the top 10 according to the P value as shown in Fig. 4. The results showed that tripterygium wilfordii treats IMN through various BPs, including response to lipopolysaccharide, response to molecule of bacterial origin, response to antibiotic, response to oxygen levels, positive regulation of cytokine production, regulation of reactive oxygen species metabolic process, positive regulation of reactive oxygen species metabolic process, response to hypoxia, response to metal ion. In the MF classi cation, the effect of tripterygium wilfordii for the treatment of IMN was mainly manifested in the following aspects: heme binding, tetrapyrrole binding, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, cytokine receptor binding, nuclear receptor activity, steroid hormone which revealed that tripterygium wilfordii has a potential application in other related diseases.
Based on the GO terms, the activity of tripterygium wilfordii was associated with numerous BPs (response to lipopolysaccharide, response to oxygen levels, positive regulation of cytokine production, response to hypoxia, etc.), a variety of MFs (oxidoreductase activity, cytokine receptor binding, nuclear receptor activity, steroid hormone receptor activity, exogenous protein binding, ubiquitin-like protein ligase binding, etc.), and diversi ed CCs (membrane raft, membrane microdomain, membrane region, external side of plasma membrane etc.), which might be basis for tripterygium wilfordii treatment in IMN.
The KEGG enrichment analysis revealed that pharmacological effects of tripterygium wilfordii on IMN were closely related to well-known IMN-associated pathways, such as AGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway. This indicates that tripterygium wilfordii may through multiple pathways to treat IMN. AGE signaling pathway was related to the autoimmune disorder, in ammation, and tissue damage in the MN rats, and AGE inhibition could reduce the in ammatory reactions and oxidative lesions in MN [24]. The IL-17 plays crucial roles in the development of in ammatory autoimmune diseases. Ifuku M demonstrated that IL-17 mRNA were overexpressed in kidney biopsy specimens of MN patients [25]. TNF-αwas demonstrated directly cytotoxic to many glomerular cell types and can promote procoagulant activity with formation of microthrombi that could contribute to renal vein thrombosis associated with MN[26]. Anti-TNF-α therapy attenuated renal immune cell in ltration in experimental MN [27]. Toll-like receptor, as classic example of pattern

Conclusion
In conclusion, the potential molecular mechanism of tripterygium wilfordii in treating IMN has the characteristics of multi-component, multi-target, and multi-pathway. AKT1, TNF, VEGFA, TP63, PTGS2, CXCL8, MAPK8, and STAT3 may be the important and direct targets of tripterygium wilfordii in the treatment of IMN. It may be related to the AGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway. Our study provides a research basis for further studies of tripterygium wilfordii in the treatment of IMN.

Declarations
Ethics approval and consent to participate No applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data are available in the manuscript and the Additional les.

Competing interests
The authors declare no con ict of interest. Authors' contributions recognition receptors, participate in autoimmune disorders. Signals generated by Toll-like receptor are transduced through NF-κB signaling and MAP kinases pathway to recruit pro-in ammatory cytokines and co-stimulatory molecules, which promote in ammatory responses [28]. Many studies have highlighted the importance of Toll-like receptor signaling pathway in the podocyte apoptosis in IMN patients [29][30].
Our study explored the potential molecular mechanism of tripterygium wilfordii in the treatment of IMN from an integrity and systematic perspective, which provided theoretical a basis for further understanding and experimental studies. It is worth nothing that our results can only give hints, and it is necessary to be veri ed by real experimental data.
SHH and WLH: study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; statistical analysis. HYJ, SXL and QJ: acquisition of data; analysis and interpretation of data, and revision of the manuscript and study supervision. WQ, GXJ and GZH: material support; analysis and interpretation of data. The authors have read and approved the nal manuscript. Figure 1 The 77 overlapping genes between the IMN and tripterygium wilfordii.  The PPI network of 77 overlapping genes.  Gene ontology analyses of the therapeutic target genes of tripterygium wilfordii for the treatment of IMN.

Figures
Each bubble represents a KEGG pathway on the vertical axis. The gene ratio is recorded on the horizontal axis. The size of each bubble indicates the number of genes enriched in each KEGG pathway. Larger the bubble is, more number of genes is involved in the pathway. Color of each bubble represents the adjusted P value of each KEGG pathway. More red the color of the bubble is, smaller its adjusted P value is.

Figure 6
The relationship between KEGG pathways and active compounds, targets of tripterygium wilfordii for the treatment of IMN.