Network Pharmacology Analysis of Traditional Chinese Medicine Formula Xiao Ke Yin Shui Treating Type 2 Diabetes Mellitus

Xiao Ke Yin Shui (XKYS) formula is a traditional Chinese medicine formula treating type 2 diabetes mellitus (T2DM). XKYS formula consists of four herbs, i.e., Coptidis rhizoma, Liriopes radix, bitter melon, and Cassiae semen. Herein, the chemical profiles of four herb extracts were investigated, and further analysis of the underlying mechanism of XKYS formula treating T2DM was performed using network pharmacology. The main components were selected for our network-based research. Targets of XKYS formula were mainly collected from two databases, SwissTargetPrediction and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the text-mining method was also implemented. T2DM relating genes and therapeutic targets were collected from five databases. Subsequently, STRING and Cytoscape were employed for the analysis of protein-protein interaction (PPI) networks. Functional annotation and pathway analysis were conducted to investigate the functions and relating pathways of target genes. The content of 12 compounds in the herb extracts was determined. With the analysis of PPI networks, a total of 76 genes were found to be important nodes and could be defined as the main target genes regulated by XKYS formula in the treatment of T2DM and its complications. Components in XKYS formula mainly regulate proteins including protein kinase B (Akt), phosphatidylinositol 3-kinase (PI3K), insulin receptor substrate (IRS), and tumor necrosis factor (TNF). XKYS formula exerts therapeutic effects in a synergetic manner and exhibits antidiabetic effect mainly via reducing insulin resistance. These findings could be guidelines in the further investigation of this formula.


Introduction
Diabetes mellitus (DM) is now recognized as a complex metabolic disorder, and type 2 diabetes mellitus (T2DM) is the most common type of diabetes [1]. Nowadays, different kinds of therapies are applied in the treatment of diabetes, including insulin and other oral medications. Such therapies may be promising in glycemic control but could also cause side-effects like hypoglycemia or gastrointestinal dysfunction [2]. us, more and more people have turned their attention to herbal medicine or diet-based therapies, seeking for safer and more cost-effective complementary medicine for T2DM [3][4][5][6].
Traditional Chinese medicine (TCM) is a rich resource, possessing data that can still be hints for the development of new drugs. e focus of the current study, Xiao Ke Yin Shui (XKYS) formula, is recorded in Bencaogangmu (compendium of Materia Medica). XKYS formula contains four herbs, namely, Coptidis rhizoma (dried rhizomes of Coptis chinensis Franch.), Liriopes radix (the dried tuberous roots of Liriope spicata ( unb.) Lour. var. prolifera Y. T. Ma), bitter melon (the immature fresh fruits of Momordica charantia L.), and Cassiae semen (the dried seeds of Cassia obtusifolia L.). All these herbs are widely used in clinical practice treating diabetes [7].
Several ingredients in these four herbs are generally accepted as the main bioactive components in the treatment of T2DM and its complications, i.e., alkaloids in Coptidis rhizoma, polysaccharides in Liriopes radix, triterpenoids and polysaccharides in bitter melon, and anthraquinone and naphthopyrone in Cassiae semen [8][9][10][11]. ereby, herb extracts were prepared aiming to yield bioactive fractions containing ingredients mentioned above. e mixture of these herb extracts may be a satisfying alternative for the treatment of T2DM.
In XKYS formula, a single component may be responsible for the therapeutic effect through different pathways, and some components may act on the same targets as well. For example, isoquinoline alkaloids from Coptidis rhizoma like berberine are generally recognized as activators of adenosine 5′-monophosphate-(AMP-) activated protein kinase (AMPK) but could also exert therapeutic effects on T2DM through inhibition of protein tyrosine phosphatase 1B (PTP1B) and peroxisome proliferator-activated receptor gamma (PPARc) [12][13][14]. Polysaccharides of Liriopes radix and extracts of Cassiae semen could ameliorate glycemic control through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway [15,16]. Triterpenoids in bitter melon are proved to be AMPK activators while polysaccharides are inhibitors of PPARc [17,18]. ese findings, however, could not fully explain the synergetic effects of XKYS formula in the treatment of T2DM and its complications.
Nowadays, researchers have been aware that the "one key, one lock" mode is insufficient to decipher the drug actions, especially in those complex diseases. Network pharmacology, however, analyzing drugs and drug targets in a systemic manner may provide us with novel insights into drug actions [19]. e key ideas of network pharmacology share much with the basic disciplines of TCM, making it a useful tool in the research of TCM [20]. In addition, rapid development of biomedical big data, like TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform), has facilitated such research [21]. us, a network-based pharmacological analysis could provide us with a comprehensive understanding towards the significance of each component, target, and pathway.
In brief, this study provided chemical profiles for the extracts of four herbs. And, network pharmacology analysis was applied to understand the underlying mechanisms of XKYS formula in the treatment of T2DM and its complications.

Materials and Reagent.
Coptidis rhizoma, Liriopes radix, fresh bitter melon, and Cassiae semen were authenticated by Professor Jiachun Chen (School of Pharmacy, Huazhong University of Science and Technology, or HUST). Voucher specimens of these herbs were deposited in Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, HUST. e four herb extracts were total alkaloids of Coptidis rhizoma (TACR), Liriopes radix polysaccharides (LRP), bitter melon extract (BME), and Cassiae semen extract (CSE), respectively. Detailed preparation methods for the extracts were reported in Supplementary Materials (Part 1). Acetonitrile (CH 3 CN) was purchased from Sigma Aldrich (USA). Water was deionized water. Hydrochloric acid, methanol, phosphate buffer, anthrone, sulfuric acid, fructose, glucose, and formic acid (HCOOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Analysis of Four Herb Extracts.
e analysis of TACR was conducted using the reported method [22]. e total carbohydrate content of LRP was examined using anthrone-sulfuric acid method according to the previous report [23].
Monosaccharide composition analysis of LRP was performed using the HPLC method after derivation with 1-pheny-3-methyl-5-pyrazolone (PMP), and the detailed method was reported in Supplementary Materials (Part 3) [24]. e mobile phase was 20 : 80 (v : v) CH 3 CN-H 3 PO 4 buffer (pH 7.0). e flow rate was 1.0 ml/min. e injection volume was 20 μl. Column temperature was held constant at 25°C. e UV detection wavelength was 250 nm. e total carbohydrate content of BME was examined using anthrone-sulfuric acid method according to the previous report [23].
HPLC analysis of BME was conducted with the mobile phase consisting of CH 3 CN (A) and water (B). e elution program was 0 min 20%A, 10 min 30%A, 30 min 50%A, and 50 min 70%A. e flow rate was 1.0 ml/min. Column temperature was held constant at 25°C. e detector was evaporative light scattering detector (ELSD), with the evaporator temperature at 60°C and nebulizer temperature at 40°C. e flow rate of nitrogen was 1.6 l/min. e standard (10, 20 μl) and sample (20 μl) dissolved in 70% methanol were subjected to HPLC analysis.
HPLC analysis of CSE was carried out according to the previous report [25].

Construction of Protein-Protein Interaction (PPI) Networks of XKYS Formula Treating T2DM and Its
Complications. To identify the corresponding targets of the main components of XKYS formula, several approaches combined with chemometric method, information integration, and data-mining were implemented. ese components, except polysaccharides, were submitted to TCMSP and Swis-sTargetPrediction server to find component-target interaction information. TCMSP mainly predicts drug-target interactions with random forest (RF) and support vector machine (SVM) method, while SwissTargetPrediction is a 2D/3D similarity measurement of small molecules [26,27]. All components, as well as the aglycon of the glycosides, were also submitted to PubMed, SciFinder, and Google Scholar for the text-mining of component-target information. And all targets were submitted to the UniProt database (http://www.uniprot.org) for validation of their gene names. Hereby, a target gene list of XKYS was obtained through in silico investigation.
Two gene lists were submitted to STRING. e STRING server generated a "Combined Score" ranged from 0 to 1 for each interaction. e higher the score is, the greater the confidence of the interaction. In STRING, an interaction >0.4 means "medium confidence" and >0.7 means "high confidence" [28]. e obtained PPI networks were intersected using Cytoscape 3.2.1, and interactions with score >0.7 were collected, generating a new PPI network, i.e., the PPI network of XKYS formula treating T2DM and its complications.

Gene Ontology (GO) Functional Annotation and KEGG Pathway Analysis.
To elucidate the function of the targets and their role in signaling transduction, the Database for Annotation, Visualization and Integrated Discovery (DA-VID) was used to analyze the GO function and KEGG pathway of the main target genes of XKYS formula in the treatment of T2DM and its complications. e biological process, cellular component, molecular function, and the pathway involved were also described.

Component-Target-Pathway Network Construction.
e network model of component-target-pathway was established using Cytoscape 3.2.1. In this network, nodes represent components (C), targets (T), or pathways (P), and edges represent the interaction of C-T or T-P. Based on the results of KEGG enrichment analysis and C-T database, the C-T-P interactions were shown to provide an overview on the mechanisms of XKYS formula in the treatment of T2DM and its complications.

Determination of Main Component Contents of Four Herb
Extracts. Four isoquinoline alkaloids, three cucurbitane-type triterpenoids, two anthraquinones, and three naphthopyrones were determined using HPLC. Results of the HPLC analysis are shown in Table 1 and Figure 1, and the structures were also reported ( Table 2). e total content of carbohydrate in LRP was determined to be 96.5% while BME 10.8%. e composition of LRP was also determined as fructose : glucose, 20.7 : 1.

Screening of Main Components from Four Herb Extracts and Construction of PPI Networks.
e screening of 19 components (Table 2) in XKYS had led to the acquisition of 216 targets, while 602 genes relating to the pathophysiology of T2DM were also collected (Supplementary Materials, Tables S1 and S2). All the gene names in two lists were uploaded to STRING, respectively. e XKYS PPI network consists of 216 proteins and 2070 interactions, whereas T2DM, 602 proteins and 11595 interactions (Figures 2(a) and 2(b)). Each interaction has a Combined Score >0.4. By intersecting these two networks, all interactions with high confidence (>0.7) were picked, generating a new PPI network.
e new network consists of 76 genes, representing the main target genes regulated by XKYS formula in the treatment of T2DM. In addition, among these genes, some genes like AKT1, mitogen-activated protein kinase 1 (MAPK1), tumor necrosis factor (TNF) were also found to be important genes involved in the progression of diabetic complications [29][30][31].
us, these genes could also be regarded as the main target genes regulated by XKYS formula in the treatment of T2DM and its complications ( Figure 2(c), Table 3).
e new PPI network contains 76 nodes and 333 edges ( Figure 2(c)). In this network, nodes represent targets, while edges, the interactions of proteins. And degree, a topological parameter describing the importance of a node, stands for the number of edges connecting to the node. e higher the degree, the more important the target in the network is.
As can be seen in Figure 2(c), important targets were painted red and located centrally in the network. AKT1, PI3KCG, MAPK1, LEP, IRS1, and PPARG were the top six genes regarding their degree.

GO Functional Annotation and KEGG Pathway Analysis.
GO functional annotation was performed on 76 target genes, and the top 20 GO terms (P < 0.01) were selected based on − log P values ( Figure 3). GO enrichment analysis indicated that these 76 target genes are responsible for glucose homeostasis, platelet activation, regulation of insulin secretion, cellar response to hypoxia, and cellular response to insulin stimulus (Figure 3(a)). ese biological processes are related to molecular functions including, steroid hormone receptor activity, protein kinase activity, protein serine/threonine (Ser/ r) kinase activity, enzyme binding, and drug binding (Figure 3(b)). And, these processes occur mainly in caveola, cytosol, plasma membrane, nucleoplasm, and receptor complex (Figure 3(c)).
KEGG pathway analysis was conducted for further exploration of these targets as shown in Figure 3(d). ese  targets were highly enriched in insulin resistance (IR), insulin pathway, adipocytokine pathway, AMPK pathway, T2DM, forkhead box protein O (FoxO) pathway, nonalcoholic fatty liver disease (NAFLD), mammalian target of rapamycin (mTOR) pathway, hypoxia-inducible factor 1 (HIF-1) pathway, glucagon pathway, and so on. ese pathways are highly relevant to the development of T2DM and its complications, and XKYS formula may exert therapeutic effects through pathways mentioned above.

Composition-Target-Pathway Network Construction.
A total of 76 genes were defined as the main target genes regulated by XKYS formula in the treatment of T2DM and its complications. ese genes were kept, and other entries were omitted in the database of C-T. e combination of C-T and T-P databases had led to a C-T-P network (Figure 4), providing us with an overview on the therapeutic effects of XKYS formula. In addition, the degree of each node is presented in Supplementary Materials Table S3. e inner cycle represents the main components of XKYS formula. Nodes painted in red were key components interacting with a larger number of targets. Among all of these 19 components, alkaloids from Coptidis rhizoma were regarded as the key components in the treatment of T2DM and its complications. e middle cycle represents the main target genes regulated by XKYS formula. When painted in red, the corresponding targets are regulated by more components and participate in more pathways. It could be observed that  Evidence-Based Complementary and Alternative Medicine

Discussion
TCM formulae are usually hard to decipher due to its mode of action, namely, "network target, multicomponents" [20]. T2DM is also a complex metabolic disorder with changes in various pathways. XKYS formula and T2DM can be considered as two networks and explained with the help of network pharmacology.
According to C-T-P network, alkaloids from Coptidis rhizoma are the main components in XKYS formula that exert antidiabetic effects. Results also indicated that around 64% of the content in TACR could be determined using the HPLC method. Among these isoquinoline alkaloids determined, berberine accounted for ∼35% of the fraction and was also proved to be a key node in the C-T-P network. Berberine has a wide range of biological activities, among which anti-hyperglycemic and anti-hyperlipidemia are two major benefits in the treatment of T2DM [8]. Berberine could activate AMPK via inhibition of mitochondrion respiratory complex I instead of the regulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ), leading to the reduction of IR and amelioration of lipid metabolism [13,32]. Momordicoside L and its analogue were detected in BME and interesting enough, and the agylcon of Momordicoside L could activate AMPK through CaMKKβ, which was distinct from the mechanism of berberine [17]. is could be a good evidence of synergetic effect presented by this formula.
LRP are highly enriched according to its total content of carbohydrate. Our previous studies had found that LRP could upregulate the insulin signaling pathway and exert therapeutic effects in diabetic rodents through activation of PI3K/Akt signaling pathway [15,23]. Aurantio-obtusin, along with its glycosides, accounted for ∼6.8% of CSE and was shown to be an important component in the C-T-P network. Previous reports had indicated that aurantioobtusin may offer therapeutic effects in hypertension through the PI3K/Akt-eNOS (endothelial nitric oxide  8 Evidence-Based Complementary and Alternative Medicine synthase) pathway, implying a possible mechanism in the treatment of diabetic complications of XKYS formula [33].
As can be seen in the C-T-P network, AKT1, PI3KCG, PI3KR1, and TNF were key genes regulated by XKYS formula in the treatment of T2DM and its complications. Proteins expressed by genes AKT1, PI3KCG, and PI3KR1 were all important members in the signaling transduction of PI3K/Akt. is signaling pathway is of great significance in the progression of T2DM due to its role in glucose metabolism [34]. e PI3K/Akt signaling pathway is also responsible for the regulation of the signaling pathway like MAPK, FoxO, and nuclear factor kappa B (NF-κB). ese pathways play important roles in the regulation of protein synthesis, cell survival, differentiation, proliferation, and apoptosis and are highly related to the proliferation and regeneration of islet β-cells [29].   Table 3.

Evidence-Based Complementary and Alternative Medicine
Tumor necrosis factor (TNF) is a cytokine that could be secreted by macrophages and adipose cells. It can induce IR by downregulation of the activity of PI3K/Akt signaling pathway. It has also been reported that MAPK and NF-κB signaling pathways are stimulated by TNF and further regulates inflammatory response, oxidative stress, and apoptosis [31].
As shown in KEGG pathway analysis, 23 out of 76 targets were found to participate in IR, ranking no. 1 according to its − log P value. Other pathways on the KEGG list were adipocytokine pathway, AMPK pathway, insulin pathway, T2DM, FoxO pathway, NAFLD, etc. And, this finding was further approved by the C-T-P network. All of these signaling pathways are all highly related to IR. us, we could make a preliminary inference from this result that the most important mechanism of XKYS formula in the treatment of T2DM may be reducing IR.
IR is a condition in which the target organs are insensitive to insulin. Long-term of unhealthy diet and lacking exercises could cause overweight or even obesity. Overweight or obese patients are in metabolic disorder states with excessive fat accumulation [35]. Some experts had put forward a hypothesis that obesity is a chronic condition of inflammation [36].
Adipocytokines like TNF and interleukin-6 (IL-6) could inhibit the binding of insulin receptor (InsR) and insulin receptor subunit (IRS). IRS would be degraded under such conditions. In addition, inflammation response and oxidative stress interact as both cause and effect, causing the damage of islet β-cells and diabetic microangiopathy and then leading to the onset of T2DM and its complications [37]. Moreover, the activity of AMPK in skeletal muscles and liver reduces in IR conditions, causing decreased oxidation of free fatty acids and a lower intake of glucose, which in turn deteriorating glycemic control [38]. e FoxO pathway is one of those key factors in the transition from IR to the damage of islet β-cells. e dysfunction of β-cells is caused by various factors, including oxidative stress and inflammatory response. e FoxO pathway is highly related to the risk factors mentioned above [39].
In addition, the pathway in cancer was also an important pathway enriched in the C-T-P network (Figure 4). For one thing, diabetes is a risk factor for carcinogenesis due to hyperinsulinemia, hyperglycemia, and fat-induced chronic inflammation [40]. For another, components in XKYS formula like isoquinoline alkaloids and cucurbitane-type triterpenoids were reported to possess antitumor activities through pathways participating in the pathophysiology of  Figure 3: GO functional annotation and KEGG analysis of 76 target genes. Biological process (a), molecular function (b), cellar components (c), and KEGG pathways (d) were sorted according to − log P values (P < 0.01). P value was calculated using a modified Fisher exact test, which ranges from 0 to 1. P value � 0 represents perfect enrichment. For each diagram, the x-axis presents GO or KEGG terms, whereas the y-axis is the − log P value of each term.

12
Evidence-Based Complementary and Alternative Medicine both diabetes and cancer [41,42]. us, such correlation was also presented in C-T-P network. It should also be noted that the PI3K/Akt signaling pathway is also regarded as a key regulator in the development of diabetic complications [29]. And, among those pathways enriched, the hypoxia-inducible factor 1 (HIF-1) pathway and vascular endothelial growth factor (VEGF) pathway are considered to be important factors involved in the development of diabetic complications [43,44]. e network-based pharmacological analysis has shown that XKYS formula could regulate the glucose and lipid metabolism and alleviate IR mainly through insulin, AMPK, adipocytokine, and FoxO pathway. And, this formula could also be used in the treatment of diabetic complications due to its effect on PI3K/Akt, HIF-1, and VEGF pathways.
is study provided an overview on the antidiabetic effects of XKYS formula in a holistic manner. However, in vivo and in vitro experiments are required to offer more information about the mechanisms of XKYS formula.

Conclusion
Components in XKYS formula mainly regulate proteins including Akt, PI3K, IRS, and TNF. XKYS formula exerts therapeutic effects in a synergetic manner and exhibits antidiabetic effect mainly via reducing IR. ese findings could be guidelines in the further investigation of this formula.

Data Availability
e data used to support the findings of this study are included within the article and the Supplementary Materials.

Conflicts of Interest
e authors declare that there are no existing conflicts of interest.