# Systems Pharmacology-Based Research on the Mechanism of Tusizi-Sangjisheng Herb Pair in the Treatment of Threatened Abortion

Authors: Ming Yang; Jianghe Luo; Yan Li; Limian Xu
Journal: BioMed Research International
 (2020)

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## Abstract

Threatened abortion (TA) is a common complication with high incidence in the first trimester of pregnancy, which will end in miscarriage if not treated properly. The Chinese herbs Cuscutae Semen (Tusizi in Chinese) and Herba Taxilli (Sangjisheng in Chinese) first recorded in the ancient classic medical bookShennong Bencao Jing are effective and widely used as an herb pair for the treatment of TA, while the active ingredients and the functional mechanism of Tusizi-Sangjisheng herb pair treating TA are still unknown. In order to exploit the relationship between those two herbs and TA, systems pharmacology analysis was carried out in this study. A total of 75 ingredients of Tusizi-Sangjisheng were collected from Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP). 12 bioactive compounds were screened, and 153 directly related targets were predicted by systematic models. Besides, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to systematically explore the potential mechanisms of Tusizi-Sangjisheng treating TA. Meanwhile, Compound-Target (C-T), Target-Disease (T-D), and Target-Pathway (T-P) networks were constructed to further quest the underlying functional mechanisms of Tusizi-Sangjisheng. As a result, 31 targets and 3 key pathways were found to be directly related to TA that includes mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase/protein kinase B (PI3K-Akt), and transforming growth factor-β (TGF-β) signaling pathways. The results in this study may provide some valuable clues about the molecular mechanisms of the efficient Chinese herb pair Tusizi-Sangjisheng in the treatment of TA.


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## Body

## 1. Introduction

Threatened abortion (TA), defined as vaginal bleeding with or without lower abdominal pain or backache with a closed cervix and an intrauterine viable fetus, is the commonest complication that occurs in early pregnancy [1], especially in 8-12 gestational weeks when the secretion of estrogen and progesterone shifts from corpus luteum to placental [2] (during the shift period, a pregnant woman is prone to go through limited corpus luteum function or an abnormality of placental progesterone production and secretion). TA affects one in five pregnancies, and about half of the cases unfortunately end in miscarriage [3, 4]. Besides, studies have shown that experiencing TA in the first trimester is associated with high risk of experiencing adverse pregnancy outcomes, such as premature rupture of membranes, placenta previa, and low birth weight [5]. TA is the cumulative result of several complicated pathogenic factors. Chromosomal abnormality is the important cause of TA that accounts for about 50-70% of cases [6, 7]. In addition, uterine malformations, cervical incompetence, polycystic ovaries, poorly controlled diabetes mellitus, maternal infections, immune dysfunctions such as antiphospholipid syndrome, and exposure to environmental toxins also have association with TA [7–9].In clinical practice, targeted and directed treatment should be used to prevent miscarriage when specific causes are identified. For example, combined aspirin and heparin are effective in TA induced by antiphospholipid syndrome while antibiotics are useful for TA induced by bacterial vaginosis [7] and surgical interventions such as cervical cerclage are alternative and efficient for cervical incompetence. However, about half of the women suffering from miscarriage have no certain causes to be identified [10], making the treatment empirical. Generally, bed rest, avoidance of sexual intercourse, and drugs for some specific manifestations are usually prescribed for a woman with symptoms of TA [2, 11]. For instance, increased uterine activity is considered to be related to TA and so tocolytic drugs like magnesium sulfate and phloroglucinol (PHL) are commonly used for treatment [12]. Besides, since low serum human chorionic gonadotropin (hCG) and progesterone level increases the risk of miscarriage, drugs like exogenous progesterone [13, 14] and estrogen [15] are usually administrated to treat TA. What is more, anti-D immunoglobulin [16] is recommended for RhD-negative women with TA. In China, TA can be mainly attributed to the deficiency of the viscera especially the deficiency of the kidney according to the theories of traditional Chinese medicine (TCM). Herbs like Cuscutae Semen (Tusizi in Chinese), Herba Taxilli (Sangjisheng in Chinese), Dipsaci Radix (Xuduan in Chinese), and Astragali Radix (Huangqi in Chinese) are used to treat TA for their functions of replenishing the kidney essence (Shen Jing in Chinese) and nourishing the liver [17, 18]. Zeng et al. [19] and Li’s [20] researches based on a data mining method have shown that Cuscutae Semen and Herba Taxilli were frequently prescribed for TA treatment and were always used in combination as an herb pair, indicating the key role of these two herbs in TA treatment and the representative value for other akin herbs to explore the potential mechanisms of action on TA treatment. Cuscutae Semen is the dry seed of Cuscuta australis and Cuscuta chinensis, which has been used for varieties of kidney conditions [21]. It has been reported to have neuroprotection properties in mouse models of Parkinson’s disease [22] and anti-inflammatory properties [23, 24] and also be effective in treating vitiligo [25]. Herba Taxilli is the dry leafy stem and branch of Taxillus sutchuenensis (Lecomte) Danser which grows on various trees and shrubs [26] and has the anti-HCV [27] and antioxidant activities [28]. Generally speaking, herbal medicines own the features of multicomponents and multieffectiveness in treating diseases which makes it hard to clearly clarify the efficiency in a molecular level. Although Tusizi-Sangjisheng is widely used for the treatment of TA, the exact mechanisms of it remain to be further elucidated.The objective of this study is to systematically explore the functional mechanism of the herb pair Tusizi-Sangjisheng in the molecular level and also to understand the relationship among those small chemicals, their related targets, and diseases. The results of this study may facilitate clarifying the mechanism of the Chinese herb pair Tusizi-Sangjisheng and offer some valuable methods for studying other akin herbs.


## 2. Materials and Methods

### 2.1. Molecular Database and ADME Screening

All compounds of Tusizi-Sangjisheng in this study were collected by using the TCMSP database (http://lsp.nwsuaf.edu.cn/tcmsp.php) which comprises more than 510 herbal entries registered in Chinese pharmacopoeia with more than 33,000 ingredients. The rational assessment of absorption, distribution, metabolism, and excretion properties (ADME) of compounds is essential to decide drug candidates. In the present study, two ADME-related models, PreOB (predict oral bioavailability) and PreDL (predict drug likeness) of the drugs, were employed to prescreen the bioactive compounds. Oral bioavailability (%F) is considered a key parameter in drug development. Oral drug absorption is mainly about two basic parameters: solubility and gastrointestinal permeability of the drug. In detail, the PreOB model, developed on the basis of a robust in-house system OBioavail 1.1 [29], was performed to predict the OB of the constituents of the herbs. The molecules with suitable OB≥30% were chosen as candidate compounds for further research. Drug likeness is a concept that is aimed at identifying where virtual or real molecules fall into drug-like chemical space based on one or more physiochemical properties. According to the mean value (0.18) of DL for all 3,206 molecules in DrugBank (http://www.drugbank.ca/), compounds with DL≥0.18 were selected as the candidate bioactive chemicals in this study. Finally, a total of 75 compounds from Tusizi-Sangjisheng were obtained and 12 bioactive compounds among them were screened.


### 2.2. Target Identification and Network Construction

Target identification for bioactive compounds is an essential step for drug discovery. Predicting drug-target and drug-pathway interactions could help understand the biological mechanisms from the perspective of network pharmacology. To obtain the related targets of these active compounds, a SysDT [30] model based on Random forest (RF) and Support Vector Machine (SVM) was employed. The UniProt database (https://www.uniprot.org/) was used to search the genes of the human species and the corresponding UniProtKB related to the predicted targets of the bioactive molecules. Information on the physiological functions of all targets was obtained from TTD (http://bidd.nus.edu.sg/group/ttd/) and UniProt (http://www.uniprot.org/) databases. Additionally, to systematically study the effects of herbal medicines and to characterize the therapeutic effects on a pathway level, three visualized networks including Compound-Target (C-T), Target-Disease (T-D), and Target-Pathway (T-P) were constructed. And the obtained target profiles were organized into several pathways by mapping to KEGG (Kyoto Encyclopedia of Genes and Genomes). All bipartite graphs were drawn by Cytoscape 3.5.1 software [31].


## 2.1. Molecular Database and ADME Screening

All compounds of Tusizi-Sangjisheng in this study were collected by using the TCMSP database (http://lsp.nwsuaf.edu.cn/tcmsp.php) which comprises more than 510 herbal entries registered in Chinese pharmacopoeia with more than 33,000 ingredients. The rational assessment of absorption, distribution, metabolism, and excretion properties (ADME) of compounds is essential to decide drug candidates. In the present study, two ADME-related models, PreOB (predict oral bioavailability) and PreDL (predict drug likeness) of the drugs, were employed to prescreen the bioactive compounds. Oral bioavailability (%F) is considered a key parameter in drug development. Oral drug absorption is mainly about two basic parameters: solubility and gastrointestinal permeability of the drug. In detail, the PreOB model, developed on the basis of a robust in-house system OBioavail 1.1 [29], was performed to predict the OB of the constituents of the herbs. The molecules with suitable OB≥30% were chosen as candidate compounds for further research. Drug likeness is a concept that is aimed at identifying where virtual or real molecules fall into drug-like chemical space based on one or more physiochemical properties. According to the mean value (0.18) of DL for all 3,206 molecules in DrugBank (http://www.drugbank.ca/), compounds with DL≥0.18 were selected as the candidate bioactive chemicals in this study. Finally, a total of 75 compounds from Tusizi-Sangjisheng were obtained and 12 bioactive compounds among them were screened.


## 2.2. Target Identification and Network Construction

Target identification for bioactive compounds is an essential step for drug discovery. Predicting drug-target and drug-pathway interactions could help understand the biological mechanisms from the perspective of network pharmacology. To obtain the related targets of these active compounds, a SysDT [30] model based on Random forest (RF) and Support Vector Machine (SVM) was employed. The UniProt database (https://www.uniprot.org/) was used to search the genes of the human species and the corresponding UniProtKB related to the predicted targets of the bioactive molecules. Information on the physiological functions of all targets was obtained from TTD (http://bidd.nus.edu.sg/group/ttd/) and UniProt (http://www.uniprot.org/) databases. Additionally, to systematically study the effects of herbal medicines and to characterize the therapeutic effects on a pathway level, three visualized networks including Compound-Target (C-T), Target-Disease (T-D), and Target-Pathway (T-P) were constructed. And the obtained target profiles were organized into several pathways by mapping to KEGG (Kyoto Encyclopedia of Genes and Genomes). All bipartite graphs were drawn by Cytoscape 3.5.1 software [31].


## 3. Results

### 3.1. Potential Active Compounds

In this study, a total of 75 compounds of the Tusizi-Sangjisheng (29 for Tusizi and 46 for Sangjisheng) were included. After screening all compounds at the criteriathatOB≥30% and DL≥0.18, 12 bioactive compounds with excellent ADME properties were obtained and the specific information is shown in Table 1.Table 1
Bioactive compounds of Cuscutae Semen and Herba Taxilli.
IDCompoundOBDLHerb nameMOL001558Sesamin56.550.83Cuscutae SemenMOL000184NSC6355139.250.76Cuscutae SemenMOL000098Quercetin46.430.28Herba Taxilli/Cuscutae SemenMOL000354Isorhamnetin49.600.31Cuscutae SemenMOL000358Beta-sitosterol36.910.75Cuscutae SemenMOL000422Kaempferol41.880.24Cuscutae SemenMOL000953CLR37.870.68Cuscutae SemenMOL005043Campest-5-en-3beta-ol37.580.71Cuscutae SemenMOL005440Isofucosterol43.780.76Cuscutae SemenMOL005944Matrine63.770.25Cuscutae SemenMOL006649Sophranol55.420.28Cuscutae SemenMOL000359Sitosterol36.910.75Herba Taxilli


### 3.2. Drug Targeting and Network Analysis

Traditionally, herbal medicines contain numerous pharmacological compounds, which offer bright prospects for the treatment of complex diseases in a synergistic manner. Network pharmacology has undergone a rapid development in recent years and emerged as an invaluable tool for describing and analyzing complex systems in pharmacology studies. Considering that the multicomponent herbs exert effects on diseases by acting to specific protein targets, drug targeting may shed light on the mechanism of herbs treating TA from the perspective of network pharmacology. In this study, after target fishing by the SysDT [30] model, a total of 153 targets were screened which were targeted by 10 bioactive compounds (two of the 12 bioactive compounds had no direct targets) from Tusizi-Sangjisheng. The detailed information is presented in Table 2.Table 2
The information of related targets of the Tusizi-Sangjisheng herb pair.
No.Target nameGene nameUniProt ID126S proteasome non-ATPase regulatory subunit 3PSMD10O75832272 kDa type IV collagenaseMMP2P08253378 kDa glucose-regulated proteinHSPA5P110214AcetylcholinesteraseACHEP223035Activator of 90 kDa heat shock protein ATPase homolog 1AHSA1O954336Androgen receptorNCOA4Q137727Apoptosis regulator BAXBAXQ078128Arachidonate 5-lipoxygenaseALOX5P099179ATP-binding cassette subfamily G member 2ABCG5Q9H22210Baculoviral IAP repeat-containing protein 5BIRC5O1539211Bcl-2-like protein 1BCL2L1Q0781712Beta-2 adrenergic receptorADRB2P0755013Caspase-8CASP8Q1479014Cathepsin DCTSDP0733915Caveolin-1CAV1Q0313516CD40 ligandCD40LGP2996517Cellular tumor antigen p53TP53P0463718Claudin-4CLDN4O1449319Coagulation factor VIIF7P0870920C-reactive proteinCRPP0274121C-X-C motif chemokine 10CXCL10P0277822C-X-C motif chemokine 11CXCL11O1462523C-X-C motif chemokine 2CXCL2P1987524Cyclin-dependent kinase inhibitor 1CDKN1BP4652725Cytochrome P450 1A1CYP1A1P0479826Cytochrome P450 1B1CYP1B1Q1667827Cytochrome P450 3A4CYP3A4P0868428DDB1- and CUL4-associated factor 5DCAF5Q96JK229Dipeptidyl peptidase IVDPP4P2748730DNA topoisomerase 1TOP1P1138731E-selectinSELEP1658132Estrogen sulfotransferaseSULT1E1P4988833ETS domain-containing protein Elk-1ELK1P1941934G1/S-specific cyclin-D1CCND1P2438535G2/mitotic-specific cyclin-B1CCNB1P1463536Gamma-aminobutyric acid receptor subunit alpha-1GABRA1P1486737Glutathione S-transferase Mu 1GSTM1P0948838Heat shock factor protein 1HSF1Q0061339Heme oxygenase 1HMOX1P0960140Inhibitor of nuclear factor kappa-B kinase subunit alphaCHUKO1511141Insulin receptorINSRP0621342Interleukin-1 alphaIL1AP0158343Interleukin-1 betaIL1BP0158444Interleukin-10IL10P2230145Interleukin-2IL2P6056846Interleukin-6IL6P0523147Interleukin-8CXCL8P1014548Interstitial collagenaseMMP1P0395649Mitogen-activated protein kinase 1Mapk1P6308550mRNA of PKA catalytic subunit C-alphaPRKACAP1761251Myc protooncogene proteinMYCP0110652MyeloperoxidaseMPOP0516453Neutrophil cytosol factor 1NCF1P1459854Nuclear factor erythroid 2-related factor 2NFE2L2Q1623655Nuclear receptor coactivator 2NCOA2Q1559656Peroxisome proliferator-activated receptor alphaPPARAQ0786957Peroxisome proliferator activated receptor deltaPPARDQ0318158Peroxisome proliferator activated receptor gammaPPARGP3723159Plasminogen activator inhibitor 1SERPINE1P0512160Poly [ADP-ribose] polymerase 1TNKSO9527161Potassium voltage-gated channel subfamily H member 2KCNH2Q1280962Prostaglandin G/H synthase 1PTGS1P2321963Protooncogene c-FosFOSP0110064Puromycin-sensitive aminopeptidaseNPEPPSP5578665RAC-alpha serine/threonine-protein kinaseAKT1P3174966RAF protooncogene serine/threonine-protein kinaseRAF1P0404967Ras association domain-containing protein 1RASSF1Q9NS2368Ras GTPase-activating protein 1RASA1P2093669Receptor tyrosine-protein kinase erbB-2ERBB2P0462670Receptor tyrosine-protein kinase erbB-3ERBB3P2186071Retinoic acid receptor RXR-alphaRXRAP1979372Runt-related transcription factor 2RUNX2Q1395073Serine/threonine-protein kinase Chk2CHEK2O9601774Serum paraoxonase/arylesterase 1PON1P2716975Sodium channel protein type 5 subunit alphaSCN5AQ1452476Solute carrier family 2, facilitated glucose transporter member 4SLC2A4P1467277Stromelysin-1MMP3P0825478ThrombinF2P0073479ThrombomodulinTHBDP0720480Transcription factor AP-1JUNP0541281Transcription factor E2F1E2F1Q0109482Transcription factor E2F2E2F2Q1420983Transcription factor p65RELAQ0420684Trypsin-1PRSS1P0747785Type I iodothyronine deiodinaseDIO1P4989586Xanthine dehydrogenase/oxidaseXDHP47989872,4-Dienoyl-CoA reductase, mitochondrialDECR1Q1669888Acetyl-CoA carboxylase 1ACACAQ1308589ATP-citrate synthaseACLYP5339690Coagulation factor XaF10P0074291Cytochrome P450 2B6CYP2B6P2081392Endothelin-converting enzyme 1ECE1P4289293Glucose-6-phosphate 1-dehydrogenaseG6PDP1141394NADPH oxidase 1NOX1Q9Y5S895NADPH oxidase 3NOX3Q9HBY096Nitric oxide synthase, endothelialNOS3P2947497Peroxisomal acyl-coenzyme A oxidase 1ACOX1Q1506798Peroxisomal bifunctional enzymeEHHADHQ0842699Sterol regulatory element-binding protein 1SREBF1P36956100Aldose reductaseAKR1B10O60218101Amine oxidase [flavin-containing] BMAOBP27338102Cyclin-A2CCNA2P20248103Estrogen receptor betaESRRBO95718104Glutamate receptor 2GRIA2P42262105Mitogen-activated protein kinase 14MAPK14Q16539106Nitric oxide synthase, inducibleNOS2P35228107Nuclear receptor coactivator 1NCOA1Q15788108Protooncogene serine/threonine-protein kinase Pim-1PIM1P11309109Serine/threonine-protein kinase Chk1CHEK1O147571105-Hydroxytryptamine 2A receptorHTR2AP28223111Alpha-1A adrenergic receptorADRA1AP35348112Alpha-1B adrenergic receptorADRA1BP35368113Caspase-9CASP9P55211114Dopamine D1 receptorDRD1Q95136115Gamma-aminobutyric-acid receptor alpha-3 subunitGABRA3P34903116Gamma-aminobutyric-acid receptor alpha-5 subunitGABRA5P31644117Muscarinic acetylcholine receptor M2CHRM2P08172118Muscarinic acetylcholine receptor M3CHRM3P20309119Muscarinic acetylcholine receptor M4CHRM4P08173120Mu-type opioid receptorOPRM1P35372121Neuronal acetylcholine receptor protein, alpha-7 chainCHRNA7P36544122Neuronal acetylcholine receptor subunit alpha-2CHRNA2Q15822123Sodium-dependent serotonin transporterSLC6A4P31645124Transforming growth factor beta-1TGFB1P01137125Aldo-keto reductase family 1 member C3AKR1C3P42330126AntileukoproteinaseSLPIP03973127Apoptosis regulator Bcl-2Bcl2P10417128Aryl hydrocarbon receptorAhrP30561129Cytochrome P450 1A2CYP1A2P05177130Gamma-aminobutyric-acid receptor alpha-2 subunitGABRA2P47869131Glutathione S-transferase Mu 2GSTM2P28161132Hyaluronan synthase 2HAS2Q92819133Inhibitor of nuclear factor kappa-B kinase subunit betaIKBKBO14920134Mitogen-activated protein kinase 8MAPK8P45983135Muscarinic acetylcholine receptor M1CHRM1P11229136Nitric oxide synthase, endothelialNOS3P29474137Nuclear receptor subfamily 1 group I member 2NR1I2O75469138Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, gamma isoformPIK3CGP48736139Prostaglandin G/H synthase 2PTGS2P35354140Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoformPPP3CAQ08209141Signal transducer and activator of transcription 1-alpha/betaSTAT1P42224142Sodium-dependent noradrenaline transporterSLC6A2P23975143Vascular cell adhesion protein 1VCAM1P193201444-Aminobutyrate aminotransferase, mitochondrialABATP80404145Alcohol dehydrogenase 1AADH1AP07327146Alcohol dehydrogenase 1BADH1BP00325147Alcohol dehydrogenase 1CADH1CP00326148Caspase-3CASP3P42574149CD44 antigenCD44P16070150Intercellular adhesion molecule 1ICAM1P05362151Tumor necrosis factorTNFP01375152Mineralocorticoid receptorNR3C2P08235153Progesterone receptorPGRP06401


#### 3.2.1. Compound-Target Network

In order to uncover the synergistic effects of multiple components and targets in Tusizi-Sangjisheng, a C-T network analysis was carried out. After excluding 2 bioactive compounds which have no directly related targets, a graph of C-T interactions was drawn (Figure1) using 10 bioactive compounds and 153 related targets. As shown in Figure 1, the blue squares represent bioactive molecules in Tusizi-Sangjisheng, and pink circles represent the corresponding targets. Among the 10 compounds, quercetin owns the highest degree (degree=108), followed by kaempferol, beta-sitosterol, and isorhamnetin, indicating the key roles of these chemicals in Tusizi-Sangjisheng. Targets like prostaglandin G/H synthase 1 (PTGS1), nitric oxide synthase (NOS), tumor necrosis factor (TNF), interleukins-6 (IL-6), and caspase have a high degree of connectivity, revealing the potential functions of treating TA.Figure 1
Compound-Target network (C-T). The blue squares represent 153 targets related to active compounds, and the pink circles represent the 10 bioactive compounds.


#### 3.2.2. Target-Disease Network

Disease-related genes interact in their products and expression patterns. Disease and disease genes are linked together to form a network through known correlations. A variety of disease network diagrams can be generated from the interdependence of cell networks based on human diseases. In these figures, if the phenotype of a disease is related to a molecule, different phenotypes will be linked [32]. In addition, disorders of human diseases should be viewed as disorders of highly connected networks within cells. These new advances and understandings provide a platform for studying the relationship between known genes and diseases, which means that different diseases may originate from the same gene [33].Existing data and theories make it possible to use network methods to study diseases from different scales. Complex network systems can visually reveal the complex relationships among diseases, genes, and pathways to a certain extent. Since the application of network models to biology, many researchers have constructed networks of different functions and properties. These network models have shown us their value in biological researches. At present, the research on diseases mainly includes protein-protein relationship networks, metabolic networks, and regulatory networks. We established a Target-Disease network to study protein-protein interactions and to understand the complex mechanism of action of TCM. This network showed that different diseases have common pathological changes and can be cured by the same compound; that is, one compound can correspond to multiple diseases.The OMIM database (http://www.omim.org/) was used to search target genes related to TA, and the search term was “threatened miscarriage” or “threatened abortion”. The target genes corresponding to the disease is compared with those corresponding to the bioactive compounds, and the common ones are the target genes of the drug in treating TA. A total of 455 targets related to TA were collected through the OMIM database. Comparing it with the 153 targets corresponding to the bioactive compounds of Tusizi-Sangjisheng, 31 common targets preventing miscarriage were obtained. As shown in Figure 2, the outer blue circles represent the targets of Tusizi-Sangjisheng and the inner pink circles represent the targets of TA.Figure 2
Target-Disease network (T-D). The outer blue circles represent targets related to Tusizi-Sangjisheng, and the inner pink circles represent the targets of TA.


#### 3.2.3. Target-Pathway Network

As concerns the effects of drugs that are related to target proteins and also signaling pathways associated with diseases, Target-Pathway network was drawn to analyze the action mechanism of Tusizi-Sangjisheng. As shown in Figure3, the network consists of 116 nodes and 381 edges, and all proteins are involved in more than one signal pathway correlation, forming a highly interconnected network.Figure 3
Target-Pathway network (T-P).


### 3.3. GO Analysis

Upload the predicted target genes to the DAVID database (Version 6.8,https://david.ncifcrf.gov/) and perform GO enrichment analysis. We found that these targets are closely related to various biological processes, such as the RNA polymerase II promoter positive regulation of transcription, positive regulation of reactive oxygen metabolic processes, and inflammatory responses to multicellular biological processes.


### 3.4. Pathway Analysis

The top three pathways with the highest degrees in Figure4 are mitogen-activated protein kinase (MAPK) signaling pathway, the phosphatidylinositol-3-kinase/protein kinase B (PI3K-Akt) signaling pathway, and the transforming growth factor-β (TGF-β) signal transduction pathway. As shown in Figure 5, the MAPK signal pathway is related to targets TNF, IL1, CACN, CASP, JNK, and MAPK14 and exerts the functions of proliferation, differentiation, and inflammation. Up to six targets are involved in the PI3K-Akt pathway as important regulators for the cell progression and survival, including CHRM1, CHRM2, p27, CHUK, Myc, and CDK2. And the TGF-β signal pathway acts on TGFB1 and Myc targets.Figure 4
Gene Ontology (GO) analysis of target genes for treatment. They-axis shows a significantly rich “biological process” category in the GO of the target gene, and the x-axis shows the enrichment score of these terms.Figure 5
Distribution of the target proteins on compressed TA pathways.


## 3.1. Potential Active Compounds

In this study, a total of 75 compounds of the Tusizi-Sangjisheng (29 for Tusizi and 46 for Sangjisheng) were included. After screening all compounds at the criteriathatOB≥30% and DL≥0.18, 12 bioactive compounds with excellent ADME properties were obtained and the specific information is shown in Table 1.Table 1
Bioactive compounds of Cuscutae Semen and Herba Taxilli.
IDCompoundOBDLHerb nameMOL001558Sesamin56.550.83Cuscutae SemenMOL000184NSC6355139.250.76Cuscutae SemenMOL000098Quercetin46.430.28Herba Taxilli/Cuscutae SemenMOL000354Isorhamnetin49.600.31Cuscutae SemenMOL000358Beta-sitosterol36.910.75Cuscutae SemenMOL000422Kaempferol41.880.24Cuscutae SemenMOL000953CLR37.870.68Cuscutae SemenMOL005043Campest-5-en-3beta-ol37.580.71Cuscutae SemenMOL005440Isofucosterol43.780.76Cuscutae SemenMOL005944Matrine63.770.25Cuscutae SemenMOL006649Sophranol55.420.28Cuscutae SemenMOL000359Sitosterol36.910.75Herba Taxilli


## 3.2. Drug Targeting and Network Analysis

Traditionally, herbal medicines contain numerous pharmacological compounds, which offer bright prospects for the treatment of complex diseases in a synergistic manner. Network pharmacology has undergone a rapid development in recent years and emerged as an invaluable tool for describing and analyzing complex systems in pharmacology studies. Considering that the multicomponent herbs exert effects on diseases by acting to specific protein targets, drug targeting may shed light on the mechanism of herbs treating TA from the perspective of network pharmacology. In this study, after target fishing by the SysDT [30] model, a total of 153 targets were screened which were targeted by 10 bioactive compounds (two of the 12 bioactive compounds had no direct targets) from Tusizi-Sangjisheng. The detailed information is presented in Table 2.Table 2
The information of related targets of the Tusizi-Sangjisheng herb pair.
No.Target nameGene nameUniProt ID126S proteasome non-ATPase regulatory subunit 3PSMD10O75832272 kDa type IV collagenaseMMP2P08253378 kDa glucose-regulated proteinHSPA5P110214AcetylcholinesteraseACHEP223035Activator of 90 kDa heat shock protein ATPase homolog 1AHSA1O954336Androgen receptorNCOA4Q137727Apoptosis regulator BAXBAXQ078128Arachidonate 5-lipoxygenaseALOX5P099179ATP-binding cassette subfamily G member 2ABCG5Q9H22210Baculoviral IAP repeat-containing protein 5BIRC5O1539211Bcl-2-like protein 1BCL2L1Q0781712Beta-2 adrenergic receptorADRB2P0755013Caspase-8CASP8Q1479014Cathepsin DCTSDP0733915Caveolin-1CAV1Q0313516CD40 ligandCD40LGP2996517Cellular tumor antigen p53TP53P0463718Claudin-4CLDN4O1449319Coagulation factor VIIF7P0870920C-reactive proteinCRPP0274121C-X-C motif chemokine 10CXCL10P0277822C-X-C motif chemokine 11CXCL11O1462523C-X-C motif chemokine 2CXCL2P1987524Cyclin-dependent kinase inhibitor 1CDKN1BP4652725Cytochrome P450 1A1CYP1A1P0479826Cytochrome P450 1B1CYP1B1Q1667827Cytochrome P450 3A4CYP3A4P0868428DDB1- and CUL4-associated factor 5DCAF5Q96JK229Dipeptidyl peptidase IVDPP4P2748730DNA topoisomerase 1TOP1P1138731E-selectinSELEP1658132Estrogen sulfotransferaseSULT1E1P4988833ETS domain-containing protein Elk-1ELK1P1941934G1/S-specific cyclin-D1CCND1P2438535G2/mitotic-specific cyclin-B1CCNB1P1463536Gamma-aminobutyric acid receptor subunit alpha-1GABRA1P1486737Glutathione S-transferase Mu 1GSTM1P0948838Heat shock factor protein 1HSF1Q0061339Heme oxygenase 1HMOX1P0960140Inhibitor of nuclear factor kappa-B kinase subunit alphaCHUKO1511141Insulin receptorINSRP0621342Interleukin-1 alphaIL1AP0158343Interleukin-1 betaIL1BP0158444Interleukin-10IL10P2230145Interleukin-2IL2P6056846Interleukin-6IL6P0523147Interleukin-8CXCL8P1014548Interstitial collagenaseMMP1P0395649Mitogen-activated protein kinase 1Mapk1P6308550mRNA of PKA catalytic subunit C-alphaPRKACAP1761251Myc protooncogene proteinMYCP0110652MyeloperoxidaseMPOP0516453Neutrophil cytosol factor 1NCF1P1459854Nuclear factor erythroid 2-related factor 2NFE2L2Q1623655Nuclear receptor coactivator 2NCOA2Q1559656Peroxisome proliferator-activated receptor alphaPPARAQ0786957Peroxisome proliferator activated receptor deltaPPARDQ0318158Peroxisome proliferator activated receptor gammaPPARGP3723159Plasminogen activator inhibitor 1SERPINE1P0512160Poly [ADP-ribose] polymerase 1TNKSO9527161Potassium voltage-gated channel subfamily H member 2KCNH2Q1280962Prostaglandin G/H synthase 1PTGS1P2321963Protooncogene c-FosFOSP0110064Puromycin-sensitive aminopeptidaseNPEPPSP5578665RAC-alpha serine/threonine-protein kinaseAKT1P3174966RAF protooncogene serine/threonine-protein kinaseRAF1P0404967Ras association domain-containing protein 1RASSF1Q9NS2368Ras GTPase-activating protein 1RASA1P2093669Receptor tyrosine-protein kinase erbB-2ERBB2P0462670Receptor tyrosine-protein kinase erbB-3ERBB3P2186071Retinoic acid receptor RXR-alphaRXRAP1979372Runt-related transcription factor 2RUNX2Q1395073Serine/threonine-protein kinase Chk2CHEK2O9601774Serum paraoxonase/arylesterase 1PON1P2716975Sodium channel protein type 5 subunit alphaSCN5AQ1452476Solute carrier family 2, facilitated glucose transporter member 4SLC2A4P1467277Stromelysin-1MMP3P0825478ThrombinF2P0073479ThrombomodulinTHBDP0720480Transcription factor AP-1JUNP0541281Transcription factor E2F1E2F1Q0109482Transcription factor E2F2E2F2Q1420983Transcription factor p65RELAQ0420684Trypsin-1PRSS1P0747785Type I iodothyronine deiodinaseDIO1P4989586Xanthine dehydrogenase/oxidaseXDHP47989872,4-Dienoyl-CoA reductase, mitochondrialDECR1Q1669888Acetyl-CoA carboxylase 1ACACAQ1308589ATP-citrate synthaseACLYP5339690Coagulation factor XaF10P0074291Cytochrome P450 2B6CYP2B6P2081392Endothelin-converting enzyme 1ECE1P4289293Glucose-6-phosphate 1-dehydrogenaseG6PDP1141394NADPH oxidase 1NOX1Q9Y5S895NADPH oxidase 3NOX3Q9HBY096Nitric oxide synthase, endothelialNOS3P2947497Peroxisomal acyl-coenzyme A oxidase 1ACOX1Q1506798Peroxisomal bifunctional enzymeEHHADHQ0842699Sterol regulatory element-binding protein 1SREBF1P36956100Aldose reductaseAKR1B10O60218101Amine oxidase [flavin-containing] BMAOBP27338102Cyclin-A2CCNA2P20248103Estrogen receptor betaESRRBO95718104Glutamate receptor 2GRIA2P42262105Mitogen-activated protein kinase 14MAPK14Q16539106Nitric oxide synthase, inducibleNOS2P35228107Nuclear receptor coactivator 1NCOA1Q15788108Protooncogene serine/threonine-protein kinase Pim-1PIM1P11309109Serine/threonine-protein kinase Chk1CHEK1O147571105-Hydroxytryptamine 2A receptorHTR2AP28223111Alpha-1A adrenergic receptorADRA1AP35348112Alpha-1B adrenergic receptorADRA1BP35368113Caspase-9CASP9P55211114Dopamine D1 receptorDRD1Q95136115Gamma-aminobutyric-acid receptor alpha-3 subunitGABRA3P34903116Gamma-aminobutyric-acid receptor alpha-5 subunitGABRA5P31644117Muscarinic acetylcholine receptor M2CHRM2P08172118Muscarinic acetylcholine receptor M3CHRM3P20309119Muscarinic acetylcholine receptor M4CHRM4P08173120Mu-type opioid receptorOPRM1P35372121Neuronal acetylcholine receptor protein, alpha-7 chainCHRNA7P36544122Neuronal acetylcholine receptor subunit alpha-2CHRNA2Q15822123Sodium-dependent serotonin transporterSLC6A4P31645124Transforming growth factor beta-1TGFB1P01137125Aldo-keto reductase family 1 member C3AKR1C3P42330126AntileukoproteinaseSLPIP03973127Apoptosis regulator Bcl-2Bcl2P10417128Aryl hydrocarbon receptorAhrP30561129Cytochrome P450 1A2CYP1A2P05177130Gamma-aminobutyric-acid receptor alpha-2 subunitGABRA2P47869131Glutathione S-transferase Mu 2GSTM2P28161132Hyaluronan synthase 2HAS2Q92819133Inhibitor of nuclear factor kappa-B kinase subunit betaIKBKBO14920134Mitogen-activated protein kinase 8MAPK8P45983135Muscarinic acetylcholine receptor M1CHRM1P11229136Nitric oxide synthase, endothelialNOS3P29474137Nuclear receptor subfamily 1 group I member 2NR1I2O75469138Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit, gamma isoformPIK3CGP48736139Prostaglandin G/H synthase 2PTGS2P35354140Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoformPPP3CAQ08209141Signal transducer and activator of transcription 1-alpha/betaSTAT1P42224142Sodium-dependent noradrenaline transporterSLC6A2P23975143Vascular cell adhesion protein 1VCAM1P193201444-Aminobutyrate aminotransferase, mitochondrialABATP80404145Alcohol dehydrogenase 1AADH1AP07327146Alcohol dehydrogenase 1BADH1BP00325147Alcohol dehydrogenase 1CADH1CP00326148Caspase-3CASP3P42574149CD44 antigenCD44P16070150Intercellular adhesion molecule 1ICAM1P05362151Tumor necrosis factorTNFP01375152Mineralocorticoid receptorNR3C2P08235153Progesterone receptorPGRP06401


### 3.2.1. Compound-Target Network

In order to uncover the synergistic effects of multiple components and targets in Tusizi-Sangjisheng, a C-T network analysis was carried out. After excluding 2 bioactive compounds which have no directly related targets, a graph of C-T interactions was drawn (Figure1) using 10 bioactive compounds and 153 related targets. As shown in Figure 1, the blue squares represent bioactive molecules in Tusizi-Sangjisheng, and pink circles represent the corresponding targets. Among the 10 compounds, quercetin owns the highest degree (degree=108), followed by kaempferol, beta-sitosterol, and isorhamnetin, indicating the key roles of these chemicals in Tusizi-Sangjisheng. Targets like prostaglandin G/H synthase 1 (PTGS1), nitric oxide synthase (NOS), tumor necrosis factor (TNF), interleukins-6 (IL-6), and caspase have a high degree of connectivity, revealing the potential functions of treating TA.Figure 1
Compound-Target network (C-T). The blue squares represent 153 targets related to active compounds, and the pink circles represent the 10 bioactive compounds.


### 3.2.2. Target-Disease Network

Disease-related genes interact in their products and expression patterns. Disease and disease genes are linked together to form a network through known correlations. A variety of disease network diagrams can be generated from the interdependence of cell networks based on human diseases. In these figures, if the phenotype of a disease is related to a molecule, different phenotypes will be linked [32]. In addition, disorders of human diseases should be viewed as disorders of highly connected networks within cells. These new advances and understandings provide a platform for studying the relationship between known genes and diseases, which means that different diseases may originate from the same gene [33].Existing data and theories make it possible to use network methods to study diseases from different scales. Complex network systems can visually reveal the complex relationships among diseases, genes, and pathways to a certain extent. Since the application of network models to biology, many researchers have constructed networks of different functions and properties. These network models have shown us their value in biological researches. At present, the research on diseases mainly includes protein-protein relationship networks, metabolic networks, and regulatory networks. We established a Target-Disease network to study protein-protein interactions and to understand the complex mechanism of action of TCM. This network showed that different diseases have common pathological changes and can be cured by the same compound; that is, one compound can correspond to multiple diseases.The OMIM database (http://www.omim.org/) was used to search target genes related to TA, and the search term was “threatened miscarriage” or “threatened abortion”. The target genes corresponding to the disease is compared with those corresponding to the bioactive compounds, and the common ones are the target genes of the drug in treating TA. A total of 455 targets related to TA were collected through the OMIM database. Comparing it with the 153 targets corresponding to the bioactive compounds of Tusizi-Sangjisheng, 31 common targets preventing miscarriage were obtained. As shown in Figure 2, the outer blue circles represent the targets of Tusizi-Sangjisheng and the inner pink circles represent the targets of TA.Figure 2
Target-Disease network (T-D). The outer blue circles represent targets related to Tusizi-Sangjisheng, and the inner pink circles represent the targets of TA.


### 3.2.3. Target-Pathway Network

As concerns the effects of drugs that are related to target proteins and also signaling pathways associated with diseases, Target-Pathway network was drawn to analyze the action mechanism of Tusizi-Sangjisheng. As shown in Figure3, the network consists of 116 nodes and 381 edges, and all proteins are involved in more than one signal pathway correlation, forming a highly interconnected network.Figure 3
Target-Pathway network (T-P).


## 3.2.1. Compound-Target Network

In order to uncover the synergistic effects of multiple components and targets in Tusizi-Sangjisheng, a C-T network analysis was carried out. After excluding 2 bioactive compounds which have no directly related targets, a graph of C-T interactions was drawn (Figure1) using 10 bioactive compounds and 153 related targets. As shown in Figure 1, the blue squares represent bioactive molecules in Tusizi-Sangjisheng, and pink circles represent the corresponding targets. Among the 10 compounds, quercetin owns the highest degree (degree=108), followed by kaempferol, beta-sitosterol, and isorhamnetin, indicating the key roles of these chemicals in Tusizi-Sangjisheng. Targets like prostaglandin G/H synthase 1 (PTGS1), nitric oxide synthase (NOS), tumor necrosis factor (TNF), interleukins-6 (IL-6), and caspase have a high degree of connectivity, revealing the potential functions of treating TA.Figure 1
Compound-Target network (C-T). The blue squares represent 153 targets related to active compounds, and the pink circles represent the 10 bioactive compounds.


## 3.2.2. Target-Disease Network

Disease-related genes interact in their products and expression patterns. Disease and disease genes are linked together to form a network through known correlations. A variety of disease network diagrams can be generated from the interdependence of cell networks based on human diseases. In these figures, if the phenotype of a disease is related to a molecule, different phenotypes will be linked [32]. In addition, disorders of human diseases should be viewed as disorders of highly connected networks within cells. These new advances and understandings provide a platform for studying the relationship between known genes and diseases, which means that different diseases may originate from the same gene [33].Existing data and theories make it possible to use network methods to study diseases from different scales. Complex network systems can visually reveal the complex relationships among diseases, genes, and pathways to a certain extent. Since the application of network models to biology, many researchers have constructed networks of different functions and properties. These network models have shown us their value in biological researches. At present, the research on diseases mainly includes protein-protein relationship networks, metabolic networks, and regulatory networks. We established a Target-Disease network to study protein-protein interactions and to understand the complex mechanism of action of TCM. This network showed that different diseases have common pathological changes and can be cured by the same compound; that is, one compound can correspond to multiple diseases.The OMIM database (http://www.omim.org/) was used to search target genes related to TA, and the search term was “threatened miscarriage” or “threatened abortion”. The target genes corresponding to the disease is compared with those corresponding to the bioactive compounds, and the common ones are the target genes of the drug in treating TA. A total of 455 targets related to TA were collected through the OMIM database. Comparing it with the 153 targets corresponding to the bioactive compounds of Tusizi-Sangjisheng, 31 common targets preventing miscarriage were obtained. As shown in Figure 2, the outer blue circles represent the targets of Tusizi-Sangjisheng and the inner pink circles represent the targets of TA.Figure 2
Target-Disease network (T-D). The outer blue circles represent targets related to Tusizi-Sangjisheng, and the inner pink circles represent the targets of TA.


## 3.2.3. Target-Pathway Network

As concerns the effects of drugs that are related to target proteins and also signaling pathways associated with diseases, Target-Pathway network was drawn to analyze the action mechanism of Tusizi-Sangjisheng. As shown in Figure3, the network consists of 116 nodes and 381 edges, and all proteins are involved in more than one signal pathway correlation, forming a highly interconnected network.Figure 3
Target-Pathway network (T-P).


## 3.3. GO Analysis

Upload the predicted target genes to the DAVID database (Version 6.8,https://david.ncifcrf.gov/) and perform GO enrichment analysis. We found that these targets are closely related to various biological processes, such as the RNA polymerase II promoter positive regulation of transcription, positive regulation of reactive oxygen metabolic processes, and inflammatory responses to multicellular biological processes.


## 3.4. Pathway Analysis

The top three pathways with the highest degrees in Figure4 are mitogen-activated protein kinase (MAPK) signaling pathway, the phosphatidylinositol-3-kinase/protein kinase B (PI3K-Akt) signaling pathway, and the transforming growth factor-β (TGF-β) signal transduction pathway. As shown in Figure 5, the MAPK signal pathway is related to targets TNF, IL1, CACN, CASP, JNK, and MAPK14 and exerts the functions of proliferation, differentiation, and inflammation. Up to six targets are involved in the PI3K-Akt pathway as important regulators for the cell progression and survival, including CHRM1, CHRM2, p27, CHUK, Myc, and CDK2. And the TGF-β signal pathway acts on TGFB1 and Myc targets.Figure 4
Gene Ontology (GO) analysis of target genes for treatment. They-axis shows a significantly rich “biological process” category in the GO of the target gene, and the x-axis shows the enrichment score of these terms.Figure 5
Distribution of the target proteins on compressed TA pathways.


## 4. Discussions

### 4.1. Bioactive Compounds of the Tusizi-Sangjisheng Herb Pair

In this study, we used systematic pharmacology to study Tusizi-Sangjisheng and found that this herb pair has a total of 12 compounds with good OB and DL properties, which are related to 153 target proteins. The most potent compounds in Tusizi-Sangjisheng is quercetin which is followed by kaempferol,β-sitosterol, and isorhamnetin. Quercetin, kaempferol, and isorhamnetin are all flavonoids that are abundant in amount and widely distributed in nature. Quercetin is one of the phytochemicals with anticancer and antioxidant activities that widely exists in nuts, teas, vegetables, herbs, and generally diet of people [34, 35]. In addition to the above effects, quercetin still has the anti-inflammatory functions by blocking the secretion of IL-6, IFN-γ, and TNF-α and increasing the decreased ratio of Bcl-2/Bax apoptotic proteins induced by lipopolysaccharide (LPS) revealing the putative value in preventing miscarriage caused by bacterial infection [36–38]. Kaempferol is a typical natural flavonol and has been reported to own many beneficial functions such as anti-inflammatory, antioxidative, antiatherogenic, hepatoprotective, neuroprotective, antidiabetic, and anticancer activities [39, 40]. It is well known for its prominent antioxidative activity [39], and the main mechanisms include decreasing the susceptibility of low-density lipoproteins (LDL) to oxidation, inhibiting the release of cytochrome C, scavenging excessive reactive oxygen species (ROS), and inhibiting the generation of ROS by regulating the level of NOS/NO, reducing the accumulation of toxic lipid peroxidation product malondialdehyde (MDA), and maintaining the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in a normal level [41–43]. Isorhamnetin is a flavonoid present in many plants and has been reported to protect against inflammatory and oxidative stress responses in various in vitro and in vivo models using LPS, inflammatory cytokines, and ischemic injury. The anti-inflammatory function of isorhamnetin is regarded to be related to inhibition of NF-κB signaling activity, and its antioxidative effect is associated with ROS blocking [44]. Besides, isorhamnetin also has the anticancer activity by acting on Akt and MAPK signal pathways [45]. β-Sitosterol, one of the several phytosterols, is a natural micronutrient in higher plants possessing numerous physiological effects including antioxidative, anticancer, anti-inflammatory, antidiabetic, and immune modulation activities [46]. Although quercetin, kaempferol, isorhamnetin, and β-sitosterol have wide pharmacological functions, the detailed correlation with TA is still unclear and needs to be further studied.


### 4.2. Main Targets of the Tusizi-Sangjisheng Herb Pair

#### 4.2.1. PTGS

PTGS, also known as cyclooxygenase (COX) [47], is a crucial enzyme for the synthesis of prostaglandins (PG) in the body, which can transform arachidonic acid into various types of PGs. There are three known subtypes of PTGS: PTGS1, PTGS2, and PTGS3. PTGS1 exists in most cells and exerts the function of maintaining and regulating the normal physiological activities by synthesizing PG. PG is closely related to physiological processes such as endometrial vascular regeneration, vascular permeability, and establishment of a placental vascular network during pregnancy [48]. COX is considered a key factor in implantation [49]. Studies have shown that the expression level of COX-2 in the women’s endometrium during the midluteal phase of women’s menstrual cycles begins to create the necessary conditions for conception. Once trophoblast invasion occurs, the COX-2 content at the invaded site will increase continuously [50], and IL-1 was found to increase COX-2 expression in endometrial cells at the implantation site [51]. The expression of COX in patients with TA/recurrent miscarriage is lower than that in normal pregnancy, which may interfere the process of embryo implantation through various PG molecules it catalyzes and participate in the pathogenic process of TA.


#### 4.2.2. NOS

NOS is the only rate-limiting enzyme that synthesizes NO. It belongs to isozymes and has three subtypes: neuronal (nNOS/NOS1), inducible (iNOS/NOS2), and endothelial NOS (eNOS/NOS3) [52], among which NOS3 is mainly expressed in vascular endothelial cells and catalyzes the production of intravascular NO. NO is a signaling molecule in the cardiovascular system that has the properties of smooth muscle relaxation, platelet inhibition, leukocyte aggregation, and also attenuation of vascular smooth muscle cell proliferation, neurotransmission, and immune defense [53]. NOS3 is also expressed in the placental tissue during pregnancy. It promotes the synthesis of NO to participate in the regulation of blood circulation in the placenta, causing a slight decrease in vascular resistance and blood pressure, which is of great significance for maintaining uterine placental blood flow. Studies have found that the expression of NOS in endothelial cells in the plasma and placenta is increased in women with preeclampsia of its compensatory mechanisms [54, 55]. The hypothesis of the pathogenesis of preeclampsia is that the expression of NOS decreased and NO synthesis reduced, resulting in increased vascular bed resistance and blocked placental blood circulation, while the successful pregnancy during the first trimester depends on the favoring invasion of trophoblasts to the endometrium and the success remodeling of the uterine spiral arteries, and NO plays important roles in this process. Paradisi et al.’s research has shown that serum NO levels in patients with TA are lower than those in normal pregnancy [56], indicating that TA may be associated with decreased NOS expression which is similar to that of preeclampsia. A reduced NO level results in the disturbance of maternal-fetal circulation and further influences the supply of blood and oxygen which may cause TA.


#### 4.2.3. TNF

TNF is a cytokine secreted by macrophages that can directly cause death of tumor cells. TNF-α and TNF-β are the two known types of TNF that can be expressed in immune cells such as T and B cells. IL-6 (interleukin 6), which belongs to the Th2-type cytokine, is a type of cytokine involved in a variety of immune inflammatory responses that could inhibit Th1-mediated immune responses. Th1 immunity causes loss of pregnancy while Th2 immunity helps to maintain pregnancy, so the ratio of Th1/Th2 is crucial for pregnancy. If the balance of Th1/Th2 is broken, TA or miscarriage may occur. TNF-α is mainly expressed by Th1 and NK cells that can kill tumor cells and also attack trophoblast cells and embryonic tissues. A high serum level of TNF-α and inadequate expression of IL-6 in endometrial tissue have been found in patients with recurrent abortion, revealing that the overproduction of TNF or underactivation of IL-6 may cause fetus damage and loss [57, 58]. In this study, the active ingredients in Tusizi-Sangjisheng act on the targets IL-6 and TNF-α to promote the expression of IL-6, increase its level in plasma and decidual tissue, and then inhibit the expression of TNF-α so as to reduce the maternal immune response against the fetus and maintain the normal development of the pregnancy.


#### 4.2.4. Caspase

Caspase is a type of protease and an important mediator of programmed cell death (apoptosis) that includes caspase-1 (containing caspase-1, 4, 5, and 11), caspase-2 (containing caspase-2 and 9), and caspase-3 (containing caspase-3, 6, 7, 8, and 10). Among them, caspase-3 is mainly expressed in the cytoplasm of trophoblast cells and could regulate cell growth and apoptosis by activating the Fas pathway. Apoptosis is essential for the normal physiology of pregnancy during implantation since apoptosis of trophoblast cells is important for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo [59, 60]. The overexpression of caspase-3 will cause abnormal apoptosis of trophoblasts, affect embryo development, and result in abortion. Meresman et al.’s study indicated that the expression level of caspase-3 in out-of-phase endometrium abortion patients is higher than that in normal, and the decreased cell proliferation and augmented cell apoptosis were also found [61]. The bioactive components of Tusizi-Sangjisheng may act on caspase-3 and suppress its expression to reduce the apoptosis of trophoblast cells.


### 4.3. Signal Pathways of the Tusizi-Sangjisheng Herb Pair

#### 4.3.1. MAPK Signal Pathway

MAPK is a class of serine/threonine protein kinases and widely present in various cells. It has important regulatory effects on gene expression. After activation, the protein migrates into the phosphorylated nucleoproteins and membrane receptors, regulating gene transcription and other life events. It consists of four separate signaling cascades: the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinases); the ERKs (extracellular signal-regulated kinases); the ERK5 or big MAPK1; and the p38MAPK group of protein kinases. When stimulated by extracellular signals, the MAPK cascade which is an evolutionarily conserved tertiary kinase cascade transduction signal will be activated by a series of complicated chemical reactions, resulting in the successive activation of MAPKKK, MAPKK, and MAPK through the phosphorylation of amino acid residues [62]. Evidences have shown that the MAPK signal pathway is activated in many processes during embryo implantation and is closely related to the invasion and proliferation of trophoblast cells and decidual stromal cells (DSCs) which play key roles in the maintenance of normal pregnancy [63, 64]. Z. Wang et al.’s research demonstrated the abnormal expressions of nucleotide-binding oligomerization domains 1 and 2 (NOD 1 and NOD 2) in villi from recurrent spontaneous abortion (RSA) patients that then inhibit the invasion and proliferation of trophoblast cells by activating the p38MAPK signal pathway [65]. In addition, inflammation (including sterile inflammation or infectious inflammation induced by bacteria such as bacterial vaginosis) and oxidative stress (smoking) could also activate the p38MAPK signal pathway and thus attenuate the normal development of trophoblast cells [66]. Furthermore, the activation of the JNK and ERK1/2 signal pathway by interleukin (IL)-33 [67] and IL-25 [68] also leads to the enhancement of invasion and proliferation of DSCs. Accordingly, for the similar pathogenesis between RAS and TA, the role of the MAPK signal pathway could also be considered by affecting the invasion and proliferation of trophoblast cells and DSCs even though there is no direct evidence. The total flavones of Cuscutae Semen were found to be effective in suppressing abortion by inhibiting MAPK pathways [69], indicating the potential mechanism of the herb pair Tusizi-Sangjisheng treating TA.


#### 4.3.2. PI3K-Akt Signal Pathway

The PI3K-Akt signal pathway consists of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB, also known as Akt) and is involved in transcription, protein synthesis, migration, apoptosis, and proliferation activities [70]. Identically, the PI3K-Akt signaling pathway also participates in the proliferation and migration of trophoblast cells that are vital for establishing and persisting normal success pregnancy. Evidence has shown that the up-/downregulation of the PI3K-Akt signal pathway is related to the proliferation and migration of trophoblast cells [71]. PI3K signaling is considered to have association with the secretion of several hormones including gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) that are important in the maintenance of normal pregnancy [72]. In addition, PI3K could also regulate insulin signaling via activating the Akt pathway [72]. Generally, obese persons with insulin resistance are often companied by reduced hypothalamic GnRH secretion and decreased fertility [73]. An experimental study also demonstrated that herbal decoctions containing herbs prevent miscarriage and are effective to promote the follicle development and fertility via activation of the PI3K-Akt signal pathway [74]. Accordingly, we assume that Tusizi-Sangjisheng may suppress miscarriage via the PI3K-Akt signal pathway by influencing the expression of sex hormones GnRH/LH and also the proliferation and migration of trophoblast cells.


#### 4.3.3. TGF-β Signal Pathway

TGF-β is a multifunctional cytokine with three isoforms (TGF-β1, TGF-β2, and TGF-β3) and acts mainly through Smad pathways [75]. Evidences have proved that TGF-β plays pivotal and dual roles in the regulation of cell growth and apoptosis which is vividly described as a “switch” [76]. During early pregnancy, the uterine cells undergo apoptosis and proliferation for the successful implantation of embryo. Those complex processes are controlled and regulated by several signal pathways, and the TGF-β signal pathway is one of them that could help to maintain normal pregnancy by balancing the proliferation and apoptosis of trophoblast cells [75]. In addition, the TGF-β signal pathway is also involved in immune and inflammatory responses by regulating indoleamine 2,3-dioxygense (IDO) expression to promote immune tolerance of the maternal-fetal interface [77], inhibiting the proliferation of human Th1 memory T cells [78]. At present, there is no direct evidence illustrating the specific relationship among herbs like Cuscutae Semen and the TGF-β signal pathway in TA. Yet we can hypothesize based on the above information that the active ingredients in the herb pair Tusizi-Sangjisheng exert their TA-preventing function by regulating the immune responses in maternal-fetal interface and controlling the normal apoptosis and proliferation of uterine cells through the TGF-β signal pathway.In normal pregnancy, the balance of the immune microenvironment at the maternal-fetal interface can ensure immune tolerance and protect the embryo from maternal immune rejection. The ability of gestational trophoblasts to proliferate, differentiate, migrate, and invade is crucial for embryo implantation, placenta formation, and embryo growth and development. Trophoblasts are the main cell types of placental tissues that differentiate into extravillous trophoblasts, syncytiotrophoblasts and cell trophoblasts. Extravillous trophoblasts can be further differentiated into cells with high infiltration capacity. Interstitial trophoblast cells and intravascular trophoblast cells then invade the uterine decidua and reshape the spiral arteries of a pregnant uterus, thereby forming placental tissues which provide nutrients for the development of the embryo. Therefore, infiltration of trophoblasts into the uterine decidua is the key step to the success of early pregnancy. If the ability of trophoblasts to proliferate, migrate, and invade is weakened, it will affect the remodeling of decidual tissue blood vessels and the formation of placenta and further develop into pregnancy-dependent complications such as abortion, preeclampsia, and intrauterine growth restriction. These processes during pregnancy are jointly regulated by many signal transduction pathways. However, the exact regulatory mechanisms are still not very clear. Three key signaling pathways mentioned above were screened by systematic methods in this study revealing that Tusizi-Sangjisheng may effectively reduce miscarriage rates via regulating the normal expression and function of those three signal pathways, correcting under- or overexpression and maintaining normal pregnancy.


## 4.1. Bioactive Compounds of the Tusizi-Sangjisheng Herb Pair

In this study, we used systematic pharmacology to study Tusizi-Sangjisheng and found that this herb pair has a total of 12 compounds with good OB and DL properties, which are related to 153 target proteins. The most potent compounds in Tusizi-Sangjisheng is quercetin which is followed by kaempferol,β-sitosterol, and isorhamnetin. Quercetin, kaempferol, and isorhamnetin are all flavonoids that are abundant in amount and widely distributed in nature. Quercetin is one of the phytochemicals with anticancer and antioxidant activities that widely exists in nuts, teas, vegetables, herbs, and generally diet of people [34, 35]. In addition to the above effects, quercetin still has the anti-inflammatory functions by blocking the secretion of IL-6, IFN-γ, and TNF-α and increasing the decreased ratio of Bcl-2/Bax apoptotic proteins induced by lipopolysaccharide (LPS) revealing the putative value in preventing miscarriage caused by bacterial infection [36–38]. Kaempferol is a typical natural flavonol and has been reported to own many beneficial functions such as anti-inflammatory, antioxidative, antiatherogenic, hepatoprotective, neuroprotective, antidiabetic, and anticancer activities [39, 40]. It is well known for its prominent antioxidative activity [39], and the main mechanisms include decreasing the susceptibility of low-density lipoproteins (LDL) to oxidation, inhibiting the release of cytochrome C, scavenging excessive reactive oxygen species (ROS), and inhibiting the generation of ROS by regulating the level of NOS/NO, reducing the accumulation of toxic lipid peroxidation product malondialdehyde (MDA), and maintaining the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in a normal level [41–43]. Isorhamnetin is a flavonoid present in many plants and has been reported to protect against inflammatory and oxidative stress responses in various in vitro and in vivo models using LPS, inflammatory cytokines, and ischemic injury. The anti-inflammatory function of isorhamnetin is regarded to be related to inhibition of NF-κB signaling activity, and its antioxidative effect is associated with ROS blocking [44]. Besides, isorhamnetin also has the anticancer activity by acting on Akt and MAPK signal pathways [45]. β-Sitosterol, one of the several phytosterols, is a natural micronutrient in higher plants possessing numerous physiological effects including antioxidative, anticancer, anti-inflammatory, antidiabetic, and immune modulation activities [46]. Although quercetin, kaempferol, isorhamnetin, and β-sitosterol have wide pharmacological functions, the detailed correlation with TA is still unclear and needs to be further studied.


## 4.2. Main Targets of the Tusizi-Sangjisheng Herb Pair

### 4.2.1. PTGS

PTGS, also known as cyclooxygenase (COX) [47], is a crucial enzyme for the synthesis of prostaglandins (PG) in the body, which can transform arachidonic acid into various types of PGs. There are three known subtypes of PTGS: PTGS1, PTGS2, and PTGS3. PTGS1 exists in most cells and exerts the function of maintaining and regulating the normal physiological activities by synthesizing PG. PG is closely related to physiological processes such as endometrial vascular regeneration, vascular permeability, and establishment of a placental vascular network during pregnancy [48]. COX is considered a key factor in implantation [49]. Studies have shown that the expression level of COX-2 in the women’s endometrium during the midluteal phase of women’s menstrual cycles begins to create the necessary conditions for conception. Once trophoblast invasion occurs, the COX-2 content at the invaded site will increase continuously [50], and IL-1 was found to increase COX-2 expression in endometrial cells at the implantation site [51]. The expression of COX in patients with TA/recurrent miscarriage is lower than that in normal pregnancy, which may interfere the process of embryo implantation through various PG molecules it catalyzes and participate in the pathogenic process of TA.


### 4.2.2. NOS

NOS is the only rate-limiting enzyme that synthesizes NO. It belongs to isozymes and has three subtypes: neuronal (nNOS/NOS1), inducible (iNOS/NOS2), and endothelial NOS (eNOS/NOS3) [52], among which NOS3 is mainly expressed in vascular endothelial cells and catalyzes the production of intravascular NO. NO is a signaling molecule in the cardiovascular system that has the properties of smooth muscle relaxation, platelet inhibition, leukocyte aggregation, and also attenuation of vascular smooth muscle cell proliferation, neurotransmission, and immune defense [53]. NOS3 is also expressed in the placental tissue during pregnancy. It promotes the synthesis of NO to participate in the regulation of blood circulation in the placenta, causing a slight decrease in vascular resistance and blood pressure, which is of great significance for maintaining uterine placental blood flow. Studies have found that the expression of NOS in endothelial cells in the plasma and placenta is increased in women with preeclampsia of its compensatory mechanisms [54, 55]. The hypothesis of the pathogenesis of preeclampsia is that the expression of NOS decreased and NO synthesis reduced, resulting in increased vascular bed resistance and blocked placental blood circulation, while the successful pregnancy during the first trimester depends on the favoring invasion of trophoblasts to the endometrium and the success remodeling of the uterine spiral arteries, and NO plays important roles in this process. Paradisi et al.’s research has shown that serum NO levels in patients with TA are lower than those in normal pregnancy [56], indicating that TA may be associated with decreased NOS expression which is similar to that of preeclampsia. A reduced NO level results in the disturbance of maternal-fetal circulation and further influences the supply of blood and oxygen which may cause TA.


### 4.2.3. TNF

TNF is a cytokine secreted by macrophages that can directly cause death of tumor cells. TNF-α and TNF-β are the two known types of TNF that can be expressed in immune cells such as T and B cells. IL-6 (interleukin 6), which belongs to the Th2-type cytokine, is a type of cytokine involved in a variety of immune inflammatory responses that could inhibit Th1-mediated immune responses. Th1 immunity causes loss of pregnancy while Th2 immunity helps to maintain pregnancy, so the ratio of Th1/Th2 is crucial for pregnancy. If the balance of Th1/Th2 is broken, TA or miscarriage may occur. TNF-α is mainly expressed by Th1 and NK cells that can kill tumor cells and also attack trophoblast cells and embryonic tissues. A high serum level of TNF-α and inadequate expression of IL-6 in endometrial tissue have been found in patients with recurrent abortion, revealing that the overproduction of TNF or underactivation of IL-6 may cause fetus damage and loss [57, 58]. In this study, the active ingredients in Tusizi-Sangjisheng act on the targets IL-6 and TNF-α to promote the expression of IL-6, increase its level in plasma and decidual tissue, and then inhibit the expression of TNF-α so as to reduce the maternal immune response against the fetus and maintain the normal development of the pregnancy.


### 4.2.4. Caspase

Caspase is a type of protease and an important mediator of programmed cell death (apoptosis) that includes caspase-1 (containing caspase-1, 4, 5, and 11), caspase-2 (containing caspase-2 and 9), and caspase-3 (containing caspase-3, 6, 7, 8, and 10). Among them, caspase-3 is mainly expressed in the cytoplasm of trophoblast cells and could regulate cell growth and apoptosis by activating the Fas pathway. Apoptosis is essential for the normal physiology of pregnancy during implantation since apoptosis of trophoblast cells is important for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo [59, 60]. The overexpression of caspase-3 will cause abnormal apoptosis of trophoblasts, affect embryo development, and result in abortion. Meresman et al.’s study indicated that the expression level of caspase-3 in out-of-phase endometrium abortion patients is higher than that in normal, and the decreased cell proliferation and augmented cell apoptosis were also found [61]. The bioactive components of Tusizi-Sangjisheng may act on caspase-3 and suppress its expression to reduce the apoptosis of trophoblast cells.


## 4.2.1. PTGS

PTGS, also known as cyclooxygenase (COX) [47], is a crucial enzyme for the synthesis of prostaglandins (PG) in the body, which can transform arachidonic acid into various types of PGs. There are three known subtypes of PTGS: PTGS1, PTGS2, and PTGS3. PTGS1 exists in most cells and exerts the function of maintaining and regulating the normal physiological activities by synthesizing PG. PG is closely related to physiological processes such as endometrial vascular regeneration, vascular permeability, and establishment of a placental vascular network during pregnancy [48]. COX is considered a key factor in implantation [49]. Studies have shown that the expression level of COX-2 in the women’s endometrium during the midluteal phase of women’s menstrual cycles begins to create the necessary conditions for conception. Once trophoblast invasion occurs, the COX-2 content at the invaded site will increase continuously [50], and IL-1 was found to increase COX-2 expression in endometrial cells at the implantation site [51]. The expression of COX in patients with TA/recurrent miscarriage is lower than that in normal pregnancy, which may interfere the process of embryo implantation through various PG molecules it catalyzes and participate in the pathogenic process of TA.


## 4.2.2. NOS

NOS is the only rate-limiting enzyme that synthesizes NO. It belongs to isozymes and has three subtypes: neuronal (nNOS/NOS1), inducible (iNOS/NOS2), and endothelial NOS (eNOS/NOS3) [52], among which NOS3 is mainly expressed in vascular endothelial cells and catalyzes the production of intravascular NO. NO is a signaling molecule in the cardiovascular system that has the properties of smooth muscle relaxation, platelet inhibition, leukocyte aggregation, and also attenuation of vascular smooth muscle cell proliferation, neurotransmission, and immune defense [53]. NOS3 is also expressed in the placental tissue during pregnancy. It promotes the synthesis of NO to participate in the regulation of blood circulation in the placenta, causing a slight decrease in vascular resistance and blood pressure, which is of great significance for maintaining uterine placental blood flow. Studies have found that the expression of NOS in endothelial cells in the plasma and placenta is increased in women with preeclampsia of its compensatory mechanisms [54, 55]. The hypothesis of the pathogenesis of preeclampsia is that the expression of NOS decreased and NO synthesis reduced, resulting in increased vascular bed resistance and blocked placental blood circulation, while the successful pregnancy during the first trimester depends on the favoring invasion of trophoblasts to the endometrium and the success remodeling of the uterine spiral arteries, and NO plays important roles in this process. Paradisi et al.’s research has shown that serum NO levels in patients with TA are lower than those in normal pregnancy [56], indicating that TA may be associated with decreased NOS expression which is similar to that of preeclampsia. A reduced NO level results in the disturbance of maternal-fetal circulation and further influences the supply of blood and oxygen which may cause TA.


## 4.2.3. TNF

TNF is a cytokine secreted by macrophages that can directly cause death of tumor cells. TNF-α and TNF-β are the two known types of TNF that can be expressed in immune cells such as T and B cells. IL-6 (interleukin 6), which belongs to the Th2-type cytokine, is a type of cytokine involved in a variety of immune inflammatory responses that could inhibit Th1-mediated immune responses. Th1 immunity causes loss of pregnancy while Th2 immunity helps to maintain pregnancy, so the ratio of Th1/Th2 is crucial for pregnancy. If the balance of Th1/Th2 is broken, TA or miscarriage may occur. TNF-α is mainly expressed by Th1 and NK cells that can kill tumor cells and also attack trophoblast cells and embryonic tissues. A high serum level of TNF-α and inadequate expression of IL-6 in endometrial tissue have been found in patients with recurrent abortion, revealing that the overproduction of TNF or underactivation of IL-6 may cause fetus damage and loss [57, 58]. In this study, the active ingredients in Tusizi-Sangjisheng act on the targets IL-6 and TNF-α to promote the expression of IL-6, increase its level in plasma and decidual tissue, and then inhibit the expression of TNF-α so as to reduce the maternal immune response against the fetus and maintain the normal development of the pregnancy.


## 4.2.4. Caspase

Caspase is a type of protease and an important mediator of programmed cell death (apoptosis) that includes caspase-1 (containing caspase-1, 4, 5, and 11), caspase-2 (containing caspase-2 and 9), and caspase-3 (containing caspase-3, 6, 7, 8, and 10). Among them, caspase-3 is mainly expressed in the cytoplasm of trophoblast cells and could regulate cell growth and apoptosis by activating the Fas pathway. Apoptosis is essential for the normal physiology of pregnancy during implantation since apoptosis of trophoblast cells is important for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo [59, 60]. The overexpression of caspase-3 will cause abnormal apoptosis of trophoblasts, affect embryo development, and result in abortion. Meresman et al.’s study indicated that the expression level of caspase-3 in out-of-phase endometrium abortion patients is higher than that in normal, and the decreased cell proliferation and augmented cell apoptosis were also found [61]. The bioactive components of Tusizi-Sangjisheng may act on caspase-3 and suppress its expression to reduce the apoptosis of trophoblast cells.


## 4.3. Signal Pathways of the Tusizi-Sangjisheng Herb Pair

### 4.3.1. MAPK Signal Pathway

MAPK is a class of serine/threonine protein kinases and widely present in various cells. It has important regulatory effects on gene expression. After activation, the protein migrates into the phosphorylated nucleoproteins and membrane receptors, regulating gene transcription and other life events. It consists of four separate signaling cascades: the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinases); the ERKs (extracellular signal-regulated kinases); the ERK5 or big MAPK1; and the p38MAPK group of protein kinases. When stimulated by extracellular signals, the MAPK cascade which is an evolutionarily conserved tertiary kinase cascade transduction signal will be activated by a series of complicated chemical reactions, resulting in the successive activation of MAPKKK, MAPKK, and MAPK through the phosphorylation of amino acid residues [62]. Evidences have shown that the MAPK signal pathway is activated in many processes during embryo implantation and is closely related to the invasion and proliferation of trophoblast cells and decidual stromal cells (DSCs) which play key roles in the maintenance of normal pregnancy [63, 64]. Z. Wang et al.’s research demonstrated the abnormal expressions of nucleotide-binding oligomerization domains 1 and 2 (NOD 1 and NOD 2) in villi from recurrent spontaneous abortion (RSA) patients that then inhibit the invasion and proliferation of trophoblast cells by activating the p38MAPK signal pathway [65]. In addition, inflammation (including sterile inflammation or infectious inflammation induced by bacteria such as bacterial vaginosis) and oxidative stress (smoking) could also activate the p38MAPK signal pathway and thus attenuate the normal development of trophoblast cells [66]. Furthermore, the activation of the JNK and ERK1/2 signal pathway by interleukin (IL)-33 [67] and IL-25 [68] also leads to the enhancement of invasion and proliferation of DSCs. Accordingly, for the similar pathogenesis between RAS and TA, the role of the MAPK signal pathway could also be considered by affecting the invasion and proliferation of trophoblast cells and DSCs even though there is no direct evidence. The total flavones of Cuscutae Semen were found to be effective in suppressing abortion by inhibiting MAPK pathways [69], indicating the potential mechanism of the herb pair Tusizi-Sangjisheng treating TA.


### 4.3.2. PI3K-Akt Signal Pathway

The PI3K-Akt signal pathway consists of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB, also known as Akt) and is involved in transcription, protein synthesis, migration, apoptosis, and proliferation activities [70]. Identically, the PI3K-Akt signaling pathway also participates in the proliferation and migration of trophoblast cells that are vital for establishing and persisting normal success pregnancy. Evidence has shown that the up-/downregulation of the PI3K-Akt signal pathway is related to the proliferation and migration of trophoblast cells [71]. PI3K signaling is considered to have association with the secretion of several hormones including gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) that are important in the maintenance of normal pregnancy [72]. In addition, PI3K could also regulate insulin signaling via activating the Akt pathway [72]. Generally, obese persons with insulin resistance are often companied by reduced hypothalamic GnRH secretion and decreased fertility [73]. An experimental study also demonstrated that herbal decoctions containing herbs prevent miscarriage and are effective to promote the follicle development and fertility via activation of the PI3K-Akt signal pathway [74]. Accordingly, we assume that Tusizi-Sangjisheng may suppress miscarriage via the PI3K-Akt signal pathway by influencing the expression of sex hormones GnRH/LH and also the proliferation and migration of trophoblast cells.


### 4.3.3. TGF-β Signal Pathway

TGF-β is a multifunctional cytokine with three isoforms (TGF-β1, TGF-β2, and TGF-β3) and acts mainly through Smad pathways [75]. Evidences have proved that TGF-β plays pivotal and dual roles in the regulation of cell growth and apoptosis which is vividly described as a “switch” [76]. During early pregnancy, the uterine cells undergo apoptosis and proliferation for the successful implantation of embryo. Those complex processes are controlled and regulated by several signal pathways, and the TGF-β signal pathway is one of them that could help to maintain normal pregnancy by balancing the proliferation and apoptosis of trophoblast cells [75]. In addition, the TGF-β signal pathway is also involved in immune and inflammatory responses by regulating indoleamine 2,3-dioxygense (IDO) expression to promote immune tolerance of the maternal-fetal interface [77], inhibiting the proliferation of human Th1 memory T cells [78]. At present, there is no direct evidence illustrating the specific relationship among herbs like Cuscutae Semen and the TGF-β signal pathway in TA. Yet we can hypothesize based on the above information that the active ingredients in the herb pair Tusizi-Sangjisheng exert their TA-preventing function by regulating the immune responses in maternal-fetal interface and controlling the normal apoptosis and proliferation of uterine cells through the TGF-β signal pathway.In normal pregnancy, the balance of the immune microenvironment at the maternal-fetal interface can ensure immune tolerance and protect the embryo from maternal immune rejection. The ability of gestational trophoblasts to proliferate, differentiate, migrate, and invade is crucial for embryo implantation, placenta formation, and embryo growth and development. Trophoblasts are the main cell types of placental tissues that differentiate into extravillous trophoblasts, syncytiotrophoblasts and cell trophoblasts. Extravillous trophoblasts can be further differentiated into cells with high infiltration capacity. Interstitial trophoblast cells and intravascular trophoblast cells then invade the uterine decidua and reshape the spiral arteries of a pregnant uterus, thereby forming placental tissues which provide nutrients for the development of the embryo. Therefore, infiltration of trophoblasts into the uterine decidua is the key step to the success of early pregnancy. If the ability of trophoblasts to proliferate, migrate, and invade is weakened, it will affect the remodeling of decidual tissue blood vessels and the formation of placenta and further develop into pregnancy-dependent complications such as abortion, preeclampsia, and intrauterine growth restriction. These processes during pregnancy are jointly regulated by many signal transduction pathways. However, the exact regulatory mechanisms are still not very clear. Three key signaling pathways mentioned above were screened by systematic methods in this study revealing that Tusizi-Sangjisheng may effectively reduce miscarriage rates via regulating the normal expression and function of those three signal pathways, correcting under- or overexpression and maintaining normal pregnancy.


## 4.3.1. MAPK Signal Pathway

MAPK is a class of serine/threonine protein kinases and widely present in various cells. It has important regulatory effects on gene expression. After activation, the protein migrates into the phosphorylated nucleoproteins and membrane receptors, regulating gene transcription and other life events. It consists of four separate signaling cascades: the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinases); the ERKs (extracellular signal-regulated kinases); the ERK5 or big MAPK1; and the p38MAPK group of protein kinases. When stimulated by extracellular signals, the MAPK cascade which is an evolutionarily conserved tertiary kinase cascade transduction signal will be activated by a series of complicated chemical reactions, resulting in the successive activation of MAPKKK, MAPKK, and MAPK through the phosphorylation of amino acid residues [62]. Evidences have shown that the MAPK signal pathway is activated in many processes during embryo implantation and is closely related to the invasion and proliferation of trophoblast cells and decidual stromal cells (DSCs) which play key roles in the maintenance of normal pregnancy [63, 64]. Z. Wang et al.’s research demonstrated the abnormal expressions of nucleotide-binding oligomerization domains 1 and 2 (NOD 1 and NOD 2) in villi from recurrent spontaneous abortion (RSA) patients that then inhibit the invasion and proliferation of trophoblast cells by activating the p38MAPK signal pathway [65]. In addition, inflammation (including sterile inflammation or infectious inflammation induced by bacteria such as bacterial vaginosis) and oxidative stress (smoking) could also activate the p38MAPK signal pathway and thus attenuate the normal development of trophoblast cells [66]. Furthermore, the activation of the JNK and ERK1/2 signal pathway by interleukin (IL)-33 [67] and IL-25 [68] also leads to the enhancement of invasion and proliferation of DSCs. Accordingly, for the similar pathogenesis between RAS and TA, the role of the MAPK signal pathway could also be considered by affecting the invasion and proliferation of trophoblast cells and DSCs even though there is no direct evidence. The total flavones of Cuscutae Semen were found to be effective in suppressing abortion by inhibiting MAPK pathways [69], indicating the potential mechanism of the herb pair Tusizi-Sangjisheng treating TA.


## 4.3.2. PI3K-Akt Signal Pathway

The PI3K-Akt signal pathway consists of phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB, also known as Akt) and is involved in transcription, protein synthesis, migration, apoptosis, and proliferation activities [70]. Identically, the PI3K-Akt signaling pathway also participates in the proliferation and migration of trophoblast cells that are vital for establishing and persisting normal success pregnancy. Evidence has shown that the up-/downregulation of the PI3K-Akt signal pathway is related to the proliferation and migration of trophoblast cells [71]. PI3K signaling is considered to have association with the secretion of several hormones including gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) that are important in the maintenance of normal pregnancy [72]. In addition, PI3K could also regulate insulin signaling via activating the Akt pathway [72]. Generally, obese persons with insulin resistance are often companied by reduced hypothalamic GnRH secretion and decreased fertility [73]. An experimental study also demonstrated that herbal decoctions containing herbs prevent miscarriage and are effective to promote the follicle development and fertility via activation of the PI3K-Akt signal pathway [74]. Accordingly, we assume that Tusizi-Sangjisheng may suppress miscarriage via the PI3K-Akt signal pathway by influencing the expression of sex hormones GnRH/LH and also the proliferation and migration of trophoblast cells.


## 4.3.3. TGF-β Signal Pathway

TGF-β is a multifunctional cytokine with three isoforms (TGF-β1, TGF-β2, and TGF-β3) and acts mainly through Smad pathways [75]. Evidences have proved that TGF-β plays pivotal and dual roles in the regulation of cell growth and apoptosis which is vividly described as a “switch” [76]. During early pregnancy, the uterine cells undergo apoptosis and proliferation for the successful implantation of embryo. Those complex processes are controlled and regulated by several signal pathways, and the TGF-β signal pathway is one of them that could help to maintain normal pregnancy by balancing the proliferation and apoptosis of trophoblast cells [75]. In addition, the TGF-β signal pathway is also involved in immune and inflammatory responses by regulating indoleamine 2,3-dioxygense (IDO) expression to promote immune tolerance of the maternal-fetal interface [77], inhibiting the proliferation of human Th1 memory T cells [78]. At present, there is no direct evidence illustrating the specific relationship among herbs like Cuscutae Semen and the TGF-β signal pathway in TA. Yet we can hypothesize based on the above information that the active ingredients in the herb pair Tusizi-Sangjisheng exert their TA-preventing function by regulating the immune responses in maternal-fetal interface and controlling the normal apoptosis and proliferation of uterine cells through the TGF-β signal pathway.In normal pregnancy, the balance of the immune microenvironment at the maternal-fetal interface can ensure immune tolerance and protect the embryo from maternal immune rejection. The ability of gestational trophoblasts to proliferate, differentiate, migrate, and invade is crucial for embryo implantation, placenta formation, and embryo growth and development. Trophoblasts are the main cell types of placental tissues that differentiate into extravillous trophoblasts, syncytiotrophoblasts and cell trophoblasts. Extravillous trophoblasts can be further differentiated into cells with high infiltration capacity. Interstitial trophoblast cells and intravascular trophoblast cells then invade the uterine decidua and reshape the spiral arteries of a pregnant uterus, thereby forming placental tissues which provide nutrients for the development of the embryo. Therefore, infiltration of trophoblasts into the uterine decidua is the key step to the success of early pregnancy. If the ability of trophoblasts to proliferate, migrate, and invade is weakened, it will affect the remodeling of decidual tissue blood vessels and the formation of placenta and further develop into pregnancy-dependent complications such as abortion, preeclampsia, and intrauterine growth restriction. These processes during pregnancy are jointly regulated by many signal transduction pathways. However, the exact regulatory mechanisms are still not very clear. Three key signaling pathways mentioned above were screened by systematic methods in this study revealing that Tusizi-Sangjisheng may effectively reduce miscarriage rates via regulating the normal expression and function of those three signal pathways, correcting under- or overexpression and maintaining normal pregnancy.


## 5. Conclusions

The putative mechanisms in the molecular level of the commonly used herb pair Tusizi-Sangjisheng treating TA were analyzed by systems pharmacology. 12 bioactive molecules withOB≥30% and DL≥0.18 of Tusizi-Sangjisheng were obtained by calculating the ADME properties. Besides, a total of 153 direct targets of 12 bioactive molecules were predicted by systematic models. By constructing a T-D network for analysis, 31 targets mainly containing PTGS1, NOS3, TNF, and caspase were found to be directly related to TA among the corresponding targets of Tusizi-Sangjisheng and TA was considered a complex disease with multifaceted causes that need to be treated from multiple angles by GO analysis. Through the T-P network enrichment analysis, Tusizi-Sangjisheng was found to act on multiple pathways by several active molecules that include MAPK, PI3K-Akt, and TGF-β signaling pathways. The mechanism of Tusizi-Sangjisheng on the treatment of TA may be through regulating the normal expression of the above three signal pathways, correcting the state of under- or overexpression, and maintaining normal pregnancy.



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*Source: 4748264-2020-07-21.xml*