Network Pharmacology and Molecular Docking Explore the Mechanism of Mubiezi-Yinyanghuo Herb Pair in the Treatment of Rheumatoid Arthritis

Objective Our previous studies have shown that the Mubiezi-Yinyanghuo (MBZ-YYH) herb pair inhibits rheumatoid arthritis (RA) cell proliferation and glycolysis, promising results with an obscure mechanism of action. Methods Therefore, it is necessary to explore the main components of MBZ-YYH and unravel the potential mechanism in RA based on network pharmacology and molecular docking methods. Components and targets of MBZ-YYH were retrieved from the TCMSP. Relevant targets of RA were searched in GeneCards, therapeutic target database (TTD), and DisGeNET databases; the common targets of the MBZ-YYH compounds and RA were obtained by comparison; and a component-target interaction network was established by Cytoscape 3.9.1. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed by PyMoL2.3.0 and AutoDock Vina1.1.2 software. Results 7 active ingredients and 58 putatively identified target genes were screened from MBZ, and 16 effective components of YYH and 230 potential targets were identified. There were 29 mutual targets between the two herbs and RA. Through the PPI network, 9 hub targets which contain JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2 were screened out. GO term enrichment analysis indicated that positive regulation of the apoptotic process, response to drugs, and response to hypoxia were significantly enriched. Based on KEGG analysis, it was mainly associated with the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway. The docking analysis revealed that the effective components showed strong binding activity with the receptors. Conclusion The effects of the MBZ-YYH herb pair on RA were coordinated by the interaction of diverse components, which may be through the IL-17 signaling pathway and the TNF signaling pathway, which target GSK3B, HK2, caspase 3, and caspase 8, inhibiting the proliferation and glycolysis of rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) and tending towards an increasing efficacy and decreasing toxicity effect on RA.


Introduction
Rheumatoid arthritis (RA) is a chronic, infammatory, and autoimmune disease characterized by infammatory changes of synovial tissue, cartilage, and joint skeleton [1].Te common clinical manifestations of RA include joint swelling, stifness, and dysfunction [2].Tis eventually leads to disability and ultimately death [3,4].RA can afect people of all ages; around 1% of them sufer from intractable pain.It not only causes great sufering for patients and family members but also brings heavy fnancial burden for individuals and society [5].Drugs that reduce joint pain or retard the progression of disease include nonsteroidal antiinfammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and steroids and biological response modifers [6].However, these drugs can produce serious side efects, including renal toxicity, gastrointestinal toxicity, hepatic damage, and myelosuppression [7][8][9][10].Moreover, it has been associated with an increased prevalence and incidence of cardiovascular diseases [11,12].
With developments in the feld of traditional Chinese medicine (TCM), the treatment of RA has entered a stage of diversifed comprehensive treatment.As an assistant and substitute medicine, TCM has gained increasing attention because of signifcant efcacy and lesser side efects.Our previous studies found that an extract of MBZ could suppress the angiogenesis of human umbilical vein endothelial cells (HUVEC) in the RA synovial cavity by inhibiting glycolysis, but the specifc mechanism is still unclear [13].MBZ has the defnite efects of dispersing swelling and healing sores.Pharmacological research has revealed that in addition to anticancer, anti-infammation, and antibacterial pharmacological efects based on the traditional efcacy, MBZ also exhibits antiulcer, antioxidation, and immune regulation efects [14].MBZ is an efective but toxic traditional Chinese medicine; therefore, the present study was carried out to search for compatible herbs that can enhance the efcacy and reduce the toxicity in treatment of RA.Kan et al. [15] showed in an interrelated study that the optimal ratio 1 : 1 of MBZ with YYH combined showed more prominent antitumor proliferation; moreover, there was no conspicuous efect on the growth of LO2 normal human liver cells, and it was speculated that the compatibility of the two herbs has the synergistic efect of enhancing efcacy and reducing toxicity.Considering this, current research has bestowed the MBZ-YYH pair on HFLS-RA to detect its efects on cell proliferation and glycolysis.Trough network pharmacology and molecular docking, we explored the action mechanism of MBZ on RA under the coordination of YYH, in expectation of providing a theoretical basis for the extensive clinical treatment of RA.Te fowchart of this study is presented in Figure 1.

Establishment of Active Ingredients and RA Disease
Target Databases of MBZ and YYH.By searching for a traditional Chinese medicine (TCM) system pharmacology (TCMSP) platform (http://tcMBZpw.com/tcMBZp.php),the processes that determine the pharmacokinetic behavior of a drug compound are its absorption, distribution in the body, metabolism, and elimination from the body (ADME).Te specifc screening criteria were oral bioavailability (OB) ≥30% and drug-like properties (DL) ≥0.18. "Rheumatoid arthritis" as keywords retrieval in the GeneCards database (https://www.genecards.org/),Terapeutic Target Database (TTD) (http://db.idrbla.net/ttd/),and DisGeNET(http:// www.disgenet.org/)databases to obtain RA-related disease targets.Te intersection between the compounds and RA disease targets was obtained by the Venn diagram.

Protein-Protein Interaction (PPI) Network of Potential
Targets of the MBZ-YYH Herb Pair and RA.Te intersection of targets obtained by Venn diagram was regarded as the potential targets of compounds for the treatment of RA.Te intersection of the other targets of MBZ and YYH was regarded as the interactional targets, and the protein-protein interaction (PPI) network of these potential targets was constructed by STRING (https://string-db.org/cgi/input.pl).Cytoscape 3.9.1 (https://cytoscape.org/)was used for visualization, and the hub targets were screened according to the degree value.

GO and KEGG Pathway Enrichment Analysis.
By the DAVID database (https://david.ncifcrf.gov/),the P value less than 0.05 was set as a condition of screening for enrichment of gene ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the key target protein.Tree modules, namely, biological process, cellular component, and molecular function, were selected for mapping.Its frst 10 entries were visualized as bubble plots using the ImageGP (https://www.bic.ac.cn/ImageGP/).

Molecular Docking.
Referring to studies in [16][17][18], the nine highest-degree targets in the "target-compound" network were regarded as receptors, and core compounds and anti-RA drugs were regarded as ligands.Te crystal structures of the 9 proteins were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb)and saved in PDB format.Tree-dimensional (3D) conformation of candidate compounds (https://pubchem.ncbi.nlm.nih.gov/) was downloaded from the PubChem database and saved in SDF format and then converted into a PDB format via Open Babel.Ligands and receptors were prepared by AutoDock Vina and PyMOL (v.2.3.0).Te original ligand and water molecules were removed from the crystal structure of the 2 Evidence-Based Complementary and Alternative Medicine receptor, nonpolar hydrogen was added, and the Gasteiger partial charge was calculated.Applying energy minimization and assigning atomic charges to process ligands, the active site of molecular docking is determined by the ligand coordinates in the target protein complex.Te ligand is set to be fexible while the receptor is rigid.All prepared receptors and ligands were saved in pdbqt format.Te afnity of the binding strength between the ligand and the target protein was assessed with AutoDock, and the conformation with the best afnity was selected as the fnal docking conformation and visualized in Pymol 2.3.0.Te stability of systems was evaluated using the root-mean-square deviation (RMSD) of the aligned protein-ligand coordinate set calculated against the initial frame.

MBZ-YYH Herb Pair Inhibited the Proliferation of RA Synovial Fibroblast Cells.
To determine the efect of the MBZ-YYH herb pair on RA cells, we treated RA cells with a 1 : 1 ratio of MBZ and YYH in a concentration gradient and a time gradient.Te results showed that the proliferation of RA cells was signifcantly inhibited by 400 μg/ml of the MBZ-YYH herb pair at 24 h (P < 0.001) (Figure 2), so we took this concentration as the best concentration for subsequent experiments.

Inhibition of RA Cell Glycolysis by the MBZ-YYH Herb
Pair.To further explore the efect of the MBZ-YYH herb pair on the glycolysis of RA cells, glucose and lactate contents and hexokinase activity were measured.Te results showed that the contents of glucose ( * * P < 0.01) and lactic acid ( * * P < 0.01) and the activity of hexokinase ( * * * P ≤ 0.001) in RA cells were recovered by the combination of MBZ and YYH, prompting that the MBZ-YYH herb pair could inhibit the glycolysis of RA cells (Figure 3).

Chemical and Pharmacological Analysis for Active Ingredient of the MBZ-YYH Herb Pair.
From the TCMSP database, a total of 31 constituents of MBZ and 130 compositions of YYH were retrieved.Trough the analysis of physical and chemical properties from TCMSP, it was found that the molecular weight (MW), octanol-water partition coefcient (AlogP), and hydrogen acceptor (Hacc) were prominently diferent between the two herbs (P < 0.05), while the hydrogen donor (Hdon), OB, and DL were not diferent.Indicating that the chemical properties of the two species were obviously diferent, however, to some extent, there is a certain intersection between them.Terefore, it is speculated that some components of this compatibility may be mutually benefcial, while others are mutual detoxication (Table 1).

Evidence-Based Complementary and Alternative Medicine
According to the threshold requirements of OB ≥ 30% and DL ≥ 0.18, the active ingredients of MBZ and YYH were screened.Considering that saponins are the main components of MBZ, oleanolic acid (OB � 29.02) was retained for target analysis to ensure accuracy.In addition, the OB values of NON (OB � 26.74%, DL < 0.04), karounidiol (OB � 26.26%, ODL � 0.77), and MOL012372 (OB � 28.3%, DL � 0.77) were similar to the mean value of the OB components of MBZ, so they were selected as candidate ingredients for further analysis (Table 2).

Network Construction of the Efective Components Corresponding Targets of the MBZ-YYH Herb
Pair for RA Terapeutic Targets.Tere were 55 and 212 targets corresponding to the active ingredients of MBZ and YYH, respectively.To further understand the role of targets, we obtained the target intersection that was regarded as a potential therapeutic target through the Venn diagram and constructed a PPI network.Te resulting PPI network was introduced into Cytoscape 3.9.1, and hub targets were selected according to the degree values which produced form network analyzer of Cytoscape (Figure 4).Aggregately, 28 targets of the two compounds intersected with RA targets (it should be noted that HK2 was included in the target set of MBZ as evidence from previous studies).Altogether 9 targets, which included the top 8 with the highest degree value with the addition of HK2 (Table 3), were used as receptors for molecular docking, and the corresponding compounds of potential targets (6 of MBZ and 12 of YYH) were used as ligands.

GO and KEGG Enrichment Analysis.
For the purpose of comprehending the molecular functions of potential targets, GO and KEGG enrichment analyses were performed.Te results showed that GO was mainly involved in protein binding, positive regulation of the apoptotic process, response to drugs, and response to hypoxia.KEGG mainly refers to EGFR tyrosine kinase inhibitor resistance, the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway (Figures 5(a) and 5(b)).It is hinted that MBZ and YYH may regulate the response of cells to drugs and the hypoxia environment through the IL-17 signaling pathway and the TNF signaling pathway.
In addition, we also conducted GO and KEGG analyses on 18 common targets of MBZ-YYH, and the results showed that it mainly involves G-protein-coupled receptor activity, extracellular ligand-gated ion channel activity, regulation of membrane and neuroactive ligandreceptor interaction, calcium signaling pathway, and regulation of actin cytoskeleton (Figures 6(a) and 6(b)).It is allusived that MBZ-YYH interaction may lead to extracellular ligand-receptor interaction through the activation of calcium ion channels by G-protein-coupled receptors on the membrane.Evidence-Based Complementary and Alternative Medicine Except ICAM1-bessisterol, which only has hydrophobic interaction, the other eight pairs all interact through hydrogen bond formation and hydrophobic force.Among them, PTGS2-Yinyanghuo C (−10.7 kcal/mol) is well paired, and Yinyanghuo C is most likely to combine with HK2 and GSK3B at the same time.It is worth noting that the efect of MBZ on HK2 is mainly achieved by MOL012372, MOL012375, MOL012377, MOL000263, and MOL000358 (all ≤−8 kcal/mol); YYH mainly acts on HK2 through MOL003542, MOL004382, and MOL004384 (all ≤−9.5 kcal/ mol) (Figure 8).

Discussion
In this study, it was found that the MBZ-YYH herb pair can inhibit proliferation and activity of hexose kinase of HFLS-RA.Further, it can restore the glucose level and reduce the content of lactic acid, suggesting that the MBZ-YYH herb pair may inhibit the glycolysis of RA cells.Tis fnding is consistent with our previous study that MBZ treated RA by glycolysis inhibition [13].Nevertheless, the mechanism of the active ingredients is still uncertain.Terefore, network pharmacology and molecular docking were used to explore the underlying mechanism.
Chemistry and pharmacology analysis shows that there are obvious diferences in the chemical properties between MBZ and YYH, but they overlap to some extent.It is hinted that there may be some components that mutual promotion and mutual-assistance, mutual restrain and mutual-detoxication.Herbs interaction is refected in the fact that each component can not only act independently but also generate new modes and mechanism of action between the components after their compatibility [19].Among the compatibility interaction mechanisms involving direct and indirect efects of components, this study may be more inclined to the latter, namely, components afect the  Network pharmacology analysis suggested that the common hub targets of the main components of herbs and RA were JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2.GO and KEGG analyses showed that the main targets involved response to drugs, response to hypoxia, IL-17 signaling pathway, and TNF signaling pathway, suggesting that the treatment of RA by the MBZ-YYH herb pair may regulate the response of cells to drugs and the hypoxia environment through the IL-17 signaling pathway and TNF signaling pathway.
During the development of RA, hypoxia alters cell biological functions by inducing mitochondrial dysfunction and promoting the switching of glycolytic pathways, leading to abnormal cell invasion and angiogenesis [20].Our previous study showed that the extract of MBZ regulated angiogenesis in RA through glycolysis inhibition, which was similar to this result [13].Although some neovascularization provides oxygen to the increased infammatory cell mass, the neovascularization network is dysfunctional and unable to restore tissue oxygen homeostasis, keeping RA joints in a hypoxic environment [21].Due to the high metabolic demand of synovial cells, the lactate content in the synovial fuid of RA is signifcantly increased and the glucose concentration is signifcantly decreased [22][23][24].Te results of the present study are in accordance with the fndings of the  Evidence-Based Complementary and Alternative Medicine abovementioned, as increased glycolytic enzyme activity in RA synovial tissue, RA synovial cell proliferation, and massive infammatory cell infltration lead to increased local capillary oxygen difusion distance and oxygen consumption, resulting in RA joint cavity hypoxia [25,26].Hypoxiainducible factor 1(HIF-1), a nuclear transcription factor that regulates cellular oxygen homeostasis and the expression of hypoxia-responsive genes, is increased under hypoxic conditions.Te gene expression of sugar transport and glycolytic enzymes was increased to promote glycolysis to adapt to the energy defciency of cells under hypoxia [27].
Te glycogen synthase kinase 3b (GSK3b), a negative regulator of glucose homeostasis, is involved in energy metabolism, infammation, and apoptosis pathways in a celltype and environment-dependent manner.Active GSK3b is associated with mitochondrial quiescence in Drosophila oocytes.However, B cells defcient in GSK3B showed higher metabolic activity and stronger proliferation [29].Te  Evidence-Based Complementary and Alternative Medicine current study identifed GSK3b inactivation as a master regulator of macrophage metabolic reprogramming in RA synovial tissue [30,31].In RA, the regulatory role of GSK3b in preventing mitochondrial hyperactivity is lost, and GSK3B-Ser9 localizes to the mitochondria and stabilizes calcium transfer at mitochondrial associated membrane contact sites, a necessary mechanism to increase ATP production and drive tissue destructive efector functions [32].While caspase-8 functions in synovial antigenpresenting cells to regulate the response to infammatory stimuli by controlling the action of RIPK3, this delicate balance maintains joint homeostasis [33].Active caspase 3 expression was increased in monocytes and synovial macrophages from RA patients compared with cells from healthy controls, and the use of caspase 3 inhibitors signifcantly blocked TNF-induced pyroptosis, which efectively alleviated arthritis in the CIA mouse model [34].In summary, the MBZ active ingredients were predicted to mainly cause apoptosis and act as a major actor, while the YYH active ingredients acted as a regulator of glucose metabolism and hexokinase activity, playing an adjuvant role in RA.Tis provides a theoretical basis for the application of MBZ combined with YYH in the clinical treatment of RA and excavates new therapeutic drugs for treatment of RA.However, it is undeniable that there are still some shortcomings in this study.Network pharmacology relies too much on the existing database, and the composition and target prediction of TCM such as MBZ may be lagging behind and the data are not comprehensive.In addition, for the drug interactions, this study only made some predictions and inferences about the indirect efects between them.For the other targets of diferent components, the interaction mechanism was not predicted due to its extensive involvement.Our next work will provide more empirical fndings and evidence.

Conclusion
In summary, MBZ and YYH act on RA through the interaction and coordination of diferent components.It may regulate the response of cells to drugs and the hypoxia environment by targeting GSK3b, HK2, caspase 3, and caspase 8 through the IL-17 signaling pathway and the TNF signaling pathway and inhibit the proliferation and glycolysis of RA FLS cells.Hence, it has a synergistic and attenuated therapeutic efect on RA.
RNA polymerase II sequence-specific DNA binding transcription factor binding mitochondrial outer membrane protease binding regulation of synaptic vesicle exocytosis extrinsic apoptotic signaling pathway caveola RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding protein serine/threonine kinase activator activity calcium ion transport into cytosol death-inducing signaling complex 0

Figure 5 :Figure 6 :
Figure 5: GO and KEGG enrichment analyses of potential targets.(a) GO enrichment analysis; (b) KEGG enrichment analysis.

Table 1 :
Comparative analysis table of the two herbs.

Table 2 :
Information table of active ingredients of two herbs.

Table 3 :
Information table of core target degree.