Study on Molecular Mechanism of Tongqi No.1 Prescription for Tubal Obstructive Infertility Based on Network Pharmacology and Molecular Docking

: In order to investigate the mechanism of action of Tongqi No. 1 Formula in the treatment of tubal obstructive infertility, a network pharmacology method was used to analyze the active ingredients and drug targets of Tongqi No. 1 Formula, and the disease targets of tubal obstructive infertility, respectively. The drug targets, disease targets, and active ingredient were then analyzed, and the results were finally validated by using molecular docking methods. It was found that 61 drug active ingredients and 215 drug targets were obtained in Tongqi No. 1 Formula. The GeneCards database, the OMIM database combined with the DRUGBANK database, was merged and duplicate values were removed, resulting in 1382 disease targets and 119 drug-disease intersecting genes. GO enrichment revealed that Tongqi No. 1 Formula has biological functions such as response to steroid hormones, regulation of apoptotic signaling pathway, positive regulation of cell death, response to growth factors, and response to estradiol. After KEGG pathway enrichment analysis, the main pathways involved are NF-KB, JAK-STAT, p13k-AKT, MAPK, etc. Molecular docking results showed that tanshinone IIA stably docked into the active pocket of AKT1 protein structure 4EJN, and the two were hydrogen bonded through amino acid residues ASN53, ASN51, VAL-271, VAL-270, SER-205, and GLN-203. The present study initially revealed that the treatment of tubal obstruction infertility with "Tongqi No. 1 Formula" is carried out through multiple components, multiple targets and multiple pathways, which provides a basis for further research


Introduction
At present, the number of infertility patients in the world reaches 186 million [1], and the infertility rate is about 15.5%, showing an increasing trend year by year.By 2020, about 25 percent of couples of child-bearing age in China were suffering from infertility, an increase from 2018.Factors that cause infertility include tubal factors, ovulation disorders and immune factors, among which tubal factors, tubal obstruction accounts for 14%-45% of all infertility causes [2] , and is the main factor that causes infertility.Modern medicine mainly uses endoscopic therapy, interventional therapy and assisted reproductive technology therapy, but there are some problems such as postoperative adhesion and easy recurrence.At present, most of the Chinese medical scientists recognized that the main pathogenesis of tubal obstructive infertility is "blood stasis", the treatment principle is to promote blood stasis and dredging collagals, clinical treatment effect is significant, shortcomings for the relevant mechanism of action still need further research.
Tongqi No.1 prescription is the experience prescription of Liu Maolin, director of Yulin Hospital of Traditional Chinese Medicine. The prescription has Chuanxiong, which has the effect of promoting blood circulation and removing blood stasis, qi and dispelling wind.Fang Zhong Sanling, Zedoary turmeric broken blood to eliminate accumulation and relieve pain, good stasis.Take pangolin to promote blood and eliminate disease, through the effect of activating collaterals.Soap thorn soft and firm scattered knot, promote mass, effusion disappear.Danshen Qi promoting blood circulation and removing blood stasis, Pueraria Gan insipid regulation, can prevent the problem of ligusticum Chuanxiong too much, fructus aurantii dissipates blood by removing blood stasis, asarum, cassia twig, Tongcao, road through the meridian activating collaterals, Wang does not leave the line of blood through the meridian, all drugs cooperate, play blood activating qi, dredge the blocking work, make the fallopian tube smooth.Since the establishment of gynecology department of Yulin Hospital of Traditional Chinese Medicine in 1989, more than 10,000 patients with tubal obstructive infertility have been cured by Director Liu Maolin.In long-term clinical work, this research team found that Tongqi No.1 formula has a significant effect on tubal obstructive infertility [3,4], which can improve the symptoms of tubal obstruction and improve the pregnancy rate.Therefore, this study used the method of network pharmacology to analyze the mechanism of action of Tongqi No.1 formula with definite clinical efficacy in the treatment of tubal obstructive infertility, which can provide a new idea for clinical application and scientific basis for the next experimental research.

Collection of components and targets of the No.I prescription
I will pass disambiguation party (rhizoma ligustici wallichii, pangolin scales, asarum, soap, ricepaperplant pith, cowherb seed, passepartout, rhizoma sparganii, rhizoma zedoariae, salvia miltiorrhiza, puerarin, acid-insoluble ash, cassia twig) in each taste traditional Chinese medicine name input TCMSP [5] (Traditional Chinese Medicine Systems Pharmacology, http://temspw.com/tcmsp.php)database, combined OB (oral bioavailability) and DL (drug-like) as the effective active ingredient and protein target screening of each drug [5], set OB>=30%, DL>=0.18, and refer to related literature for supplement.When the screening was completed, the target proteins corresponding to each drug were standardized using the Uniprot (http://www.www.uniprot.org/) database, and the corresponding genes of the target proteins were retrieved using the UniprotKB database. The search condition was set as Organism: Homosapiens (Human) for protein and gene transformation and construction of a composition-target protein database.

Target collection for tubal obstructive infertility
Through GeneCards (https://www.genecards.org), OMIM (http://www.omim.or) and TDD (http://bidd.nus.edu.sg/group/cjttd)),With "fallopian tube complication infertility" as the key word,The DRUGBANK database (https://www.drugbank.ca) was used to find clinical first-line targets of Western drug action for the treatment of tubal obstructive infertility as a supplement [6] .Duplicate values were merged and deleted from the disease database to obtain the final disease target protein, and its final results were collated through the Uniprot database.

Composition of Tongqi No.1 Formula --PPI network construction of tubal obstructive infertility related targets
In order to study the correlation between the drug target of Tongqi No.1 prescription and the target gene of tubal obstructive infertility disease, Tongqi No.1 prescription was compared with the target gene of tubal obstructive infertility. The intersection gene of Tongqi No.1 prescription was selected and STRING11.0 [7] (http: the rest of the data were set as Homosapiens and highestconfidence (>0.9), and PPI was constructed.CytoScape3.7.1 software is used to visualize the results, and MCODE plug-in is used to describe the biological processes and corresponding functions of the core target.

Enrichment analysis of core target function and pathway of Tongqi No.1 formula component -tubal obstructive infertility.
The Metascape platform [8,9] (http://metascape.org/gp/index.html) has two features, first, a comprehensive explanation of its annotation function, and second, the timely update, once a month, is achieved. If the core targets are entered into the Metascape platform with a setting of P<0.01, the main biological processes and metabolic pathways of the core targets could be enriched and analyzed. The results were visualized using the online website platform of Microbiology Letter (www.bioinformatics.com.cn) to further elucidate the mechanism of action of Tongqi No. 1 Formula for the treatment of tubal obstructive infertility.

Tongqi No.1 formula component -tubal obstructive infertility target -pathway network map construction
The composition of Tongqi No.1 prescription --target of tubal obstructive infertility --pathway network diagram was constructed using Cytoscape3.7.1 to analyze the effective components and target parameters of Tongqi No.1 prescription. According to the obtained results, the main core targets and main active components of Tongqi No.1 prescription were determined.

Molecular docking verification of active ingredients and key targets
Based on the previous research content, PyRx software was used to verify the molecular docking of key active ingredients and action targets of Tongqi Formula I, and the best docking results were visualized through pymol, and the other molecular docking results were drawn heat map.

Acquisition of active ingredient targets for Tongqi Recipe I.
Initial extraction of pharmaceutical chemical components: There were 7 species of ligusticum ligusticum, 8 species of asarum asarum, 11 species of saponaria saponata, 4 species of Fructus vermicelli, 4 species of luputong, 5 species of tricolor, 3 species of zedoary rhizome, 65 species of salvia miltiorrhiza, 4 species of pueraria rhizoma, 15 species of Fructus aurantii, 7 species of cassia twig and 7 species of pangolin. A total of 61 active compounds were obtained by deleting many of the same compounds, only one of which was kept and no corresponding target compound was removed.See Table 1.

Analysis of active ingredients and drug target network of Tongqi Prescription I
The 61 active ingredients of Tongqi I formula correspond to 215 target genes. Using Cytoscape3.7.1 software, the relationship between compounds and targets is made into a network and the network diagram is drawn, as shown in Figure 1.The result is analyzed and the degree value is calculated by using the plug-in NetworkAnalyzer in the software. The larger the degree value is, the more important the node is in the network.The statistical results showed that there were 275 nodes in the network. According to the degree value, the top 5 active ingredients in the network nodes were quercetin, β-sitosterol, kaempferol, luteolin and stigmasterol, respectively, and the corresponding drug targets were KCNH2, PGR, AR, RELA and PGR.Through the analysis of the active ingredients and drug targets of Tongqi No.1 prescription, it can be concluded that the therapeutic effect of Tongqi No.1 prescription may play a role through multiple active ingredients corresponding to multiple targets.

Disease target collection for tubal obstructive infertility
The disease database was merged and duplicates were removed, and the final results were 86 collated through the Uniprot database, resulting in 1382 disease targets, as shown in Figure 2 (1).

Composition of Tongqi No.1 Formula -PPI network construction related to tubal obstructive infertility
Venny2.1.0 website was used for online analysis to obtain the intersection target genes of disease genes and drug targets, as shown in Figure 2 (2).
By using STRING database, the intersection genes were included to construct the drug-disease PPI network. The PPI network had 119 nodes and 2358 edges, with an average interval of 0.701 and an average node degree of 39.6.Topological analysis results showed that target proteins with high degree values included luteolin, quercetin, isovitexin, etc., which were visualized on Cytoscape3.7.1, as shown in Figure 3.  After PPI network is obtained, module is obtained by using MCODE plug-in in CytoScape3.7.1, as shown in Figure 3. According to the P value, descriptions of the functions of the top 3 biological processes in the PPI network and Module are obtained, as shown in Table 2.

Enrichment analysis of target function and pathway
Through retrieval on Metascape data platform, signal pathway analysis was carried out on targets related to tubal obstructive infertility treated by Tongqi No.1 formula, and the results were visualized using Weisheng platform.The results showed that the occurrence of tubal obstructive infertility was closely related to the function of multiple targets.GO enrichment analysis was performed on the top 10 items and bubble map was drawn. Tongqi Formula 1 mainly participated in biological processes including regulation of apoptotic signaling pathway, positive regulation of cell death, response to growth factors, response to estradiol and other biological functional targets for the treatment of tubal obstructive infertility mainly concentrated in protein kinase activity and nuclear receptor binding.It mainly involves adhesion, platelet a granules, Bcl-2 family protein complex and so on.GO enrichment results were shown in Figure 4 (1), and it was found that AGE-RAGE signaling pathway, p53 signaling pathway, JAK-STAT signaling pathway, NFκB signaling pathway, etc., were the main pathways for Tongqi Formula 1 treatment of tubal obstructive infertility. KEGG analysis results were shown in Figure 4 (2), and target pathway enrichment results were shown in Table 3.   AKT1,CCND1,CCNA2,CCNB1,CCND2,CDKN1A,CHEK1,MAPK14  ,E2F1,IGFBP3,IL1A,IL6,CXCL8,MDM2,MYC,SERPINE1,MAPK1,R  AF1,RB1,RELA,TP53,CHEK2   hsa04115  p53 signaling  pathway  16  -23.28  BAX,CCND1,BCL2,BCL2L1,CASP3,CASP8,CASP9,CCNB1,CCND  2,CDKN1A,CHEK1,IGFBP3,MDM2,SERPINE1,TP53,CHEK2   hsa04630  JAK-STAT  signaling pathway  18  -20.33  AKT1,CCND1,BCL2L1,CCND2,CDKN1A,EGF,EGFR,IFNG,IL2,IL4  ,IL6,IL10 Figure 5.The built-in NetworkAnalyzer subfunction of the software was used to analyze the network topology parameters of "Tongqi No.1 prescription" for tubal obstructive infertility, and the core components and core action targets were obtained. According to the analysis of the results, it is predicted that the main components of the treatment of tubal obstructive infertility are quercetin, followed by kaempferol, β-sitosterol, etc. The main active components are shown in Table 4.

Construction of the pathway network diagram of Tongqi No.1 formula component -tubal obstructive infertility
The connection degree of AKT1 in the network was 18, the mediality was 0.03, and the tightness was 0.45. It was predicted that AkT1 was the most important target of Tongqi No.1 formula in the treatment of tubal obstructive infertility.The next are BCL2, MAPK1, RELA, CCND1, CASP3, PPKCA, BAX, ESR1 and PTGS2, as shown in Table 3.

Molecular Docking
The binding energy of ligand and receptor is inversely proportional to the required energy. Binding energy <0kcal/mol indicates that the receptor and ligand can spontaneously bind, while binding energy <-5kcal/mol indicates that the receptor and ligand have good binding energy activity.The main active ingredients of Tongqi No.1 formula and its important targets were respectively docked. Tongqi No.1 formula had 12 main active ingredients and 10 important targets, a total of 120 docked times.The results are shown in the form of heat map, as shown in Figure 6. The binding energy of AKT1 and tanshinoneiia (-11.4kcal/mol) was the best. The molecular docking results showed that tanshinone IIA could stably bind to the active pocket of AKT1 protein structure 4EJN, and the two could bind through amino acid residues ASN53, ASN51, VAL-271, VAL-270,SER-205 and GLN-203 were hydrogen-bonded, and their binding results were visualized by pymol, as shown in Figure 7. Except HIFIA protein had poor docking with its active components, other active components had good docking with proteins.

Discuss current research
Currently, it is believed that tubal obstruction caused by inflammation is the most common pathogenic factor in tubal obstructive infertility.Inflammation or scar formation in the fallopian tube can cause fallopian tube obstruction, resulting in abnormal peristaltic function of the fallopian tube, and eventually infertility [10]. At present, most traditional Chinese medicine experts believe that the disease that causes tubal obstructive infertility is caused by "blood stasis", and its pathogenesis is that blood stasis obstructs cell collaterals and eventually leads to infertility. Therefore, the method of promoting blood stasis and smoothing collaterals and dispersing nodes should be used throughout the treatment, with the same treatment of specimens [11]. Based on more than 50 years of clinical and basic research, Tongqi I prescription was established to treat tubal obstructive infertility, and the clinical effect is remarkable.
Through this study on the molecular mechanism of "Tongqi No. I prescription" in the treatment of tubal obstructive infertility, the results showed that Tongqi No.1 prescription contained 61 active ingredients corresponding to 215 target genes, and the main active ingredients included quercetin, urushiflavin, kaempferol, etc. Topological analysis of 119 predicted therapeutic targets in PPI showed that STAT3, VEGFA, AKT1, TP53, HIFIA, IL-6, CASP3, TNF, PTGS2, EGF, EGFR and MMP9 were important targets for Tongqi No.1 prescription in the treatment of tubal obstructive infertility. According to KEGG cluster analysis and target pathway analysis, Tongqi No.1 prescription has multi-target and multi-pathway effects in the treatment of tubal infertility.Among them, the most involved pathways were JAK-STAT signaling pathway (hsa04630), NF-κB signaling pathway (hsa04064), estrogen signaling pathway (hsa04915) and P53 signaling pathway (hsa04115).
Modern medicine believes that inflammation can lead to local inflammatory exudation, edema, hyperplasia, and eventually obstruction of the fallopian tube.In the process of inflammation and infection, plasma proteins and immune cells will appear at the site of infection and injury, which play the role of mediating inflammatory response to eliminate invading pathogens and promote tissue repair [12]. At the same time, the intracellular signaling pathway will be activated to further promote inflammatory response. In this process, inflammatory cells will produce many inflammatory mediators, the most important pro-inflammatory factors are IL-6 and TNF-a [13]. IL-6 is a kind of glycoprotein with various biological activities [14], which is mainly secreted by macrophages or epithelial cells. It phosphorylates AKT in macrophages, keeps macrophages viable, enhances the contraction activity of fibroblasts, and eventually causes severe scar. Various chlamydia can enter host cells using different but related cell surface receptors that may share a common downstream signaling process for scarring via G proteins, particularly through the P13K-AKT pathway [15]. Bai Xue et al. [16] found that the mrna expression of pro-inflammatory factor IL-6 in patients with tubal inflammatory obstructive infertility was higher than that of normal people, suggesting that IL-6 could participate in chronic inflammatory response of the body to further promote fibrosis of the inflammatory injury machine, and the final outcome would be tubal obstruction. Schols [17], a foreign scholar, confirmed that IL-6 is closely related to systemic inflammatory response, and Ulich [18] found that IL-6 may participate in the process of transforming acute inflammation into chronic inflammation, making the acute episode of tubal inflammation turn into chronic episode, and further lead to local tissue adhesion of the tubal. Further leads to granulation tissue, fibrous tissue growth, resulting in tubal hyperplasia, fibrosis, and eventually the formation of tubal obstructive infertility. Tumor necrosis factor a (TNF-a) is a protein in essence, and fibrinous adhesion is closely connected with it, and it is also the crossing center of different signal transduction pathways. It activates fibroblasts and has strong adhesion to white blood cells while participating in the inflammatory and immune responses of the body, thus aggravating the damage of fallopian tubes.TNF-a can also mediate the release of IL-6, IL-8 and other inflammatory factors [14,19]. JendroMC et al. [20] have shown that macrophages infected with chlamydia can escape the killing effect of Tcell, which is beneficial to the survival of chlamydia in cells and the establishment of continuous infection. The amount of TNF-A produced is proportional to the damage to the fallopian tubes. When IL-6 and TNF-a act synergistically, inflammatory mediators network can be formed. When the inflammatory mediators network is formed, vascular endothelial cells can be indirectly or directly damaged and a large number of inflammatory substances can be exudated, thus aggravating the damage to the fallopian tube [21]. Relevant studies [22,23] have shown that: The main causes of tubal obstructive infertility are related to TNF-a and IL-6. TNF-a can synthesize IL-6 through mediated immune response, and IL-6 can damage the epithelium and mucosa of the tubal by participating in the chronic inflammatory response of the body, thus causing tubal obstructive infertility.As epidermal growth factors and receptors, EGF, EGFR and ERBB2 can participate in cell proliferation, apoptosis and local adhesion formation.Epidermal growth factor (EGF) can promote MAPK phosphorylation, but this study was conducted in bovine oviduct epithelial cells and was not found in human oviduct epithelial cells.The EGFR pathway can also regulate Notch signaling, generate a variety of cilia in the fallopian tube, regulate the fallopian tube microenvironment, and facilitate fertilization and embryo survival.Tubal epithelial cells are activated with the participation of TP53 proteins and bal-2 family apoptosis proteins. Severe inflammation can lead to aggregation of glycoproteins and mucus, accelerate the apoptosis of tubal epithelial cells, and cause the loss of tightly connected tubal epithelium, which is not conducive to fertilization [15].
In addition, cytokines interact with immune cell surface receptors to trigger inflammatory signals, which are mainly NF-KB, JAK/STST and MAPK signaling pathways. For NF-κB signaling pathway, studies have shown that in tubal obstructive infertility patients reproductive tract infection produced a variety of inflammatory factors, can activate TLR2 and TLR4 in the body, when TLRs activated, the signal will be transmitted along the cell to NF-κB, activate downstream NFκB molecules, and at the same time TLRs and MyD88 activation, Inflammatory cytokines such as TNF-A accumulate and inhibit the expression of factors such as IL-10. The expression of inflammatory cytokines leads to the continuous activation of NF-κB, which again releases a large number of inflammatory cytokines, forming a cascade of worsening reactions. The persistence of the above reactions will lead to the damage of the tubal tissues, the loss of cilia in large numbers in the tubal group, the atresia of the tubal umbrellas, the adhesion to the surrounding tissues, and even the formation of tubal scar due to the sustained inflammatory damage, and eventually the loss of its function leading to infertility [24].
The main active ingredient in formula I can play a role. Quercetin can delay inflammatory factors and reduce neutrophils. At the same time, it can also lower neutrophils, thus playing an antiinflammatory role [25]. Lurushetin can inhibit the activation of NF-κB and phosphorylation of p38MAPK, which can downregulate the activity of mast cells and inhibit the interaction between mast cells and activated T cells, thus playing an anti-inflammatory role. Studies have reported that urushetin can play an anti-inflammatory role by using NF-KB, JAK-STAT, p13k-AKT, MAPK and other pathways to restrict the expression of toll-like receptor 4, TNF-a, interleukin, chemokines and other inflammatory mediators [26,27]. The anti-inflammatory effects of luteolin [28] are mainly directed at transcription factors, such as signal transduction and the regulation of transcription activator 3 (STAT3), and can alter various signaling pathways involved in inflammation, or its antiinflammatory effects can inhibit the release of PGE2 and the expression of NF-κB in the nucleus and bind to DNA.Further down-regulation of COX-2 expression was associated with [28,29]. Studies have shown that the imbalance between the coagulation system and the fibrinolytic system is related to repeated infection and chronic inflammation, which increases the blood viscosity and leads to the occurrence of tissue adhesion. Tanshinone IIA can directly block the abnormal activation of NF-KB and regulate the expression of VCAM-1 and ICAM-1 induced by inflammatory factors to achieve clinical anti-inflammatory purposes [30]. Kaempferol prevents NF-κB from entering the nucleus, thereby reducing the release of inflammatory mediators [31]. JNC et al. [32] found that β-sitosterol plays an anti-inflammatory role by reducing the synthesis of NO, inhibiting the activity of IL-6 in macrophages, and reducing the secretion of IL-1, TNF-a and other inflammatory factors.
In summary, this study found that the possible mechanism of Tongqi No.1 prescription in treating tubal obstructive infertility is as follows: 1.Through NF-κB, JAK-STAT, p13k-AKT, MAPK and other pathways, the activation of NF-κB can be inhibited, the expression of toll-like receptor 4, TNF-a and other inflammatory mediators can be inhibited, and the activity of mast cells can be downregulated, and the interaction between mast cells and activated T cells can be weakened to play the anti-inflammatory role.2. The abnormal activation of NF-κB may play a role in blocking, and then regulate the expression of VCAM-1 and ICAM-1 induced by inflammatory factors to achieve clinical anti-inflammatory purposes [32]. By reducing the synthesis of NO, it can also act on IL-6 in macrophages to decrease its IL-6 activity and reduce the secretion of IL-1, TNF-a and other inflammatory factors.It can be seen that Tongqi No.1 prescription treats tubal obstructive infertility through multiple components, multiple targets and multiple approaches, which provides new clinical guidance for the treatment of tubal obstructive infertility and also provides theoretical support for the treatment of tubal obstructive infertility. However, the dosage of Tongqi No.1 prescription is not taken into account in this study.And the changes of TCM after decocting, its metabolites in vivo were not included in the analysis.Further experimental verification will be carried out in the future to further study the main molecular mechanism of Tongqi I formula.