A network pharmacology and molecular docking study on treatment mechanism of Bacterial Dysentery of Huanglian-Huangqin-Huangbo herb pair

Background: Bacillary dysentery (BD) is one of the most common epidemic infectious diseases. Hundreds of millions of people are infected with BD each year among the world. The patients usually have the following symptoms: abdominal pain, diarrhea, intestinal ora imbalance, etc. Antibiotic are widely used for the treatment in clinical practice. However, due to the overuse of antibiotics, the bacterial resistance is increasingly serious and the medical works are facing with the risk that the antibiotics would lose ecacy. Apart from chemical medicines, traditional Chinese medicines (TCM) are also well accepted for BD treatment, especially in Asian countries. Huanglian-Huangqin-Huangbo herb pair (HHH) is typical and commonly used to treat symptoms such as abdominal pain, diarrhea, and intestinal ora imbalance caused by BD. Also, the HHH has antibacterial, anti-inammatory, and antidiarrheal effects. In this study, we are committed to ascertain the potential active compounds of HHH and the onset mechanism for the treatment of BD. Methods With the help of the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (Traditional Chinese Medicine Systems Pharmacology Database, TCMSP) and PubChem database to search and screen the chemical components and targets of Coptis, Scutellaria, Phellodendron, the gene names were corrected through the Uniprot database, and used the CTD database, TTD database, GeneCards database and DRUGBANK database to obtain BD-related disease targets. The online drawing platform Bioinformatics was used to analyze the "active compound-disease" intersection target, and utilized Cyoscape 3.7.2 software to construct a visualized Chinese medicine-active compound-target network and protein interaction network in order to screen the potential key active compounds and key targets; GO function analysis and KEGG pathway enrichment analysis of the target were carried out through the Metascape database platform, and Cyoscape 3.7.2 software was used to construct a gene-pathway network to screen potential pathways and their mechanism of action. Molecular docking of the key active compounds of the HHH with the key target of BD. Results A total of 331 potential active compounds were screened for the HHH, among which 87 key active wogonin, β-sitosterol, isofumarine, and tetrahydroberberine be Act on BD through 34 potential intersection targets such as IL-6, AKT1, PTGS2, TNF, CASP3, VEGFA, etc. GO gene function analysis yielded a total of 20 biological process (BP) items, 7 cell composition (CC) items, and molecular function (MF) items (P<0.01), mainly involving lipopolysaccharide reaction, reactive oxygen metabolism process, cell factor receptor binding, inorganic substance response, membrane raft, cytokine receptor binding and other biological processes. KEGG pathway enrichment analysis identied 14 signaling pathways (P<0.01), mainly related to cancer signaling pathways, IL-17 signaling pathways and other key pathways. The results of molecular docking HHH owed that the core active components such as quercetin, β-sitosterol, wogonin, isofumarole, baicalein and other core active compounds have good binding effects with the core targets of TNF, IL-6, PTGS2, and BCL2 (binding energy <-5 KJ/mol).


Background
Bacillary dysentery, also known as BD, which is an intestinal infectious disease caused by dysentery bacillus (Shigella Castellani) and the features of BD are that it has rapid transmission rate, wide epidemic range, obvious seasonality [1] and people are likely to be infected. Also, BD is internationally recognized as one of most important infectious diseases [2] with its disease incidence and case mortality ranking rst in diarrheal diseases worldwide. According to statistical data, there are about 165 million people who get infected with BD each year in the world and about 1.1 million cases die, which is more common in developing countries with poor sanitary conditions such as the Africa [3]. In China, the top ve diseases with most morbidity are viral hepatitis, tuberculosis, syphilis, gonorrhea and bacterial and amoebic dysentery , respectively [4].
The Shigella is the major pathogen of the onset of BD and there are four common serogroups including Sh. exneri (group A), Sh.sonnei (group B), Sh.dysenteriae (group C) and Sh.boydii (group D), which type Sh.dysenteriae has strong cytotoxin and it could act on the intestinal mucosa to lead to its congestion, edema, ulcers and even necrosis, causing intestinal dysfunction, inducing fever, nausea and vomiting, abdominal pain, diarrhea, tenesmus, purulent bloody stools and other clinical symptoms. What is worse, it could also cause systemic complications, such as electrolyte imbalance, epileptic seizure and hemolytic uremic syndrome [5][6]. Antibiotics are the drugs of choice in the clinical treatment of BD and quinolones (nor oxacin,o oxacin,cipro oxacin,etc.),sulfonamides(trimethoprimsulfamethoxazole, etc.), cephalosporins (ceftriaxone, cefotaxime, etc.), and penicillin (ampicillin, etc.) are commonly used [7] , however, all of them have been proved to serious drug resistance and are gradually facing the risk of becoming invalid and no available drug to choose [8][9].
As a traditional Chinese medicine (TCM) therapy with most characteristics in China, some TCM masterpieces such as Treatise on Febrile Diseases, Puji Fang and so on have records on the treatment of BD. Those treatment could be dialectically classi ed as: damp-heat dysentery, cold-dampness dysentery, epidemic toxic dysentery, yin de ciency dysentery, aesthetic cold dysentery, and rest dysentery [10]. By searching the database of TCM recipes with the keywords of "dysentery" and "bacillary dysentery", we found that the recipe under the guidance of TCM theory such as Sanhuang Tablets [11] , Huanglian Jiedu Decoction [12] , Shaoyao Decoction [13] , Pulsatilla Decoction [14] , Gegen Qinlian Decoction [15] have a signi cant effect on the clinical treatment of BD and a variety of dosage form products such as tablets, pills, powders, capsules and injections have been developed [16].
Huanglian-Huangqin-Huangbo herb pair (HHH), consisting of Coptis chinensis Franch. (Huanglian in Chinese), Scutellaria baicalensis Georgi. (Huangqin in Chinese) and Phellodendron amurense Rupr. (Huangbo in Chinese), is commonly used in clearing away internal heat and dampness, purging heat and detoxifying in the TCM clinical practice which could clear the internal heat of upper, middle, lower energizer in human body [17]. Nowadays, there are many research which show that HHH has an inhibitory effect on pathogenic bacteria such as Shigella, Escherichia coli, Staphylococcus aureus and Salmonella [18][19][20]. These bacteria are corresponding to the pathogeny of BD. However, the active compounds and potential targets of HHH in the treatment of BD have not been found, and the further research on its molecular mechanism is not enough yet.
Network pharmacology is a new discipline which bases on the theory of systems biology. Through analyzing the network of biological systems, selecting speci c signaling nodes for multi-target drug molecular design, emphasizing the multi-pathway regulation of signaling pathways, network pharmacology could re ect the characteristics of "multi-component, multi-target, and multi-pathway" of traditional Chinese herbal compounds [21]. In this study, we predicted the possible targets of BD which are likely to be stimulated by different active compounds in HHH. Meanwhile, we researched for potential important ingredients and key targets and explored the overall regulatory mechanism of multiple components, multiple targets, and multiple pathways of HHH action.

Methods
Active compounds and targets of HHH Through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP,https://tcmspw.com/molecule.php?qn=1925), by searching Huanglian, Huangqin and Huangbo to collect the potential targets of HHH, then screening the compounds using lters with OB (oral bio-availability) ≥30% and DL (drug likeness) ≥0.18 to obtain the active compounds database of HHH. Furthermore, by using Pub chem (https://pubchem.ncbi.nlm.nih.gov/) to grasp the molecular formula, CAS number and download the two-dimension structure in SDF format. By utilizing Uniprot database(https://www.uniprot.org/uploadlists/) to obtain the formula gene names in order to build up a database of active compound and potential targets of HHH.
Therapeutic targets of HHH on BD Using the online drawing platform Microbiology Information Platform( http://www.bioinformatics.com.cn/ ), the potential targets of HHH on the active compounds and BD were compared and analyzed, and their intersections was taken as the potential target of HHH on the treatment of BD. Then we corrected the target names to o cial gene symbol.

Network Construction
We upload the therapeutic target protein of HHH on BD to the String database (https://string-db.org/ ) in the form of Gene symbol, then we performed the protein-protein interaction (PPI) analysis, the species was set as "Homo sapiens", the data was exported as TSV les, then imported into Cytoscape3.7.2, the node size and color were set to re ect the connectivity degree, the degree of the node indicates the number of connections between this node and other nodes in the network, the network was analyzed for topological attributes with the help of the function "Network analyze" in Cytoscape3.7.2, and the hub targets which degree value are greater than the median were con rmed . Enrichment analysis using Metascape database (https://metascape.org/gp/index.html) after importing the HHH-BD intersectional targets. Then selected "OFFICEGENESYMBOL" option, chose the "Homosapiens" option of species, selected "Homosapiens" for the background and ltered with P < 0.01 as the condition to obtain the biological process of HHH on the treatment of BD and related pathways.
From the "drug-active compound-target" network constructed by HHH active compounds and BD related targets (Fig.2 Analysis of the "active compound-gene target-pathway" network of HHH The "active compound-gene target-pathway" network of HHH was generated using Cytoscape3.7.2 by 87 related active compounds, 34 intersection gene targets and 14 important pathways (Fig. 7).

Molecular docking results
To further verify the effect, the screened active compounds namely quercetin, β-sitosterol, scutellarin, wogonin, isocorypalmine and the following potential target proteins: TNF (PDB:1JH5), IL-6 (PDB:4O9H) and PTGS2 (PDB:5KIR) were used for molecular docking. The results (Tab. 2) showed that the binding energy of the target protein and the corresponding active component were less than -5 kJ/mol, indicating that the target proteins have good binding property. Three proteins were predicted successfully to be the targets in treatment of BD, quercetin, β-sitosterol, scutellarin, wogonin, and isocorypalmine. Three targets and the lowest binding energy of ve ligand components were used to draw the docking mode diagram of the components and targets by PyMol 2.3.4 (Fig.8). The binding sites of β-sitosterol-PTGS2 were ASP-133 amino acid residues, β-sitosterol-TNF exerted effects through ASN-102 and GLN-93 amino acid residues, and the binding sites of Baicalein-PTGS2 were HIS-207 and ASN-382 amino acid residues.
Tab.2 Results of molecular docking between core compounds and core targets

Discussion
Based on the theory of evidence-based medicine of TCM, the form of compound is often applicated and treated clinical of various diseases, but due to the complexity of Chinese native medicine ingredient, existing methods mainly studied around some index ingredients and pharmacological effects. It cannot show the TCM`s mode of systemic effects, "plural ingredients, plural targets" completely, thus to clarify the network relations of different active compound and effect targets is the key to the development of TCM modernization [22]. Network pharmacology research is based on systems biology theory, which can analyze and predict the interaction between components and targets, so as to more comprehensively explain the multi-pathway regulation effects of different components on signal pathways, it is a powerful method to study the systemic effects of TCM compounds [23]. For further analysis, the TCMSP database provided 331 compounds which obtained from HHH in the study. According to the principle of ADME, OB re ects the process of drug absorption, distribution, metabolism and excretion, and DL re ects the similarity between compounds and known drug molecules. It is generally believed that only drugs with OB ≥ 30% and DL ≥ 0.18 are worthy of development [24][25].
Therefore, 87 active compounds were further screened based on these two indicators in this study, and including 14 active compounds of Huanglian, 36 active compounds of Huangqin, and 37 active compounds of Huangbo. By analyzed the PPI network we found that, Huanglian s associated main target genes were AKT1, CDKN1A, IL-6, RELA, BAX, BCL2, CASP3, NOS2, PTGS2, VEGFA, and Huangbo`s associated main target genes were PTGS2, NOS2, BAX, BCL2, CASP3, CYP1A2, PON1, TNF. In addition, there were 34 targets shared with BD Disease targets, namely IL-6, AKT1, PTGS2, TNF, CASP3, VEGFA and BCL2. For example, Among them, Shigella ora was positively correlated with environmental factors such as IL-6 and TNF-α [26]. PTGS, also known as cyclooxygenase (COX), it divided into two subtypes, namely COX-1 and COX-2. Previous studies have shown that in ammatory response related to PTGS closely [27]. The speci cal binds between TNF-α and receptors can induce in ammatory response [28]. And AKT1 is the hub target of PI3K-Akt signaling pathway, which has not only played an important role in the process of promoting cell proliferation and inhibiting apoptosis, but also participated in the process of in ammation. It is essential in intestinal in ammation [29].
Nowadays, it is still unclear of the exact pathogenesis of BD. The main pathogenic process is that the bacteria go through the gastrointestinal barrier to the colon, then invade colonic epithelial cells and they begin to grow and diffuse into adjacent infected cells, cause in ammation, epithelial cell death, ulcers, increase uid in the colon that produce pus and mucus, etc [30]. It has been reported that Shigella can secrete speci c virulence factors, thus disrupting the integrity of the epithelial barrier. In this way, the ability of pathogens to penetrating into colon tissues in the body can be enhanced, it nally induced in ammatory reactions, destroyed colon epithelial cells and produced clinical symptoms [31]. The basic clinical treatment of intestinal diseases is to prevent in ammation from happening and mechanism of action may be realized by regulating Th17/Treg balance and controlling intestinal bacteria [32]. mediating immune in ammatory response [33].
Among the 87 active compounds selected by HHH, such as quercetin, β-sitosterol, scutellarin, wogonin, and isocorypalmine, scutellarin can effectively inhibit the growth of Shigella bacteria, signi cantly reduce the content of IL-6 and TNF-α in serum of CIM mice, and slow down the occurrence of in ammatory reaction [26]. Wogonin has anti-in ammatory effect because it can inhibit the in ammatory process of Il-6, TNF-α and other important cytokines [34]. Quercetin can eliminate in ammation in vivo and in vitro by inhibiting in ammatory cytokines such as IL-6 and TNF-α and in ammatory enzymes. Moreover, quercetin has an inhibitory effect on various bacteria such as Escherichia coli and Salmonella, especially its inhibitory effect on gram-negative bacteria is stronger than gram-positive bacteria [35][36]. The βsitosterol play an anti-in ammatory role by signi cantly inhibit the expression of IL-6 and COX-2 [37]. Berberine has an obvious inhibitory effect on dysentery bacillus [38]. scutellarin, wogonin, quercetin and other avonoids and their natural plant extracts can also maintain and repair the integrity of the intestinal mucosa, adjusting the composition of the intestinal micro ora. For example, inhibiting the harmful bacteria such as e.coli while promoting the growth of bene cial bacteria such as Bi dobacterium, Bacteroidetes, which regulates intestinal immunity, reduce proin ammatory factor, reduce in ammatory bowel disease caused by a variety of intestinal microecological imbalance [39]. By PPI network, we further screening core active compounds of quercetin, β-sitosterol, scutellarin, wogonin, isocorypalmine, and core target gene TNF, IL-6 and PTGS2. By using molecular docking, obtained the core binding energy of active compounds and core target gene are less than − 5 KJ/mol, and these data were further proved the accuracy and reliability of the results in this study.
Currently, no correlation between these compounds and bacillary dysentery has been reported. However, they play a more important role in other diseases. For example, obacunone can inhibit VSMC proliferation, reduce intimal hyperplasia, and inhibit the occurrence of venous bridge restenosis by down-regulating the phosphorylation level of Akt [40], epiberberine has hypoglycemic effect [41], cantharidin has antitumor effect [42], and acacetin can protect the nerve function of rats HAD acute cerebral infarction [43]. In addition, some potential targets were also found, such as EGFR, IFNG, IL-10, MAPK1, RELA, SOD1, etc. It has been reported that EGFR is associated with non-small cell lung cancer [44]. IFNG activates macrophages, neutrophils, endothelial cells, platelets, as well as complement systems and coagulation systems, and then inducing the release of bioactive substances such as tumor necrosis factor α prostaglandin and kinin, and is widely recognized as a regulator of tumor immune surveillance and tumorigenesis [45]. MAPK1 plays an important role in the pathological process of preeclampsia [46]. RELA is essential for maintaining liver homeostasis against TNF-α driven immunotoxicity [47]. Therefore, the development of potentially active compounds as a new drug for the treatment of BD and the elucidated target gene as a new mechanism of action for the treatment of BD has very important research signi cance and innovation, which will be carried out in the next work plan.

Conclusion
Sum up, this study found that except for compound known to work on BD in HHH, such as quercetin, scutellarin, β-sitosterol, Berberine, there may still exist in other compound more involved in BD compounds of internal like obacunone, epiberberine, Coptisine hydrochloride, cantharidin, salviarin, acacetin. The speci c effects of these compounds will need to be con rmed by further experimental studies at the cellular and animal levels.

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding author on request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.