Yin-Zi Yue,Li Zeng,Xiao-Peng Wang,Yang Zong,Shuai Yan,*

1Nanjing University of Chinese Medicine, Nanjing, China.2Suzhou Hospital of Traditional Chinese Medicine,Suzhou,China.

Background

Adhesive intestinal obstruction (AIO) is a common acute abdominal disease in abdominal surgery [1],which can lead to retention of intestinal contents.Its clinical manifestations include abdominal distension,abdominal pain, nausea, vomiting, obstruction of anal exhaust and bowel movement, etc.Over 40% of intestinal obstruction is caused by abdominal adhesion after operation, of which 75% is adhesive small intestinal obstruction [2].In the United States, more than 350,000 adhesiolysis operations are performed annually, resulting in more than 960,000 days of care and 2.3 billion USD in medical expenses,affecting the quality of life of patients, and imposing a heavy medical burden on the government[3].Traditionally,it is believed that most recurrent AIOs need to be removed by surgery.However, even minimally invasive laparoscopic surgery cannot escape the vicious circle of "operation-obstruction-reoperation-reoccurrence" [4].Therefore, it is still necessary to study the formation mechanism and prevention strategies of AIO.

According to the theory of traditional Chinese medicine(TCM),Qi stagnation and blood stasis are the pathological basis of AIO, and the principles of treatment are promoting blood circulation and removing blood stasis [5].Radix et Rhizoma Rhei(Dahuang) (RERR) andSemen Persicae(Taoren) (SP)are the core drug pair commonly used in the treatment of AIO.RERR and SP drug pair comes from the classical prescriptions of Chinese medicine Didang soup and Taohe Chengqi decoction inShanghanlun(an ancient book of Chinese medicine published in the 3rd Century A.D.) and Biejia Jian pills, Dahuang Shuchong pill, and Dahuang Mudan decoction inJingui Yaolue(an ancient book of Chinese medicine published in the 3rd Century A.D.).However, the therapeutic mechanism of RERR and SP in the treatment of AIO is still unclear.

Network pharmacology can explain the potential mechanism of multi-component, multi-target, and multi-pathway action of TCM through the analysis of multiple complex networks and multi-level interconnection [6].In this study, we examined the material basis and molecular mechanism of RERR and SP in the prevention and treatment of AIO through the network pharmacology approach.

Methods

Constructing the chemical composition database of RERR and SP

The chemical constituents of RERR and SP were retrieved from the traditional Chinese medicine system pharmacology analysis platform (TCMSP;http://ibts.hkbu.edu.hk/LSP/tcmsp.php) using the keywords "Radix et Rhizoma Rhei" and "Semen Persicae"[7].

Screening of active components and target proteins

Oral bioavailability (OB) is one of the most important pharmacokinetic parameters in drug absorption,distribution,metabolism,and excretion.It indicates the speed and degree of absorption of active ingredients or active groups of oral drugs by the body[8,9].With the help of the TCMSP data platform, the chemical constituents of RERR and SP which corresponded to an OB > 30% and drug-likeness (DL) > 0.18 were screened as active ingredients and related target proteins.

Gene name identification of the target protein and construction of the compound-target network

The corresponding gene names of the target proteins were searched by UniProt, HCBI, PubMed, and other databases.Cytoscape 3.2.1(http://www.cytoscape.org/)was used to construct a compound-target network [10]and analyze the value of the Degree parameter between compounds and targets.

Construction of protein interaction and screening of its core network

Protein-protein interaction core network refers to the study of the correlation between compounds and disease-related proteins from the perspective of biochemistry, signal transduction, and genetic networks [11].To further understand the mechanisms of RERR and SP compounds and disease targets at the protein level, the selected compounds were uploaded to STRING10.5 to obtain the protein interaction relationship, and then the obtained data were imported into Cytoscape 3.2.1 to screen for common target genes between compounds and diseases to obtain the core network map of protein interaction.

Signaling pathway analysis of core targets

To further understand the functions of the core genes screened and their roles in the signaling pathways, the selected targets for the compatibility treatment of AIO with RERR and SP were imported into Database for Annotation, Visualization and Integrated Discovery(DAVID) [12].(https://david.ncifcrf.gov/home.jsp).All target gene names were corrected to their official names by entering the list of target gene names and restricting species to humans.The thresholdP< 0.05 was set for gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)metabolic pathway enrichment analysis.The results were visualized using the Omishare Tools(http://www.omicshare.com/tools/index.php/) on-line mapping website.

Results

Table 1 Basic information of 15 active compounds in RERR and SP

RERR, Radix et Rhizoma Rhei (Dahuang); SP, Semen Persicae (Taoren); OB, Oral bioavailability; DL,Drug-likeness;MW,Molecular weight;GA,Gibberellin.

Figure 1 RERR-SP compound-target network

Figure 2 Core network diagram of protein interaction between RERR-SP and AIO

Screening of active compounds from RERR and SP

A total of 158 compounds in RERR and SP were retrieved from the TCMSP, of which 92 were from RERR and 66 were from SP.Fifteen active compounds were screened with OB > 30% and DL > 0.18, of which 6 came from RERR and 9 from SP.The basic information of 15 active compounds in RERR and SP is shown in Table 1.

Compound-target interaction network

The compound-target network comprised 69 nodes(10 compound nodes, 59 target nodes) and 93 edges(Figure 1).The green node represents the compound molecule of SP, blue represents the compound molecule of RERR, and the red node represents the drug target.Each edge represents the interaction between the compound molecule and target.Five of the 15 compounds were not involved in the network construction.In a network, the degree of a node(Degree) represents the number of routes connected to the node in the network.According to the topological properties of the network, the larger Degree nodes are selected for analysis.These nodes with more connecting compounds or targets play pivotal roles in the whole network and may be the key compounds or targets.The main components of RERR screened were aloe-emodin, eupatin, catechin, rhein.Gibberellins(diterpenoids) were the main components of SP after screening.

In this network, each compound interacted with an average of 9.3 targets, and each target interacted with an average of 1.58 compounds.Therefore, there is an interaction between one compound and multiple targets in the RERR and SP drug pair.Additionally,xxx there was the phenomenon of different compounds acting together on the same target, which reveals the mechanism of interaction between multi-components and multi-targets.Figure 1 shows that molecular 000471 aloe-emodin has 17 potential targets, followed by molecular 000096 catechin with 11 potential targets,molecular 002268 rhein with 7 potential targets, and molecular 001340 new compound gibberellin (GA)120 with 7 potential targets.

Construction and screening of protein interaction core network between rhubarb, peach kernel, and AIO

Figure 2 shows that the protein interaction core network comprised 84 nodes and 1004 edges,in which nodes represent proteins, and each edge represents the interaction among proteins.Among them, the nodes with a Degree value greater than 45 accounted for 20.69% of the total.The top five were peroxisome proliferator-activated receptor-γ (PPAR-γ), tumor necrosis factor (TNF), ubiquitin C (UBC), E1A binding protein of 300 kDa (EP300), and glucocorticoid receptor gene (NR3C1).PPAR-γ and TNF may play inflammatory roles in the pathogenesis of AIO.UBC and EP300 are involved in regulating signal transduction and cell cycle activation as well as regulating the dynamic balance of inflammation and collagen deposition.NR3C1, as a glucocorticoid receptor gene, may regulate AIO-related microRNAs and play an anti-inflammatory role in AIO.It is suggested that the mechanism of RERR and SP in treating AIO may be related to their anti-inflammatory,immunomodulatory,and anti-fibrotic effects.

Figure 3 GO enrichment analysis of targets in biological processes

Figure 4 GO enrichment analysis of cell compositions

Figure 5 GO enrichment analysis of molecular function

Target pathway analysis

Through the analysis of GO function enrichment in DAVID, 516 GO items were obtained (P< 0.05).Among them, 354 items were biological processes, 60 items were molecular functions, and 112 items were cell compositions.As shown in Figures 3-5, the targets of active components from RERR and SP are mainly concentrated in biological processes such as signal transduction, anti-apoptotic, inflammatory response,and oxidative stress; in molecular functions,transcription factor binding, protein binding, and histone deacetylase binding rank first; and in cell components, nucleus, plasma membrane, and cytoplasm account for the most significant proportion.KEGG pathway enrichment screening obtained 102 signaling pathways (P< 0.05).Figure 6 shows the most significant of the 30 pathways.TNF signaling pathway,NOD-like receptor pathway,NF-κB pathway,MAPK signaling pathway, and T cell receptor signaling pathway play key roles in the pathogenesis of AIO.

Figure 6 Top 30 pathway by KEGG enrichment analysis of RERR and SP for AIO

Discussion

There are still many challenges in the clinical treatment of AIO.The pathogenesis of AIO is a complex process of multiple factors where different factors interact with and influence each other.Therefore, the improvement of the treatment technology of AIO needs further study and understanding of the pathogenesis of AIO.TCM has unique insights into the pathogenesis and treatment of AIO.At present, research on the pathogenesis of AIO focuses on intestinal barrier damage, bacterial translocation, inflammatory response, immune imbalance,and so on.

RERR and SP are the core drug pair commonly used in the clinical treatment of AIO.It was found that Chinese medicinal formulae Taohe Chengqi decoction(containing RERR and SP) could significantly relieve the symptoms of abdominal pain and distention in AIO patients, promote the recovery of gastrointestinal function, reduce the levels of white blood cells,C-reactive protein, and TNF-α, accelerate the recovery of illness, and reduce the probability of transit surgery[13].Bingbinget al.[14] conducted a retrospective analysis of 970 cases with AIO in which based on routine treatment, Chinese medicinal formulae Dachengqi decoction was injected by gastric tube; of these, 618 cases were cured or improved without surgical treatment.Relevant experimental studies showed that Dachengqi decoction reduced plasma and small intestinal tissue diamine oxidase,malondialdehyde, and superoxide dismutase in rats with incomplete intestinal obstruction, reduced bacterial translocation and endotoxin absorption, and improved intestinal mucosal barrier function in the prevention and treatment of AIO [15, 16].Therefore,the mechanism of RERR and SP in the treatment of AIO may be related to the regulation of immune and inflammatory signaling pathways.In this study, we first used the related technology of network pharmacology and found that the mechanism of RERR and SP on the treatment of AIO may mainly focus on compounds,targets,and signaling pathways.

The screened compounds were analyzed.It was found that the main active components of RERR were anthraquinones and their compounds.Aloe emodin,zeranol, catechin, and rhein had higher OB and DL values.Aloe emodin, catechin, and rhein have been reported to have high immunomodulatory,anti-inflammatory, anti-tumor, and anti-fibrotic activities [17-19].The screened components of SP are not as well-known as amygdalin, amygdalase, and volatile oil, but mainly gibberellins, such as GA119,GA120, GA121, and GA122.These compounds were isolated from SP for the first time by Nakayamaet al[20].Recent study has shown that these compounds possess antioxidant activity and can regulate cell growth and development [21].In conclusion, the screening process can be comprehensive and show the entire range of active ingredients.

The main proteins of RERR and SP were PPAR-γ,TNF, UBC, EP300, and NR3C1.PPAR-γ is a ligand-dependent transcription factor, one of the subtypes of PPAR.It belongs to the type II nuclear receptor superfamily and plays an important role in anti-inflammation [22].Studies have shown that the PPAR-γ ligand inhibits the production of inflammatory cytokines in mouse macrophages by inhibiting NF-κB and MAPK signaling pathways [23], suggesting that PPAR-γ may mediate the role of these pathways in the development of AIO.UBC is a ubiquitin-binding enzyme involved in the formation of polyubiquitin chains and regulates signal transduction and other cellular processes, such as DNA damage repair and activation and regulation of natural and acquired immune systems.Study has reported that UBC13 can significantly inhibit the expression of interleukin(IL)-6, IL-1β, TNF-α, and cyclooxygenase-2 in RAW264.7 cells [24].EP300 is an important multifunctional transcription co-activator, which plays a key role in many physiological processes, such as cell differentiation, cell cycle regulation, and cell apoptosis [25].The functions of UBC13 and EP300 network proteins are related to chemotaxis and activation and inflammation of immune cells,especially T cells, and are closely related to the early formation of AIO[26].

Pathways related to signal transduction function included the TNF signaling pathway, NOD-like receptor pathway, NF-κB pathway, MAPK signaling pathway, and T cell receptor signaling pathway, which affect immune and inflammatory regulation.AIO induces inflammatory immune response [27], which is mediated by specific cells of the adaptive immune system.Recent studies have confirmed that intraperitoneal trauma or hypoxia can activate the secretion of cytokines by peritoneal macrophages to exert immune effects, and mediate the activation of T cells mediated by antigen presenting cells [28].A clinical study showed that Chinese medicinal formulae Dahuang Mudan decoction (containing RERR and SP)significantly reduced serum inflammatory factors in patients with bowel obstruction (TNF-α and IL-6 levels), improved intestinal capillary microcirculation,and relieved edema of the intestinal wall [29].Abdominal mast cells and intestinal macrophages can activate the expression of inflammatory factors,triggering the cascade release effect and activating multiple pathways.It has shown that Chinese medicinal formulae Jiawei Taohe Chengqi decoction(including RERR and SP) can significantly reduce the level of monocyte chemoattractant protein-1 [30].These results suggest that RERR and SP may play a multi-target role by activating immune and inflammatory pathways.

The target of the active ingredients of RERR and SP is to prevent and cure AIO mainly by regulating immunity, activating immune effect, decreasing the expression level of inflammatory cytokines,accelerating apoptosis of intestinal macrophages/T lymphocytes, and antagonizing inflammatory reaction and tissue fibrosis to repair the intestinal mucosal barrier.The results described above reveal the molecular mechanism of RERR and SP and provide a preliminarily explanation.

Conclusion

The inflammatory-immune signaling pathway is of high significance in the prevention and treatment of AIO by RERR and SP.It is involved in activating immune effects, inducing Th17/Treg balance, and antagonizing inflammatory response and tissue fibrosis to repair the intestinal mucosal barrier.However, this study also had some shortcomings.Although network pharmacology reduces part of the workload, it may also omit some unknown chemical components.Further experiments are needed to support the results of network pharmacology.Second, the method of network pharmacology does not consider the relationship between the emperor and minister, the dosage and manner of preparation and administration of TCM, nor does it consider the metabolic effect of human liver enzymes and intestinal flora on TCM.In the follow-up experimental study, combined with in vivo and in vitro experiments, the mechanism of RERR and SP in preventing and treating postoperative AIO will be further studied.