Use of Network Pharmacology to Investigate the Mechanism of the Compound Xuanju Capsule in the Treatment of Rheumatoid Arthritis

Hindawi BioMed Research International Volume 2021, Article ID 5568791, 14 pages https://doi.org/10.1155/2021/5568791

Research Article Use of Network Pharmacology to Investigate the Mechanism of the Compound Xuanju Capsule in the Treatment of Rheumatoid Arthritis

Wenyang Wei, Wanpeng Lu, Xiaolong Chen, Yunfeng Yang, and Mengkai Zheng

Academic Research and Development Center of Zhejiang Strong Pharmaceutical Co., Ltd., Hangzhou, 310053 Zhejiang, China

Correspondence should be addressed to Mengkai Zheng; [email protected]

Received 24 February 2021; Accepted 24 July 2021; Published 10 August 2021

Academic Editor: Nadeem Sheikh

Copyright © 2021 Wenyang Wei et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. To clarify the therapeutic mechanisms of compound Xuanju capsule-treated rheumatoid arthritis (RA) based on network pharmacology tactics. Method. The TCMSP, TCMID and STITCH databases were used to screen the active ingredients and targets in the compound Xuanju capsule; the OMIM, TTD, PharmGKB and GeneCards databases were applied to screen the RA-related disease targets. Then, the obtained targets were imported into Cytoscape 3.7.1 software to construct the active ingredient-target network and the RA-related disease-target network. The active ingredient-target PPI network, the RA-related disease-target PPI network and the common target PPI network were built by using the STRING platform and Cytoscape 3.7.1 software. The GO and KEGG analyses of the common targets were analyzed by using the Metascape and Bioinformatics online tools. Results.A total of 51 active ingredients and 513 corresponding ingredient targets were harvested from the compound Xuanju capsule; 641 RA-related disease targets were obtained. After two PPI networks were constructed and merged, 116 RA-related targets of compound Xuanju capsules were identified and analyzed. 116 RA-related targets of compound Xuanju capsules are mainly involved in the biological processes and molecular functions, such as the cytokine-mediated signaling pathways, the response to lipopolysaccharide and the blood vascular development, the cytokine activity, the cytokine receptor binding and the receptor regulator activity. Furthermore, 116 RA-related targets of compound Xuanju capsules are concentrated in signaling pathways such as the IL-17, TNF, Th17 cell differentiation, Toll receptor and RA signaling pathway. Conclusion. The compound Xuanju capsule had the action characteristics of multiple components, multiple targets, and multiple pathways in the treatment of RA, which might primarily reduce the release of proinflammatory factors (such as IL-6 and TNF-α) and increase the production of anti-inflammatory factors (such as IL-10) by regulating inflammation-related signaling pathways (such as IL-17), thereby alleviating the inflammatory damage and improving the bone tissue repair.

1. Introduction mon therapeutic drugs for RA include the nonsteroidal anti- inflammatory drugs, the disease-modifying antirheumatic Rheumatoid arthritis (RA), a common autoimmune dysfunc- drugs, glucocorticoids and biologic agents. While these drugs tion disease characterized by the chronic inflammation of the are taken, there are also the long-term side effects such as joint synovium and the systemic vasculitis, can severely increased risk of infection and potential fatal liver damage impair the physical function and the quality of life [1]. [5]. Traditional Chinese Medicine (TCM) has been used to Worldwide, approximately 1% of the population is affected treat RA for more than three thousands of years in China. by RA, and the RA prevalence in women is about three times Modern pharmacological researches demonstrated that the higher than that in men [2]. The pathogenesis of RA is rela- effective active ingredients in Chinese medicine could tively complicated and related to immune imbalance, genetic weaken the activity of RA and protect joints from deformity factors, environment factors, and so on, in which the partic- [6]. In TCM theory, RA belongs to the category of therapeu- ipation of cytokines plays an important role [3, 4]. The com- tic concept “Bi Zheng ”, which is related to the deficiency of 2 BioMed Research International vital qi as well as the invasion of “wind (feng)”, “cold (han)”, 2. Methods “damp (shi)”, “stasis (yu)” and other evil qi [7, 8]. TCM can regulate the body function as a whole through a multifaceted 2.1. Screening of Active Ingredients of the Compound Xuanju approach and multiple mechanisms [9], therefore, it usually Capsule. The active ingredients of black ants (XJ), Herba Epi- adopts the principles of warming the meridian, dispersing medii (YYH), Fructus Lycii (SCZ) and Fructus Cnidii (GQZ) the cold and relieving the pain in RA treatment. in the whole prescription were collected by the Traditional The compound Xuanju capsule, as a Chinese patent Chinese Medicine Systems Pharmacology (TCMSP) database medicine, is composed of black ants (XJ), Herba Epimedii and analysis platform (http://lsp.nwu.edu.cn/tcmsp.php), (Epimedium brevicornu Maxim., YYH), Fructus Cnidii Traditional Chinese Medicine Integrated Database (TCMID, (Cnidium monnieri (L.) Cuss., SCZ), and Fructus Lycii http://www.http://megabionet.org/tcmid), Natural Products (Lycium barbarum L., GQZ). The black ants, as the primary Database of Traditional Chinese Medicine of Shanghai Insti- drug, can nourish the kidney and liver, invigorate the circu- tute of Organic Chemistry (http://www.chemcpd.csdb.cn) lation of blood and remove blood stasis, drive wind, and and the related literature of the compound Xuanju capsule disperse cold [10]. Herba Epimedii, as the subject drug, [21, 22]. According to the integrative absorption, distribu- can tonify the kidney, strengthen yang, and dispel wind tion, metabolism and excretion (ADME) model, bioavailabil- and dampness [11]. Fructus Cnidii, as the adjuvant drug, ity (OB) and drug-likeness (DL) were regarded as the can warm the kidney, invigorate yang, and dissipate cold screening criteria for active ingredients. The active ingredi- [12]. Fructus Lycii, as the envoy drug, can tonify the liver ents were remained which were satisfactory with criteria of OB ≥ 30% DL ≥ 0:18 and kidney and firm bones [13]. The combined use of four and for further study [23]. drugs has a good therapeutic effect on RA in clinical prac- 2.2. Prediction and Visualization of Active Ingredient- tice by warming and tonifying kidney-yang and removing Potential Targets. To obtain the target of the each active wind and cold [14]. In addition, the compound Xuanju ingredient in the compound Xuanju capsule, the potential capsule could inhibit Th17 cells to secrete IL-17, while the targets were predicted from the TCMSP and STITCH data- secretion of Th17 and IL-17 played an important role in bases (https://string-db.org). And then, the targets corre- the pathogenesis of RA [15]. Furthermore, earlier sponding to the active ingredients screened from the two researches have shown that the compound Xuanju capsule target databases were standardized in the UniProt database had the immunomodulatory and anti-inflammatory effects (http://www.uniprot.org), with the properties set to [16]. It had a significant inhibitory effect on the phagocytic “reviewed” and “human” [24]. Subsequently, these standard- function of the reticuloendothelial system and the delayed ized targets were merged and the duplicated items were allergic reaction in mice and significantly inhibited the removed. Finally, an active ingredient-target network was increase in the amount of abdominal exudate and the num- constructed using Cytoscape 3.7.1 software, and the network ber of white blood cells induced by carboxymethyl cellulose topology analysis was carried out. The degree, a topological stimulation. At the same time, it could also inhibit the for- parameter, was applied to screen the kernel active ingredients mation of cotton ball granulation tissue. Modern pharma- and the corresponding targets of the compound Xuanju cological studies have also found that black ants contained capsule. rich formic acid and a variety of trace elements, had obvi- fl ff ous anti-in ammatory e ect, could reduce the tissue dam- 2.3. Prediction and Visualization of RA-Related Disease age, and eliminate free radicals to promote bone Targets. With “Rheumatoid arthritis” to be the keyword, formation [17]; icariin, the core component of Epimedii, RA-related targets were retrieved from three public online fl could inhibit the in ammatory response and the synthesis databases, namely, the Therapeutic Target Database (TTD, fl fi of in ammatory substances and enhance the nonspeci c http://bidd.nus.edu.sg/group/cjttd), the PharmGKB database immune function and cellular immunity [18]; osthole, a (https://www.Pharmgkb.org), the Online Mendelian Inheri- bioactive coumarin compound, was mainly contained in tance in Man (OMIM) database (http://www.omim.org) fi Fructus Cnidii and could signi cantly inhibit the release and the GeneCards database (https://www.genecards.org) fl α fl of in ammatory cytokines TNF- and IL-6 and the in am- [25, 26]. These targets were also sent to the UniProt database matory mediator NO [19]. However, the pharmacodynamic for normalization, and then, the duplicate targets were basis and more molecular mechanism of compound Xuanju removed. Eventually, the RA-related disease target network capsule-treated RA are still unknown. was built by using Cytoscape 3.7.1 software as well. The network pharmacology method of TCM based on the analysis of network big data can effectively uncover the 2.4. Construction of the Protein-Protein Interaction (PPI) potential compatible action mechanism of drugs, thus pro- Network and Screening of the Common Targets. The active viding a powerful tool for accelerating the modernization of ingredient targets of the compound Xuanju capsule and the TCM research and development [20]. In the present study, RA-related disease targets were analyzed using the STRING the method of TCM network pharmacology was adopted to database, and “Homo sapiens” was limited. Two PPI net- further analyze the potential active ingredient, related targets works of the active ingredient targets of the compound and molecular signaling pathways of compound Xuanju Xuanju capsule and the RA-related disease targets were built capsule-related RA, with the aim of providing more theoret- and merged through Cytoscape 3.7.1 software. Afterward, ical basis for the effectiveness of compound Xuanju capsule- the common network (the intersection part of two PPI net- treated RA. works) was further analyzed by using Cytoscape 3.7.1 BioMed Research International 3

Table 1: 51 active ingredients and the number of the corresponding potential targets of the compound Xuanju capsule.

Chemical component Target number OB (%) DL Tocopherol 10 32.29 0.70 Citral 8 22.52 0.52 Formic acid 236 33.26 0.00 Beta-sitosterol 7 15.00 0.81 24-Epicampesterol 2 37.58 0.71 Linoleyl acetate 3 42.10 0.20 Sitosterol 11 36.91 0.75 DFV 10 32.76 0.18 Chryseriol 14 35.85 0.27 8-Isopentenyl-kaempferol 12 38.04 0.39 Kaempferol 65 41.88 0.24 Anhydroicaritin 28 45.41 0.44 Yinyanghuo A 4 56.96 0.77 Yinyanghuo C 5 45.67 0.50 Yinyanghuo E 4 51.63 0.55 8-(3-Methylbut-2-enyl)-2-phenyl-chromone 24 48.54 0.25 Icariin 9 41.58 0.61 Icariside A7 1 31.91 0.86 Luteolin 52 36.16 0.25 Magnograndiolide 4 63.71 0.19 Quercetin 153 46.43 0.28 Poriferast-5-en-3beta-ol 7 36.91 0.75 Ammidin 8 34.55 0.22 Diosmetin 10 31.14 0.27 Xanthoxylin N 8 35.51 0.21 Prangenidin 6 36.31 0.22 Ar-curcumene 2 52.34 0.65 Cnidimol B 1 68.66 0.26 Cnidimol F 3 54.43 0.28 Cniforin A 5 55.89 0.47 Cniforin B 1 36.70 0.60 O-Acetylcolumbianetin 11 60.04 0.26 Isogosferol 1 30.07 0.25 o-Isovalerylcolum bianetin 6 64.03 0.36 Stigmasterol 34 43.83 0.76 Sitosterol alpha1 7 43.28 0.78 Cycloartenol 2 38.69 0.78 Mandenol 2 42.00 0.19 Atropine 18 45.97 0.19 Campesterol 12 37.58 0.71 24-Methylidenelophenol 1 44.19 0.75 Daucosterol 16 36.91 0.75 Glycitein 23 50.48 0.24 CLR 9 37.87 0.68 Fucosterol 7 43.78 0.76 Lophenol 6 38.13 0.71 6-Fluoroindole-7-dehydrocholesterol 2 43.73 0.72 7-O-Methylluteolin-6-C-beta-glucoside 3 40.77 0.30 4 BioMed Research International

Table 1: Continued.

Chemical component Target number OB (%) DL Cycloeucalenol 1 39.73 0.79 Lanost-8-en-3beta-ol 4 34.23 0.74 Obtusifoliol 2 42.55 0.76

Figure 1: The active ingredient-target network of the compound Xuanju capsule. The blue diamonds depict the targets; yellow rounds delineate the herbs; green, orange, pink, and red rounds delineate XJ’s, YYH’s, SCZ’s, and GQZ’s active ingredients, respectively; and other rounds portray the common ingredients. software, and the common targets were obtained. Addition- .com.cn). The ClueGO plug-in in Cytoscape 3.7.1 software ally, we further screened targets whose degree value ranked was applied for constructing the visual results of the target in the top 20 as the core targets of compound Xuanju proteins involved in signal pathways. capsule-related RA. 3. Results 2.5. Enrichment Analysis of the Common Targets. The Gene Ontology (GO) [27] biology process annotation and the 3.1. Collection of Active Ingredients of the Compound Xuanju Kyoto Encyclopedia of Genes and Genomes (KEGG) [28] Capsule. In the present study, we searched all the reported pathway enrichment analysis of the common targets were chemical components in the compound Xuanju capsule carried out through the Metascape online database (http:// through TCMSP, TCMID, and Natural Products Database metascape.org), which got the potential signal pathways, of Traditional Chinese Medicine of Shanghai Institute of related biological processes, cell components, and molecular Organic Chemistry. A total of 478 ingredients were collected, functions of the active components in the compound Xuanju among which 46 was found in black ants, 130 in Epimedium, capsule. The results of GO enrichment analysis and the bub- 114 in Fructus Cnidii, and 188 in wolfberry. According to the ble diagram of KEGG pathway enrichment were drawn using screening criteria of the ADME, including the OB and DL, 87 the Bioinformatics online tools (http://www.bioinformatics active ingredients of the compound Xuanju capsule were BioMed Research International 5

Table 2: The top 10 active ingredients and the corresponding target degree values of the compound Xuanju capsule.

Chemical component Degree Component target Degree Formic acid 237 PTGS2 12 Quercetin 155 PTGS1 10 Kaempferol 66 PRKACA 10 Luteolin 53 GABRA1 9 Stigmasterol 36 RXRA 8 Anhydroicaritin 29 AR 8 8-(3-Methylbut-2-enyl)-2-phenyl-chromone 25 PRSS1 8 Glycitein 24 SCN5A 8 Atropine 19 NCOA2 8 Daucosterol 17 ESR1 8

COL4A1 CD34 TACR1 THY1 PDE4B CXCR4 C5AR1 MAPK10DKK1CELA1 CXCL14PLOD2 CASP3 ESR2 IL11COL9A3 SLC9A2 ALOX5NLRP12 IGFBP1 CIITA HFE CXCR2 FASLGIL1RL1 DCN EBP ITGB7 MMP20MTHFRSAA1 BMP6 CXCR3 CP CHST4 FGF2 NFKBIAMATN1 IFNB1TNFRSF13B ICAM1TYROBP FCGR2B HSPD1 F5 CILP NOS2 TYR P2RX7 PRTN3CCL2 BCL2L1 TAC1 HTR2A GSTT1 CD40 NOD2 ESR1IGFBP2 SERPINC1 KLRK1 IL12B SIRT1 IL7R TAPBP HAS2 VDR BMP4 ADAMTS4 NFKB1 GPSM3 CCL4 MMP10 CPOX SAA4 ANXA1 SLC6A14 PSTPIP1TSPO CTLA4 IL1RN PLAU IL1F10 IL16 ADAMTS3 DDX25 NGFCALCA ALB FSTL1 LIF TNIP1 TNC TNFSF12 OSM PLA2G2DC1S TGFBR2MICAAKR1B1 VIM CD19 CREB1 HLA-DPB1 TGFB1FGFR1 SMAD2IL23A PTHLH FLT4 FAP CAMK2GFCER2 FTL GRP NFKBIL1 LEPR PLA2G5 ADAMTS1 CHI3L2 MUC5AC CCR6 LACC1 IL15 PNOC LTBRLRP5 ID2 IER3 GALNS COL11A1 GZMB TNFAIP6 PTH1R HIP1 CRP IL22 SERPINE1 CDH11 TNXB PIK3R2 HGD CAV1 CXCL11TNFRSF11B CAPN2 CRAT IL7 TRAF6 LDLRLIMK1 POMCTDRD6 P4HTM SOX5 FCGR2APDIA3 BBC3 CFP MMP12 TLR9 SLC19A1XDH CTHRC1CD86 IL2RA HOXD9 BDKRB1 COL9A1 HRH2 CR2 CHAT SOCS3 TAT GPI CCL27 OSCAR LTA TRPV2 ITIH2 PDGFRB DPEP1 LTFPDE4A CD80 PADI4 JAM3 HIF1A TREM2 CYLD SELE CLTC VEGFD ADAMTS9 PTPRJ IL6ST ITGB2 SAAL1 VEGFC MAFB TRPV1 CCL18 HMGB1 SLPI STAT4EPRS1 DNASE1L3 IDO1 IL17R FMOD GLA SLC22A4 VSIR PLA2G2A FOS CD163 SOX9 NFKB2 CSF1RADAMTS13 MME CD244 NLRP1 CCL5 ACTG1 SPP1 ICAM2 HGF PTGER1 IFNG SQSTM1 THBS1 ACKR3 ITGA9 RIPK3 ADAM17 JAK1 IL12A HAPLN1 MMP8 IRF5 TYMS CCR5 TCN2 PDYN SST TMSB4X JUN BMPR1A COL1A1 CCR2 HLA-DRB1 TLR4 MUC1 MYC VCAM1 IBSP MMP19 HAS3 SLC1A1 CASP4 TREX1 GAL IL3 APOB TDRD1 NR3C1 ABCG2 MS4A1 PDGFRA IL2 GC FPR1 TNFSF11 TF UGDH NPY COL4A2 FAM122B IGFBP3F8PSMB8 HTRA2 ELN CSF3 LTB4R CXCL16 GPX4 MYCN TIMP3 F2RL1 AP2A1 MMP11 HP IL18TIE1 MATN3 CNR1 CD81 TNFRSF11A CYP3A4 MAPKAPK5 JAK2 HEXD SERPINF2 IRAK1 MTOR CYBB BMP7 HSPA5 CASP1 TYRO3 ASAH1 HRH4DHODH RPS6KA3 G6PD CSF2RA C4BPA GGT1CD40LG SELL ITGB1 KIT EGR1 CAT RA VTN RNGTT CXCL5 RELA CD58 LMNB2 CEMIPMC3R ATF2 A2M CRHBP UGT1A1 CCR1 TGFA CTSH STK16 EDNRACTSL HSPE1 FOXP3 BAX HTRA1 CASC3 GDF5 MAPK3 CCN5 PRKCZ ABCG5 OPRK1 MAEL SUCNR1 PNPIL36RN MVK PTPN22 TNFSF10 SFTPD PTGS1TINAGL1 LCE3B ANGPT2 HDAC6 ESRRA FUT1 IL33 SLC36A2 IL1RAPL2 IL17RA ANG CRH PPARG ITGA4 F10 CHI3L1 RIPK1 CEBPDNMBR IL6 RABEP2 IL1B TNFRSF13CMMP2 PTGIRATF1 GSTM1 GCH1 TIMP1 SUMO1RUNX2CYP24A1 VCP SOD1 C3 RARS1 BRD2 MMP1 ENO1 MEFV ASPN GAPDH NGFR CD247 F2RL2 FN1 CTSG LMNA TAGAP FLT1 NAMPT PTGES IL13 CYBA KDR PTGS2 MMP9 CXCR1 IL4R TNFSF13 MT2A PIK3CG CTSK PDE4D GRN EGLN1 ADAMTS7 IL5 JAK3 SLC22A5 SCGB1A1 ICAM3 TXNRD1 ELANE MIF TNFRSF18 COL11A2 TIMP4 SPARC ALOX12 BMP2 PLA2G4ACSF2 MUC17 GTF2I IL4 VEGFA ANGPT1 F3 IL1RAP NTAN1 HBEGF MMP3 ROCK2 TINAG TEK HLA-DRB4 HMGCR CD36 CHRNA7HLA-B TIMP2 AMBP PTGER3 CAST CCL3 EGF TRPS1 CD44 STAT3 IGHE IL1R2 MPO DUSP19 MMP7 ACE CD4 DHFR PCSK9 DPP4 TRPV4 HLA-DPA1 PRG4 SELP PARP1 VIP COMP LEP FPR2 MAPK14 PLAURSLC9A1 NFE2L2 AKT1 NAA80CTNNB1COX5A CXCL2 MAPK12 IL18BP TDRD7STING1MCL1EFNB1 EDN1OPRL1 CXCL12 CD14 CYP7B1 PTPN2 IL17A TGFB3 KITLGARG2BMP5PIWIL2 CTSB TNFRSF1BCYP27B1 FASMMP14SLC22A2 GSTP1 MUC3AFCGR3A BGLAPCDH5PPARACEBPB CCN6 ADAMTSL1 ERAP2CXCL10 MAP3K1 MYCL MAPK8 PTK2 ACP5 S1PR1 TRAPPC2 ANKH STAT1SLC11A1 CXCL1 KLF2IFNGR1 PTGER2TNFTGFB2 PROCRCD209 IFNA2 CD79A PLA2G1B CARD14 MAP2K7 MUC3B COPAIL18RAP GGH ADORA3 LPIN2 BCL6 SYK COL9A2NOTCH2 MUC2 COL3A1 FOSL1PSMB9 SLC2A1 SLC9A3 HLA-CS100A12PLCG2 CFIROM1 GPBAR1 MAZ HMOX1 COL2A1 SCNN1B PRKCD IL2RBSGPL1 BCL2L11 FST CFLAR NFATC1 PTH PIK3R1 ADAMTS12MUC7 PDGFB ERAP1 ADAMTS5 C4A TRAF3IP2 ANXA2 IGF1 ANKRD55 IL1R1 ERBB2 TFRC CXCL8 HAS1 STEAP4 NLRP3IL6R TLR2 CTSS LCE3C C5 TNFRSF1ATP53 CCN2 ACAN PLA2G2F CSF1 ANGPTL4 IL10 ICOS SERPINF1 IL1AOPRD1 CNMD IL23R MMP13MFGE8 FGFR3 PDPNMDM2 SOD3 FGF1TNFSF13B MAPK1 PIWIL1

Figure 2: The RA-related disease-target network. The red round represents the disease (RA), and the blue rounds delineate targets related to RA. 6 BioMed Research International

UGT3A1

LCMT1 PCOLCE SYT7 PLOD1

RAB9A ACPP PDXP

NFKBIA HPN NUF2 P4HA2 TRAPPC4 IKBKB SOD1 CASK C8G IRF1 FGFR3 PKIA COL3A1 PIM1 MMP9 HAS2 SLPI PYCR2 PPARD CHEK1 ICAM1 NKX3-1 LTF HIF1A MYL6B ASNA1 BCL2L1 KDR FKBP1A PAFAH1B3 PHOSPHO1 GAST PAEP PPP3CA EIF2AK3 RASSF1ARF1 MRI1 ELK1 CHUK XIAP E2F2 SP7 PARP1 CHEK2 BAK1 MCL1 CD40LG BIRC5 PNPLA2 CTLA4 PSMB3 KRAS PLA2G2A PSMB6 CTSB ALOX5 COL1A1 PRDX2 TOP2A CALM1 TGFB1 CTSL IL1A NR3C2 CDK2 BAX MDM2 CDKN1A CCNA2 CASP9 ERBB2 IL2 CCNB1CDKN2A PRKAB1 CASP8 FOXP3 PGR HRH1 NOS3 SLC6A3 SCN5A PTGS1 CDK4 RRM1 ERBB3 ADRB2 PTER RPS6KA3 CDC25BTYR NCF1 RHOB E2F1 PDE3A DUOX2 PRKCBRUNX2 HSP90AA1 FFAR2 IGFBP3 LTA4H IL4 GLO1 MMP2 CCND1 PNP LYZ HSPB1 PTEN PTGES JUN BCL2 CHRM3 MMP1 CAV1 PCNA CASP7 MAPK14 MET AMER1 CPA1 FOS CTNNB1 GRIN2A CMPK1 F13A1 CXCL10 ATP5A1 RB1 SERPINE1 DPP4 PNMT PLA2G2ETRPA1 HSF1 PIK3CGFABP4 DHODH FFAR1 GM2A GJA1 DNMT3A ADRA1B UCP3MMP13 EGFR VEGFA EGF RXRB NOS2 MYC F7 IGF2 UCK2 ADRA2A UCP2 SRC ALAS1 PSMA6 NT5M PRKACA TPI1 IFNG HSPA5 MAPK3 PLAU PDE11A ADRA1D ANXA1 SMAD2 CRP HMOX1MPO HPRT1 TOP1 ADRA1AOPRM1 TP53 FOLH1 GLB1 IL6 AKT1 CDK1 UCP1 CASP3 MTAP CHRM2 CASP1 CXCL8SP1 LAP3 PSMD3 PLA2G1B MAPK1 IL10 MTHFD2 ADRA2B APP TNF HCK CHRM5 GAPDH ALDH4A1 DRD1 SPP1 ESR1 GSK3B DRD2 MAPK8 NFE2L2 AKR1B1PRSS3INSR PPP1CC DCXR LAS1L CHRM1 TAC1 ARF6IL1B CKB CHRNA7 GCG NR3C1 GART CHRM4 MAOA GSTM4 GSR STAT1 ODC1 KCNH2 PYCR1 AFG3L2SEC14L2 ADRB1 HTR1A PSMA1 DUSP23 CXCL2 F3 GOT2 GSTM1 RXRA VCAM1 NR1H2 HTR2A HK2 ESR2 CDO1 GPHN CCK ADRA2C ACHE SERPINA1 APOA1 PON1 OTC GABRA1 AR ALDOA NPEPPS CXCL11 APOB NQO1 PKM GLRA2 OPRD1 BCHE NDUFS1 DECR1 GSTA1 MAOB RAF1 SELE RASA1 GNPDA1 SLC6A4 GRIA2 CAT MPI ABCC1 SEC14L3 GLRA1 POR GLUL PTPN1 APOE NCOA2 NDUFS3 PPIH ARG1 AHSA1 OAT CLDN4 MMP3 NTHL1 EIF6 PPARA PRSS1 CYP2B6 SCARB1 SHMT1PSAT1 ADSS GSTA3 NCOA1 HAL GNMT PTGS2 ADSSL1 SLC6A2 HTR2C GSTA2 GPT PCPYGL AHCY PLAT NR1H3 AMD1 BHMT MMACHC F10 GSTM2 PPIA GCLM RELA ADCY2 MDH2 NDUFS8ASS1 PNLIP THBD CYP1A1 LDHB CTRB1 GLRA3 SEC14L4 GABRA6 NR1I2 CAD GPI SPRGLUD1 KCNMA1 NR1I3 ABCG1 PFAS PGLS CKM GAMT AMY2A GRIA1 ADH7 GOT1 MMP8 DAO ENSG00000160200 NOS1 ADH1A UGT1A8 HTR1B CTH ABAT CA3 ACACA MTHFD1 GABRA5 CCL2 ALAS2 ABCG5 PPARG CIAO1 TF ME2 MTR LDHA GSTA5 CYP1A2 CYP7A1 AKR1C2 RBKS PDE5A CTSD AGXT PTGER3 GABRA3 GSTP1 SDHA CYP11A1 RUNX1T1 GPT2 CELA1 ALPP LCAT PDF GABRA2 PDHB GSTZ1 CA2 SLC2A2 BHMT2 GALK1 MMP12 ADH4ALDH9A1 CETP CYP51A1 SREBF2 COX5A HMGCR TST EHHADH ASL MGAM PPCDC MSMO1 GUSB NFS1 CYP2C8 SHMT2 GLYATL1 ADH1B CSN1S1 UROD SLCO2B1 MAT2B RTCA PHYKPL AHR ABCG8 HSD17B2 CBR1 CYP7B1 PIPOX GATM DHCR24 ABCB11 UGT1A9 KYNU PYGM SLCO1B3 HSD3B2 ALDH1B1 GCAT LPL SREBF1 SLC2A4 XDH ADH1C HSD17B8 NSDHL AZIN1 HAO1 ALDH5A1 GALE CYP39A1 CES2 GALT UROC1 CPT1A SLCO1B1 ACAT2 CYP1B1 ATP5B GNAT1 HIBCH UGT1A3 EBP CA1 ALDH2 SULT1E1 AKR1C3 UGT1A7 ME1 SLC25A10 ABCA1 HSD3B1 FAXDC2 DIO1 CCBL1 CYP3A4 CYP8B1 CMBL ACADSB ABCG2 ME3

METAP2

HSD17B7

Figure 3: The PPI network of the active ingredient targets that consists of 500 nodes and 9488 edges. The yellow rounds represent the protein targets acted by the active ingredient, and the black lines denote the interaction relationship between the protein targets. obtained after duplicated targets were removed. In addition, gets had higher degree values. The top 10 active ingredients three antirheumatic active ingredients of Xuanju were found and the corresponding targets are shown in Table 2. from literatures, namely, citral, formic acid, and β-sitosterol [29]. Including all the above active ingredients, a total of 90 3.3. RA-Related Disease Targets and the Visualized Network. active ingredients of the compound Xuanju capsule were Searching for RA-related targets through TTD, PharmGKB, obtained. OMIM and GeneCards databases, when “Homo sapiens” targets were remained and the duplicated targets were removed, 3.2. Active Ingredient-Potential Targets and the Visualized a total of 641 targets were obtained. The above targets were Network. STITCH and TCMSP databases were used to pre- submitted in the Cytoscape 3.7.1 software to construct the dict the targets of the active ingredients. After the “Homo RA-related disease-target network (Figure 2). sapiens” targets were remained and the duplicated targets were removed, 51 active ingredients and 513 corresponding 3.4. PPI Network Topology Analysis. The STRING online action targets were screened, as shown in Table 1. The above database was used for PPI network topology analysis. The targets were imported into Cytoscape 3.7.1 software to con- analyzed PPI relationship data results (.tsv format ﬁle) were struct the active ingredient-target network diagram imported into the Cytoscape 3.7.1 software, and then, the (Figure 1). The network contained 570 important nodes visualized PPI network of the active ingredient targets and 936 edges. Among them, the formic acid, quercetin, (Figure 3) and the PPI network of RA-related disease targets kaempferol, luteolin, and stigmasterol had higher degree (Figure 4) were obtained. Among them, the PPI network of values, PTGS2, PTGS1, PRKACA, GABRA1 and RXRA tar- the active ingredient targets involved 500 nodes (interaction BioMed Research International 7

MUC17

GALNT12 FUT1

MUC3A CHST4 WISP3 COPA

MUC7 MS4A1 CXCL14 PIK3R2 JAM3 HLA-DRB1 CRHBP ICAM2 MAPK10 TYRO3 IL33 NTAN1 IL7R KDR TNFSF13 TRPV2 CARD14 IL17A P2RX7 MEFV F2RL1 TRAPPC2 OPRD1 ATF2PTGES HIP1 MAP3K1 SFTPDID2 TACR1 SGPL1 FPR2HRH4 PTGER1 PSTPIP1 FCER2 CXCL16 NOTCH2LTB4RPTGER3 GRP EGR1 SCGB1A1 CD58 CD81 C5AR1 IL12A FCGR2B CR2 AP2A1LDLR CD247TNFRSF18FCGR3A IL2RB CD163 IL1RAPL2TAGAP SELL ACP5 ICAM3 EFNB1 MAZ CD244 TNFRSF11ACXCR1 BDKRB1JAK3 ACKR3 MMP11 PROCR KLRK1ASAH1IL4R SLC11A1CCR5IL36RN SUCNR1 PTGER2 PADI4 TNFRSF13B TYROBP MUC5ACS100A12IL22CXCR3MME GPSM3C1S LPIN2 ICOS CCL3CCL18 PNOC CDH11 ANGPT1 CD79ATNFSF12CD40LG F2RL2 MMP19 IRF5 FLT4 S1PR1 C5MMP10BMPR1A GPBAR1 BCL6PLA2G2A IL23RCD86CXCL11PDPN IL3 IL11SELPTEK ITGA9 TNXB CD209HMGB1 CD19ITGB2TFRCCCR1 VIMADAMTS1PLAUR EDNRA LACC1 IL1RAPIL18BP CD36 PDGFRA CDH5CRH PTGIR RIPK3 CD14 SYK CXCL10CD34CSF1RCXCR2 FPR1 ANKRD55 MAFB FAS ENSG00000196689 BMP7 PTPRJDKK1 IL2RA KITLG IL13IL5OSCARPDYNCTSSFASLG F3 C4A PTPN22 EDN1MMP13 TIE1 HRH2 IFNG TNFSF13B MUC1 ICAM1PIK3R1VCAM1SELE ADAM17 ADORA3AMBP CELA1 IL18RAP IL15IL18NFKB1 JAK2PRKCZIL1RNGSTM1IL1F10SST CNR1 GGT1 WISP2HAS1 LCE3B CCL2 IL1R1 IL1R2CD80 RUNX2 ANXA1 ALOX12 IL17RAGPR29CXCL5 CCR2 FGFR3 PLA2G1B CFI DPEP1 PIK3CG CIITA IFNB1 CXCL1 IL1BCTSKJAK1 ITGB7 PRG4 HLA-BHLA-CSTAT4 CSF2RAFOXP3CLTCSTAT3 CXCR4CCL4 NPYCEBPBCD44 ITGB1FGFR1BGLAP COL9A1 BMP5 TNIP1 THY1 CXCL12IFNA2CSF1MMP1 DPP4ACAN BMP6 GDF5 TLR4 NLRP3 IL10IL6 LTA IGF1 ITGA4SMAD2PTHLH MICA TNFSF10FCGR2AIDO1 CCL27 TNF ELN ASPN ADAMTS13 F10 LCE3C SPP1 MT2A IL6R IL7FSTL1 CCL5CXCL8 TP53 CRP COL1A1ANGPTL4 PTPN2 TLR2 CSF2IL2 PTGS2 PTHADAMTS5HGFFGF2 SERPINF2COMPPTH1R CTLA4 NFATC1POMCIL4 AKT1MMP9 IL1A TIMP1 GAL CD40 TRAF6CYBB OSMSTAT1VEGFAPLAUOPRL1CSF3 VTN COX5AHAS2 NLRP12 PRTN3 MPO EGF HTR2APTK2 SLC9A3 TNFRSF1B TREM2IFNGR1 CASP1NFKB2 ALBCFP CREB1 FST BMP2 C10orf54 IL12BPSMB8 NOD2NR3C1 TNFRSF1A SIRT1KIT MMP2LEPFIGF VEGFCTAC1SPARC TGFA MAPK8TNFSF11 HFECTGF FMOD KLF2 IL6ST SUMO1 C3 OPRK1 FOSL1CEBPD NFKBIAMDM2RELACD4IGFBP1MYCMAPK1 FN1 A2M TNC COL9A2 NGFRTRAF3IP2 IER3 IRAK1 MCL1NOS2 TNFRSF11B TGFB2SOX5RPS6KA3HBEGF NFKBIL1 ADAMTS4 CYLD IL23A TLR9 SAA1ELANE ACTG1PDGFBSOX9LECT1 NMBR CYP7B1 GZMB CTNNB1MMP3LRP5PPARGCALCAPTGS1 DCN MATN3 MC3R PLOD2 CTSH ANG GAPDH HLA-DPB1 FLT1 SERPINC1ITIH2 CILP TINAG ROM1 IL16 IL1RL1 HMOX1 MIF CAV1 THBS1 COL2A1 IBSP HAS3 JUN ERAP1 NLRP1 CFLARPRKCD TIMP2MAPK3 GTF2I SOCS3 HIF1A LIF CP COL3A1COL11A1 RNGTT GPX4 HLA-DPA1MMP14MMP7MAP2K7 VIPADAMTS9 TIMP4ADAMTS3 MVK SQSTM1 PPARABCL2L11ESR1 PLA2G4AANGPT2TIMP3 GC COL9A3 FOS CASP4 HDAC6 SOD1 MTOR CXCL2F5 CTSG CTSL SAA4 BBC3 ALOX5 LEPR PCSK9CYBATGFBR2 HAPLN1 SLC22A4 PLCG2 ERAP2 AKR1B1GSTP1 VDRHP ACE COL4A2 ANXA2 MAPK12 TNFAIP6TGFB3 CTHRC1 TYR DUSP19 CYP24A1 LTBR RIPK1 MMP20 COL4A1 TAPBP NAMPT SLPI ATF1 GGHNFE2L2 LMNA FGF1 CASC3 BCL2L1 XDH ARG2 HSPA5 MMP12SLC2A1 FAP F8 APOB BMP4 TRPS1EGLN1 CTSB IGFBP3C4BPA ROCK2 COL11A2 CASP3 PDIA3HMGCR ABCG2 CHI3L1 TSPO PARP1 GRN MYCN ESR2CAMK2G HTRA2CYP3A4 SOD3 CAPN2 SERPINF1 MAPKAPK5BRD2 HTRA1 GLA PDGFRB PSMB9 SLC22A5 BAX PLA2G2D LTF MFGE8 ADAMTS12 MATN1 DHFR ENO1 VCP CHAT PDE4A SLC9A1TF STEAP4 IGFBP2 CRAT TMSB4X PIWIL1 PIWIL2 ADAMTS7 ABCG5 HSPD1HSPE1CYP27B1TXNRD1 MTHFR UGDH ERBB2 ADAMTSL1SERPINE1 SLC6A14 TGFB1 G6PD GALNSFTLESRRA CHRNA7 CHI3L2 CAT TYMSCAST PNP LMNB2 MAPK14 CEMIP PDE4B PLA2G2F DHODH MYCL TRPV4UGT1A1 TDRD1 NGF TAT LIMK1 GPI SLC36A2 GCH1PDE4DHGD SAAL1 TDRD6 MMP8 TREX1 TNFRSF13CPLA2G5 TDRD7 DNASE1L3 SLC19A1 CPOX TCN2 DDX25 EBP SLC1A1 MAEL P4HTM ANKH

SLC22A2

HOXD9

Figure 4: The PPI network of RA-related disease targets that consists of 625 nodes and 19389 edges. The red nodes represent the protein targets worked on the RA, and the black lines denote the interaction relationship between the protein targets. proteins) and 9488 edges (interaction relationships); the PPI 3.6. GO Enrichment Analysis. GO enrichment analysis network of RA-related disease targets involved a total of 625 includes molecular function (GO-MF), biological process nodes (interaction proteins) and 19389 edges (interaction (GO-BP), and cellular component (GO-CC). The top 10 relationships). GO analysis results were selected according to the degree of importance and signiﬁcance, as shown in Figure 6. The 3.5. Screening of the Common Targets. In the Cytoscape 3.7.1 GO-MF analysis indicated that the core targets of compound software, the CytoNCA plug-in was applied to merge two PPI Xuanju capsule-related RA mainly aﬀected the cytokine networks and obtain a visualized intersection target network activity, the receptor regulator activity, the receptor ligand (Figure 5), which consisted of 116 common nodes and 2241 activity, cytokine receptor binding, phosphatase binding, edges. As shown in Figure 5, these key targets were highly transcription factor binding, and other molecular functions correlated in the potential target network relationship of in the treatment of RA. The GO-BP analysis showed that compound Xuanju capsule-treated RA, including IL-6, the core targets of compound Xuanju capsule-related RA VEGFA, TNF, AKT1, MAPK3, CXCL8, IL10, IL1β, JUN could be involved in a majority of biological processes in and CASP3. The 116 common targets were ranked according the treatment of RA, such as the cytokine-mediated signaling to the degree value, and the top 20 core targets of compound pathways, the response to lipopolysaccharide, the response to Xuanju capsule-related RA are shown in Table 3. molecule of bacterial origin, the response to bacterium, the 8 BioMed Research International

PIK3CG HAS2 COX5A GSTM1

FGFR3 HTR2A F10 MMP12 COL3A1 ESR2 TAC1 IL1A OPRD1 PTGER3 FOS MMP13 CXCL8 MMP1 CXCL11 JUN RUNX2 MPO CTSB ANXA1 CAV1 SPP1 RPS6KA3 FOXP3 SOD1 SELE MMP3 TNF NFE2L2 RELA CAT MAPK8 TP53 CHRNA7 VCAM1 IFNG NFKBIA AKR1B1 VEGFA BAX CD40LG MAPK3 MMP9 PARP1 MAPK1 CTLA4 PLAU PTGS2 CTNNB1 ERBB2 PNP COL1A1 STAT1 IL1B CRP MMP8 CCL2 AKT1 CXCL2 IL4 SMAD2 IL10 CXCL10 MDM2 ICAM1 EGF ABCG2 XDH IL6 HIF1A MYC NR3C1 IL2 PTGES CASP1 BCL2L1 SERPINE1 CASP3 TYR MAPK14 ESR1 GAPDH MMP2 CTSL HSPA5 PLA2G2A PPARG TGFB1 KDR HMOX1 IGFBP3 ALOX5 DPP4 PPARA LTF PTGS1 GSTP1 MCL1 APOB CYP3A4 NOS2 PLA2G1B GPI TF SLPI F3

CELA1

EBP HMGCR DHODH ABCG5 CYP7B1

Figure 5: The common target PPI network that consists of 116 nodes and 2241 edges. The 116 rounds represent the potential protein targets of compound Xuanju capsules against RA, and the black lines denote the interaction relationship between the protein targets. The color and size of the round indicate the size of the degree value; the degree value corresponds to the round color from yellow to purple. cellular response to lipid, the positive regulation of cell motil- ClueGO plug-in in Cytoscape 3.7.1 software, the visualiza- ity, the positive regulation of cellular component movement, tion results of the signal pathway of the target proteins the positive regulation of cell migration, the blood vessel involved were constructed. It was found that the efficacy of development, and the positive regulation of locomotion. the compound Xuanju capsule was mainly achieved by regu- The GO-CC analysis indicated that the core targets of com- lating the IL-17 signaling pathway, the TNF signaling path- pound Xuanju capsule-related RA were primarily enriched way, the Th17 cell differentiation pathway, the Toll receptor in the membrane rafts, the membrane microdomain, the signaling pathway, and so on, as shown in Figure 8. cytoplasmic vesicle lumen, the secretory granule lumen, the extracellular matrix and so on. 4. Discussion 3.7. KEGG Pathway Analysis. The KEGG pathway analysis Network pharmacology is a research method to clarify the was carried out to further explore which pathways were disease occurrence and development from the perspectives related to the screened common targets. Among the top 20 of systems biology and biological networks, through the col- pathways (sorted by the degree value), there were mainly lection of ingredients and action targets; an interaction net- the IL-17 signaling pathway, the TNF signaling pathway, work is constructed to further investigate the regulation the Th17 cell differentiation pathway, the Toll receptor sig- effect of TCM on different signals, thereby revealing the naling pathway, the RA pathway, the fluid shear stress and action mechanism of TCM [30]. Based on the network phar- atherosclerosis pathway, and so on (Figure 7). Using the macology tactics, this study deeply explored the potential BioMed Research International 9

Table 3: The information of top 20 common targets.

UniProt Betweenness Closeness Neighborhood Gene Protein Degree ID centrality centrality connectivity IL6 Interleukin-6 P05231 93 667.9930402 0.833333333 89.12839505 Glyceraldehyde-3-phosphate GAPDH P04406 91 719.0841193 0.815602837 85.92631575 dehydrogenase VEGFA Vascular endothelial growth factor A P15692 89 413.5753955 0.793103448 84.52425102 TNF Tumor necrosis factor P01375 89 335.4934403 0.798611111 84.68661002 RAC-alpha serine/threonine-protein AKT1 P31749 87 524.2608782 0.798611111 80.85548315 kinase MAPK3 Mitogen-activated protein kinase 3 P27361 84 410.4623204 0.777027027 76.40073952 TP53 Cellular tumor antigen p53 P04637 81 307.2138965 0.746753247 73.70696197 CXCL8 Interleukin-8 P10145 78 218.2893964 0.732484076 70.86636431 PTGS2 Prostaglandin G/H synthase 2 P35354 78 206.0168462 0.737179487 69.98848441 EGF Proepidermal growth factor P01133 76 261.8569934 0.737179487 65.03746714 MMP9 Matrix metalloproteinase-9 P14780 76 433.8806129 0.71875 68.81580781 CCL2 C-C motif chemokine 2 P13500 75 157.4214188 0.72327044 67.00969048 IL10 Interleukin-10 P22301 74 145.584267 0.71875 65.80203946 IL1β Interleukin-1 beta P01584 74 158.014336 0.71875 65.60821648 MAPK8 Mitogen-activated protein kinase 8 P45983 73 129.355471 0.709876543 65.62343779 JUN Transcription factor AP-1 P05412 73 122.1960842 0.709876543 66.15527831 CASP3 Caspase-3 P42574 72 107.386397 0.701219512 64.73650484 MAPK1 Mitogen-activated protein kinase 1 P28482 71 166.1161984 0.705521472 61.56264833 MYC Myc proto-oncogene protein P01106 64 147.9851483 0.67251462 54.86995265 MMP2 72 kDa type IV collagenase P08253 62 74.82792554 0.66091954 52.92741783 mechanism of the Chinese patent medicine compound [33]. Luteolin can reduce the proliferation of fibroblast-like Xuanju capsule in the treatment of RA. 51 active ingredients synovial cells in RA by inhibiting the NF-κB and JAK/STAT of compound Xuanju capsules, such as formic acid, querce- signal pathways [34]. Stigmasterol is a natural plant product tin, kaempferol, luteolin and stigmasterol, were screened with anti-inflammatory activity, which can inhibit the signal through multiple large databases. It was predicted that there pathway of the inflamed joints, improve the antioxidant sta- were 116 potential targets of the compound Xuanju capsule tus, and transfer inflammation [35]. Anhydroicaritin has the in the treatment of RA, such as IL-6, GAPDH, VEGFA, function of repairing the bone tissue; in addition, it can pro- TNF and AKT1. Five potential mechanism pathways of com- mote the angiogenesis by increasing the expression of proan- pound Xuanju capsule-treated RA were analyzed and sum- giogenic factors such as VEGFA and McP-1, thus providing marized, namely, the IL-17 signaling pathway, the TNF more nutrients for bone formation [36]. Therefore, the signaling pathway, the Th17 cell differentiation pathway, above-mentioned active ingredients might be all the medici- the Toll receptor signaling pathway and the RA pathway. nal material bases of the compound Xuanju capsule in the Among the 51 active ingredients selected in this work, treatment of RA, which had an important reference value formic acid, quercetin, kaempferol, luteolin, stigmasterol, for subsequent clinical and experimental research. anhydroicaritin and so on were the top 10 active ingredients Each node in the network represents the protein corre- according to the degree value, which might be the main active sponding to the action target; thus, 116 common nodes ingredients of the compound Xuanju capsule against RA. involved in the intersection network indicated that there Researches have demonstrated that formic acid as one of were a total of 116 potential action targets of the compound the biologically active substances of black ants was the mate- Xuanju capsule in the treatment of RA. Additionally, the size rial basis for its anti-inflammatory, analgesic, antibacterial and color of the nodes represent the degree value of the target and antirheumatic effects and immune regulation and pre- protein, and the connection between the nodes represents the vention of arteriosclerosis [17, 31]. Quercetin can improve potential interaction between the target proteins [37]. The morning stiffness, arthralgia, and other clinical symptoms target protein in the network relationship is greatly corre- of RA patients and reduce disease activity scores [32]. lated with the node size, the node color, and the connections, Kaempferol can inhibit the migration and invasion of which increase with them [38]. Hence, IL-6, VEGFA, TNF, fibroblast-like synovial cells in RA by blocking the activation AKT1, MAPK3, CXCL8, IL10, IL1β, JUN, CASP3 and other of the MAPK pathway and inhibit the reorganization of the targets showed an obvious correlation in the network rela- actin cytoskeleton during cell migration, thereby reducing tionship of compound Xuanju capsules in the treatment of the arthritis symptoms of collagen-induced arthritis rats RA, as shown in Figure 5. This demonstrated that the 10 BioMed Research International

47.00

40.00 40 38.00 36.00 35.0035.00 35.00 35.0035.0034.00

21.00 20 19.00 20.00 19.00 18.00 18.00 18.00 17.0017.0017.0017.0017.00 17.00 16.00 Enrichment score Enrichment 13.00 11.0011.00 10 10.00 8.00 9.00

0 caveola vesicle lumen vesicle membrane raf membrane cytokine activity cytokine membrane region membrane extracellular matrix extracellular phosphatase binding phosphatase response to bacterium to response plasma membrane raf membrane plasma endopeptidase activity endopeptidase receptor ligand activity activity ligand receptor secretory granule lumen secretory granule cellular response to lipid to response cellular membrane microdomain membrane blood vessel development cytokine receptor binding cytokine receptor cytoplasmic vesicle lumen vesicle cytoplasmic receptor regulator activity activity regulator receptor transcription factor binding factor transcription fcolin-1-rich granule lumen granule fcolin-1-rich serine-type peptidase activity response to lipopolysaccharide to response positive regulation of locomotion of regulation positive positive regulation of cell motility of regulation positive protein homodimerization activity homodimerization protein serine-type activity endopeptidase positive regulation of cell migration of regulation positive cytokine-mediated signaling pathway signaling cytokine-mediated response to molecule of bacterial origin molecule of to response positive regulation of cellular compontent movement movement compontent cellular of regulation positive Biological process Cellular component Molecular function BP CC MF

Figure 6: GO enrichment analysis of the common targets. The top 10 GO functional terms were selected (P <0:05). BP: biological processes; CC: cellular components; MF: molecular functions. compound Xuanju capsule had higher binding activity with ulation of angiogenesis which can promote the infiltration of these targets which could be used as the potential action tar- inflammatory cells into the joint and lead to synovial hyper- gets. It had shown that TNF-α was increased in the synovium plasia and progressive bone destruction [43, 44]. AKT1 is and cartilage of RA patients, which could promote the carti- believed to be closely related to the production of RA syno- lage destruction by inhibiting the collagen synthesis, stimu- vial fibroblasts [45]. The MAPK gene participates in signal- lating the fibroblasts and chondrocytes to produce the ing pathways that can reduce the oxygen free radicals, prostaglandins and collagenase, stimulating the chondro- inhibit the cell proliferation, promote the apoptosis, reduce cytes to secrete metalloproteinases, inducing the differentia- the inflammatory response, and inhibit the angiogenesis, tion of peripheral blood mononuclear cells into osteoclasts, thus effectively inhibiting RA progression [46]. JUN, closely etc. [39]. Meanwhile, TNF-α can increase the production of associated with the proliferation, differentiation, maturation, the proinflammatory factors (IL1β, IL-6, etc.) and chemo- and function of the osteoblast, plays an important role in kines (CXCL8, etc.) through the cascade reaction and further maintaining the dynamic balance of bone formation and aggravate the inflammatory response [40]. IL-6 is a major bone resorption [38]. The results of the current study showed inflammatory mediator and induced osteoclast precursors that the known active ingredients of the compound Xuanju to differentiate into the real osteoclasts during bone metabo- capsule could primarily act on 116 targets in the treatment lism [41]. Thus, IL-6 is the main factor for further destruc- of RA. Among the 116 targets, IL-6, VEGFA, TNF, AKT1, tion of bone and cartilage. As the negative regulator of the MAPK3, CXCL8, IL10, IL1β, MAPK8, JUN and other targets proinflammatory cytokines, 1L-10 can reduce the release of had higher degree values, indicating that these targets might the proinflammatory cytokines [42]. One of the earliest be the core targets of the compound Xuanju capsule against observed phenomena in synovitis is the formation of new RA, and the corresponding action mechanism was character- blood vessels. VEGF is the key molecule involved in the reg- ized by multiple components and multiple targets. BioMed Research International 11

Top 20 of pathway analysis −log10 (P value) AGE-RAGE signaling pathway in diabetic complications 40 Fluid shear stress and atherosclerosis IL-17 signaling pathway Pathways in cancer TNF signaling pathway 35 Chagas disease (American trypanosomiasis) Leishmaniasis Proteoglycans in cancer T17 cell diferentiation Toxoplasmosis 30 Pertussis

Pathway Toll-like receptor signaling pathway Rhenumatoid arthritis

Bladder cancer 25 Infuenza A Hepatitis B Infammatory bowel disease (IBD) Salmonella infection 20 Apoptosis Endocrine resistance 20 25 30 35 40 Enrichment Count 15 25

20 30

Figure 7: KEGG pathway enrichment analysis of the common targets. The top 20 pathways were identiﬁed. The color represented P value, and the size of the spot represented count of genes.

The GO and KEGG pathway enrichment analysis were conducted on 116 common targets. The GO results found that the cytokine activity and the cytokine receptor binding were closely related to the occurrence of RA in terms of molecular functions. In addition, the signal pathways and vascular development mediated by the cytokines in biological processes had a certain impact on RA; this was also corrobo- rated with the results that the compound Xuanju capsule might act on 166 potential core targets in the treatment of RA. KEGG pathway enrichment analysis results demonstrated that the main signaling pathways regulated by the compound Xuanju capsule were the IL-17 signaling pathway, the TNF signaling pathway, the Th17 cell differentiation pathway, the Toll receptor signaling pathway, the RA pathway, the liquid shear stress and atherosclerosis pathways, etc. It had shown that the TNF signaling pathway was the key pathway derived from the enrichment analysis and an important pathway in the inflammatory response [47]. The occurrence and development of RA are related to the abnormal expression of IL-6, IL1β, TNF and PTGS2 in the TNF Figure 8: KEGG signal pathway enrichment analysis of core targets signaling pathway [48]. IL-17 not only causes the inflamma- in the compound Xuanju capsule treating RA. KEGG terms are ff represented as nodes, and the size of the node indicates the size of tion but also stimulates the osteoclast di erentiation to cause the degree value; the degree value corresponds to the node size bone and cartilage damage. At the same time, IL-17 can also from small to big. stimulate the production of many types of chemokines and aggravate the inflammatory response. Furthermore, IL-17 12 BioMed Research International can induce the prostaglandins to enter the inflammatory sites method were mutually confirmed with the observation through the cyclooxygenase-2 and then damage the synovial results of the compound Xuanju capsule in the clinical treat- surface and articular cartilage [49]. Therefore, the IL-17 sig- ment of RA [14, 15]. naling pathway plays an important role in the course of RA is a chronic inflammatory autoimmune disease with RA. The RA pathway includes the T cell receptor signaling the complex pathogenesis [35]. The results of the present pathway, the Toll receptor signaling pathway, the VEGF sig- study suggested that RA was related to the abnormality of naling pathway, the osteoclast differentiation, and the Th17 the IL-6-based multitarget and IL-17 pathway-based multi- cell differentiation, all of which are related to the immune signal pathways; thus, the pathogenesis of RA had the charac- regulation and inflammation. Among them, the abnormal teristics of multitarget and multipathway. To some extent, it T cell-mediated immune response is the main pathogenesis explained the phenomenon that there was no good effect in of RA. In the diseased synovial tissue, a large number of acti- the single target treatment of the disease in clinic. TCM has vated T lymphocytes gather around the blood vessels. The the characteristics and advantages of multicomponent, mul- activated T lymphocytes interact directly with synovial mac- titarget, and multipathway in the treatment of diseases. rophages, secrete the cytokines, and stimulate the synovial Therefore, it is still a research hotspot to find drugs for RA cells to release collagenase and protease, which further treatment. caused the cartilage and bone damage [38]. The Toll receptor In conclusion, based on the network pharmacology signaling pathway directly triggers the intracellular bacteri- methods, the current study found that the compound Xuanju cidal mechanism or induces the production of immune capsule, in the treatment of RA, might mainly act on 116 tar- inflammatory factors on the basis of identifying pathogenic gets based on IL-6 through 51 active ingredients such as for- microorganisms to expand nonspecific defenses. Research mic acid, quercetin and kaempferol, and then regulated the manifested that the Toll receptors were significantly IL-17 signal pathway, the TNF signal pathway, the Th17 cell expressed in the pathological tissues of RA patients [50]. differentiation pathway, the Toll receptor signaling pathway, The VEGF signaling pathway inhibits the expression of cyto- the RA pathway, etc., to further affect the release of the pro- kines in arthritis, reduces the production of collagen, inhibits inflammatory factors and the anti-inflammatory factors, the proliferation of synovial cells, and weakens the invasion thereby alleviating inflammatory damage and improving ability [41]. Th17 and Treg cells are two subtypes of CD4+ bone tissue repair. The above results reflected the mechanism T cells. Among them, Th17 cells can highly secrete inflam- and advantages of the compound Xuanju capsule in treating matory cytokines such as IL-17, IL-6 and IL-22, mediating RA with multiple components, multiple targets, and multiple inflammatory reactions and autoimmune reactions in the pathways, and also confirmed that inhibiting the secretion of body [51]. Abnormal activation of Th17 cells will cause the inflammatory factors was one of its major mechanisms. imbalance of Th17/Treg cells and play a key role in autoimmune diseases such as RA, especially in local inflammatory Data Availability response and bone destruction, while long-term local inflammation will induce and worsen the destruction of RA articu- We have presented all our main data in the form of figures lar cartilage [52]. Cells in the body can not only sense the and tables. The datasets supporting the conclusions of this stimulation of various chemical factors in the external envi- article are included within the article. ronment but also live in various mechanical environments, such as the pressure of fluid as well as shear stress caused Conflicts of Interest fl fl by the uid ow. In the interaction between the extracellular fl matrix and the signal molecules on the cell membrane, these The authors declare that there are no con icts of interest. mechanical stresses can be sensed by cells, thereby activating ’ intracellular signaling pathways, transforming the mechani- Authors Contributions cal signals into the intracellular biochemical signals, and thus Wenyang Wei was responsible for data analysis and original affecting cell shape, adhesion and migration, gene expression, diagram preparation. Wenyang Wei, Wanpeng Lu, and Xiao- differentiation, and even cell death [53]. These mechanical long Chen wrote the manuscript. Yunfeng Yang supervised factors play a decisive role in the changes of cellular functions the whole research. Wenyang Wei and Mengkai Zheng such as cardiovascular disease, bone formation, and organ revised the manuscript. development. The present study results indicated that the main signaling pathways regulated by the compound Xuanju Acknowledgments capsule included the IL-17 signaling pathway, the TNF signaling pathway, the Toll receptor signaling pathway, the The authors thank the members of their laboratory and their ff Th17 cell di erentiation pathway, the RA pathway and the collaborators for their research work. liquid shear stress and atherosclerosis pathway. Furthermore, the inflammatory signal pathways were the main pathway. References This suggested that the compound Xuanju capsule might mainly regulate the inflammatory pathways to protect the [1] J. A. Sparks, “Rheumatoid arthritis,” Annals of Internal Medi- bone tissue and improve the clinical symptoms of RA cine, vol. 170, no. 1, pp. 1–16, 2019. patients such as joint swelling and pain. Moreover, the above [2] W. Wang, X. Wang, X. Tang, Q. Jiang, and Y. Fan, “Classifying research results predicted by the network pharmacology rheumatoid arthritis by traditional Chinese medicine Zheng: a BioMed Research International 13

multi-center cross-sectional study,” Journal of Traditional clinical application,” Osteoporosis International, vol. 29, Chinese Medicine, vol. 39, no. 3, pp. 425–432, 2019. no. 3, pp. 535–544, 2018. [3] D. S. Pisetsky, “Advances in the treatment of rheumatoid [19] L. Kong, Y. Yao, Y. Xia, X. Liang, Y. Ni, and J. Yang, “Osthole arthritis: costs and challenges,” North Carolina Medical Jour- alleviates inflammation by down-regulating NF-kappa B signal, vol. 78, no. 5, pp. 337–340, 2017. naling pathway in traumatic brain injury,” Immunopharma- – [4] D. Giannini, M. Antonucci, F. Petrelli, S. Bilia, A. Alunno, and cology and Immunotoxicology, vol. 29, no. 3, pp. 349 360, I. Puxeddu, “One year in review 2020: pathogenesis of rheu- 2019. matoid arthritis,” Clinical and Experimental Rheumatology, [20] Q. Tao, J. du, X. Li et al., “Network pharmacology and molec- vol. 38, no. 5, pp. 387–397, 2020. ular docking analysis on molecular targets and mechanisms of ” [5] K. Gou, R. Zeng, X. Ren et al., “Anti-rheumatoid arthritis Huashi Baidu formula in the treatment of COVID-19, Drug effects in adjuvant-induced arthritis in rats and molecular Development and Industrial Pharmacy, vol. 46, no. 8, – docking studies of Polygonum orientale L. extracts,” Immunol- pp. 1345 1353, 2020. ogy Letters, vol. 201, no. 5, pp. 59–69, 2018. [21] Q. Jin, J. Lu, R. Gao, J. Xu, X. Pan, and L. Wang, “Systemati- “ cally deciphering the pharmacological mechanism of Fructus [6] L. Zhang, Z. Cao, Y. Yang, X. Tan, J. Mao, and L. Su, Tradi- ” tional Chinese medicine on treating active rheumatoid arthri- Aurantii via network pharmacology, Evidence-Based Comple- tis: a protocol for systematic review and meta-analysis,” mentary and Alternative Medicine, vol. 2021, 14 pages, 2021. Medicine, vol. 99, no. 24, article e20642, 2020. [22] C. Qian, P. Chen, W. Zheng et al., “To explore the potential [7] S. Lu, Q. Wang, G. Li, S. Sun, Y. Guo, and H. Kuang, “The mechanism of Shanhaidan granules in the treatment of erectile dysfunction based on network pharmacology,” Chinese Phar- treatment of rheumatoid arthritis using Chinese medicinal – plants: from pharmacology to potential molecular mecha- macological Bulletin, vol. 36, no. 6, pp. 864 870, 2020. nisms,” Journal of Ethnopharmacology, vol. 176, pp. 177–206, [23] S. K. Kim, S. Lee, M. K. Lee, and S. Lee, “A systems pharmacol- 2015. ogy approach to investigate the mechanism of Oryeong-san formula for the treatment of hypertension,” Journal of Ethno- [8] X. Li and S. Zhang, “Herbal compounds for rheumatoid arthri- pharmacology, vol. 244, article 112129, 2019. tis: literatures review and cheminformatics prediction,” Phy- “ totherapy Research, vol. 34, no. 1, pp. 51–66, 2020. [24] P. Zhong, L. Song, M. Gao et al., Network pharmacology- based strategy for predicting active ingredients and potential [9] H. Wang and M. Wang, “Nanometer preparation of tradi- targets of Gegen Qinlian decoction for rotavirus enteritis,” Evi- tional Chinese medicine for rheumatoid arthritis,” Zhongguo dence-Based Complementary and Alternative Medicine, Zhong Yao Za Zhi, vol. 34, no. 1, pp. 3908–3916, 2019. vol. 2020, Article ID 2957567, 12 pages, 2020. [10] Q. Wang, C. Zhou, Y. Xiao, Z. Wu, W. Wei, and J. Cai, “Effects [25] Y.-Y. Shi, Y.-Q. Li, X. Xie et al., “Homotherapy for heteropathy of Xuanju extract on benign prostatic hyperplasia,” Chinese active components and mechanisms of Qiang-Huo- Sheng-Shi Journal of Andrology, vol. 34, no. 3, pp. 31–36, 2020. decoction for treatment of rheumatoid arthritis and osteoar- “ [11] L. Wang, Y. Li, Y. Guo et al., Herba Epimedii: an ancient Chi- thritis,” Computational Biology and Chemistry, vol. 89, article nese herbal medicine in the prevention and treatment of oste- 107397, 2020. oporosis,” Current Pharmaceutical Design, vol. 22, no. 3, [26] H. Qian, Q. Jin, Y. Liu et al., “Study on the multitarget mech- pp. 328 –349, 2016. anism of Sanmiao pill on gouty arthritis based on network [12] W. Zhang, D. Ma, Q. Zhao, and T. Ishida, “The Effect of the pharmacology,” Evidence-Based Complementary and Alterna- Major Components of Fructus Cnidii on Osteoblasts In Vitro,” tive Medicine, vol. 2020, Article ID 9873739, 11 pages, 2020. Journal of Acupuncture and Meridian Studies, vol. 3, no. 1, “ – [27] M. Ashburner, C. Ball, J. Blake et al., Gene Ontology: tool for pp. 32 37, 2010. the unification of biology,” Genetics, vol. 25, no. 1, pp. 25–29, [13] K.-H. Lu, C.-T. Liu, R. Raghu, and L.-Y. Sheen, “Therapeutic 2000. potential of Chinese herbal medicines in alcoholic liver dis- [28] M. Tanabe and M. Kanehisa, “Using the KEGG database ease,” Journal of Traditional and Complementary Medicine, ” – resource, Current Protocols in Bioinformatics, vol. 1, vol. 2, no. 2, pp. 115 122, 2012. pp. 1.12.1–1.12.43, 2012. “ [14] L. Wang, C. Wang, X. Wang, and Y. Zhang, Clinical study of [29] Z. Wu and B. Wu, “Prospect of treatment of chronic diseases compound Xuanju capsule combined with methotrexate in the by Polyrhachis vicina Roger,” Bulletin of Biology, vol. 1995, ” treatment of refractory rheumatoid arthritis, Chinese Tradi- no. 5, pp. 3–6, 1995. tional Patent Medicine, vol. 40, no. 2, pp. 299–304, 2018. [30] H. Li, T. Lv, B. Wang et al., “Integrating network pharmacol- “ ff [15] C. Wang, X. Wang, Y. Zhang, and L. Wang, E ect of com- ogy and experimental models to investigate the mechanism pound Xuanju capsules on Th17 cells/interleukin 17 in of Huanglian Jiedu decoction on inflammatory injury induced ” patients with rheumatoid arthritis, Pharmacology and Clinics by cerebral ischemia,” Evidence-Based Complementary and – of Chinese Materia Médica, vol. 32, no. 6, pp. 198 201, 2016. Alternative Medicine, vol. 2021, Article ID 2135394, 15 pages, [16] W. Jia, J. Xue, Y. Wang, and H. Huo, “Study on immunoregu- 2021. fl ff latory and anti-in ammatory e ects of compound Xuanju [31] G. Song, “Medicinal use and nutrition of ants,” Food and Drug, ” capsule, Chinese Traditional and Herbal Drugs, vol. 2003, vol. 2005, no. 9, pp. 63–65, 2005. no. 2, pp. 62–65, 2003. [32] H.-R. Kim, B.-M. Kim, J. Y. Won et al., “Quercetin, a plant [17] Y. He and G. Li, “New progress in pharmacological research of polyphenol, has potential for the prevention of bone destruc- ant preparation,” Lishizhen Medicine and Materia Medica tion in rheumatoid arthritis,” Journal of Medicinal Food, – Research, vol. 14, no. 2, pp. 113 115, 2003. vol. 22, no. 2, pp. 152–161, 2019. [18] Z. Wang, D. Wang, D. Yang, W. Zhen, J. Zhang, and S. Peng, [33] D. Pan, N. Li, Y. Liu et al., “Kaempferol inhibits the migration “The effect of icariin on bone metabolism and its potential and invasion of rheumatoid arthritis fibroblast-like 14 BioMed Research International

synoviocytes by blocking activation of the MAPK pathway,” [47] W. Mo, Z. Su, and Y. Liang, “Study on the mechanism of Mill- International Immunopharmacology, vol. 55, pp. 174–182, ettia speciosa Champ. in the treatment of rheumatoid arthritis 2018. based on network pharmacology,” Journal of Guangxi Medical – [34] L. Lou, Y. Liu, J. Zhou et al., “Chlorogenic acid and luteolin University, vol. 37, no. 5, pp. 843 849, 2020. synergistically inhibit the proliferation of interleukin-1β- [48] B. Osta, G. Benedetti, and P. Miossec, “Classical and paradox- induced fibroblast-like synoviocytes through regulating the ical effects of TNF-α on bone homeostasis,” Frontiers in activation of NF-κB and JAK/STAT-signaling pathways,” Immunology, vol. 5, p. 48, 2014. Immunopharmacology and Immunotoxicology, vol. 37, no. 6, [49] B. W. Kirkham, A. Kavanaugh, and K. Reich, “Interleukin- – pp. 499 507, 2015. 17A: a unique pathway in immune-mediated diseases: psoria- [35] M. Ahmad Khan, A. Sarwar, R. Rahat, R. Ahmed, and S. Umar, sis, psoriatic arthritis and rheumatoid arthritis,” Immunology, “Stigmasterol protects rats from collagen induced arthritis by vol. 141, no. 2, pp. 133–142, 2014. fl ” inhibiting proin ammatory cytokines, International Immu- [50] R. Yao, Y. Wang, and H. Cai, “Research progress on the corre- nopharmacology, vol. 85, article 106642, 2020. lation between Toll-like receptor and rheumatoid arthritis,” [36] X. Lai, X. Huang, and Y. Zeng, “Protective effect of anhydroi- Chinese Journal of Immunology, vol. 31, no. 9, pp. 1287– caritin against peritonitis in mice,” Chinese Journal of Cellular 1289, 2015. – and molecular immunology, vol. 29, no. 10, pp. 1036 1039, [51] T. Wang, X. Sun, J. Zhao et al., “Regulatory T cells in rheuma- 2013. toid arthritis showed increased plasticity toward Th17 but [37] S. Reng, L. Ni, D. Meng et al., “Mechanisms of Cortex retained suppressive function in peripheral blood,” Annals of phellodendri-Herba tuberculate speranskia in treatment of the Rheumatic Diseases, vol. 74, no. 6, pp. 1293–1301, 2015. ” rheumatoid arthritis based on network pharmacology, Chi- [52] A. Paradowska-Gorycka, A. Wajda, K. Romanowska-Próch- nese Journal of New Drugs and Clinical Remedies, vol. 38, nicka et al., “Th17/Treg-related transcriptional factor expres- – no. 12, pp. 757 766, 2019. sion and cytokine profile in patients with rheumatoid [38] Q. Liang, J. Yan, Y. Feng, F. Li, T. Xie, and X. Fang, “Effect and arthritis,” Frontiers in Immunology, vol. 11, article 572858, mechanism of YAO medicine compound containing Cissus 2020. pteroclada on rheumatoid arthritis in rats and its Q-marker [53] S. Chien, “Molecular basis of rheological modulation of endo- ” prediction, Chinese Traditional and Herbal Drugs, vol. 50, thelial functions: importance of stress direction,” Biorheology, – no. 19, pp. 4705 4712, 2019. vol. 43, no. 2, pp. 95–116, 2006. [39] H. Shi, D. Wang, R. Wu, and H. Li, “Role of tumor necrosis factor-α mediated nuclear factor kappa B signaling pathway in antiogenesis in rheumatoid arthritis,” Medical Recapitulate, vol. 18, no. 15, pp. 2397–2400, 2012. [40] T. Morita, Y. Shima, K. Fujimoto et al., “Anti-receptor activa- tor of nuclear factor κB ligand antibody treatment increases osteoclastogenesis-promoting IL-8 in patients with rheumatoid arthritis,” International Immunology, vol. 31, no. 5, pp. 277–285, 2019. [41] W. Ding, L. Han, K. Qian, and C. Lin, “Mechanism of Caulis Sinomenii on rheumatoid arthritis based on network pharmacology,” China Medical Herald, vol. 17, no. 17, pp. 15–20, 2020. [42] A. Saxena, S. Khosraviani, S. Noel, D. Mohan, T. Donner, and A. R. A. Hamad, “Interleukin-10 paradox: a potent immuno- regulatory cytokine that has been difficult to harness for immunotherapy,” Cytokine, vol. 74, no. 1, pp. 27–34, 2015. [43] Y. Zhang, H. Qiu, H. Zhang, L. Wang, C. Zhuang, and R. Liu, “Vascular endothelial growth factor A (VEGFA) polymor- phisms in Chinese patients with rheumatoid arthritis,” Scandi- navian Journal of Rheumatology, vol. 42, no. 5, pp. 344–348, 2013. [44] H. A. Elshabrawy, Z. Chen, M. V. Volin, S. Ravella, S. Virupannavar, and S. Shahrara, “The pathogenic role of angiogenesis in rheumatoid arthritis,” Angiogenesis, vol. 18, no. 4, pp. 433–448, 2015. [45] X. Xie, Y. Wang, S. Luo, Z. Zhao, and X. Li, “Pathogenesis of rheumatoid arthritis,” World Latest Medicine Information, vol. 19, no. 71, pp. 109–111, 2019. [46] L. Shi and W. Sun, “Mechanism of Shaoyao Gancao decoction in treatment of rheumatoid arthritis based on network pharmacology,” Chinese Herbal Medicines, vol. 51, no. 24, pp. 6246–6257, 2020.