Exploration of the Mechanism and Active Targets of Chinese Herbal Formula, JTTZ, for the Treatment of Type 2 Diabetes with Obesity and Hyperlipidemia Based on Network Pharmacology

Previous studies have indicated that the JTTZ formula exhibits clinical benet in T2D with obesity and hyperlipidemia such as lowering blood glucose, blood lipids, weight, and ameliorating symptoms as well as regulating islet function. However, their mechanism of action remains unclear. T2D with obesity and hyperlipidemia is associated with a severely poor management duo to diculty in achieving the clinical goals and lack of effective multi-targeted therapies. In this study, we explored its potential mechanisms and therapeutic targets by network pharmacology. The active ingredients and targets of JTTZ were obtained in the TCMSP, TCMID, TCM Database@Taiwan, PubChem and Swiss Target Prediction. And the therapeutic targets were searched from TTD, DrugBank Database and DisGeNET. Then, topology analysis were used as secondary screens to identify key hubs of the network. Finally, the data was integrated by Cytoscape software to construct a common network module. PPI networks were visualized to identify the interaction of the candidate targets. GO and KEGG pathway analysis were implemented. Rerult: 110 active compounds and 166 candidate targets of JTTZ against T2D with obesity and hyperlipidemia were obtained to construct compound-targets network. And, the therapeutic targets AKT2, RELA, NFKB1 and GSK3B were identied. GO and KEGG pathway analysis indicated that the biological processes related to inammatory response, insulin secretion, steroid and bile acid metabolism, and 13 pathways mainly including adipocytokine signaling pathway, cAMP signaling pathway and cGMP-PKG signaling pathway were enriched. Our data established that JTTZ intervenes with adipose tissue dysfunction via regulating to the adipocytokine (leptin and adiponectin), AMPK signaling pathway, cAMP and cGMP-PKG signaling pathway, inhibits systematic inammatory response by NF-κB and MAPK signaling pathway, and ameliorates insulin resistance through PI3K/AKT2 pathway, all of which could thus offer a promising therapeutic strategy. In addition, AKT2, RELA, NFKB1 and GSK3B were identied to be regarded as potential therapeutic targets as well. 34). On the one hand, WAT play an important role in energy storage in the form of free fatty acids (FFA), which would maintain lipid metabolic homeostasis based on triacylglycerol (TAG) hydrolysis by lipoprotein lipase (LPL) activity to move non-esteried fatty acids (NEFA) into adipocytes and triacylglycerols (TAGs) lipolysis via cAMP activates protein kinases A (PKA) and cGMP activates protein kinases G (PKG) leading to phosphorylation of perilipin and hormone-sensitive lipase (HSL)(35, 36). In addition, the activated AMPK exerts the antilipolytic effects on adipocytes by regulating HSL controlled by PPARγ(37, 38). PPARγ is key regulator of adipocyte differentiation and glucose homeostasis, whose agonist commonly used to treat diabetes(39). And BAT are important in thermogenesis via generating ATP within a large number of mitochondria for energy production(35). GO and KEGG pathway enrichment analysis also showed JTTZ could ameliorate T2D patients with obesity and hyperlipidemia via AMPK signaling pathway, cAMP signaling pathway and cGMP-PKG signaling pathway. On the other hand, adipose tissue is a remarkably complex endocrine organ to secret a number of adipocyte-derived factors called as adipokines, such as leptin and adiponectin derived from WAT, insulin-like growth factor 1 (IGF-1) derived from BAT, and a variety of proinammatory cytokines like tumor necrosis factor (TNFα), interleukin-1β (IL-1β) and interleukin-6(IL-6)(34). Our present study showed adipocytokine signaling pathway had the most maximum BC and occupied an important position in pathway-targets network. The past studies revealed that both leptin and adiponectin could promote increased insulin sensitivity and glucose tolerance via activities of AMPK and exerting anti-inammatory effects through IGF-1 and the inducement of M2 macrophages, respectively(40–42). Moreover, expanded adipose tissue synthesizes


Introduction
The prevalence of type 2 diabetes (T2D) and obesity has continued to increase in over ve decades, which causes major threats to human health globally (1)(2)(3). In 2019, it was estimated that 463 million people live with diabetes, in which T2D was account for 90% of all diabetes, and it was predicted to double by 2045 (4,5). 60% of patients with T2D are obese (body mass index, BMI ≥ 30 kg/m2) ,and 70-80% have hyperlipidemia (5,6). Insulin resistance in liver, skeletal muscle, and adipose tissue accompanied by dysfunction of pancreatic islet β cells is the common underlying abnormality of T2D and obesity and hyperlipidemia. However, causes of T2D and obesity remain largely unclear (2,6). At present, metformin is the rst-line drug for the treatment of T2D by reduced hepatic glucose output and improvement of obesity-induced metabolic in ammation (7). Although there is several well-established drug classes for treating T2D, it is still a considerable unmet need for a drug that reverses diseases progression base on combined treatment strategies (8).
Our previous studies showed that JTTZ fomula, the Chinese herbal formula, could safely and signi cantly decrease the blood glucose, blood lipids, and body weight, enhance islet β cell function, improve insulin resistance for T2D patients with obesity and hyperlipidemia, which has been explored via a multicenter randomized, positive-controlled clinical trial with a total of 450 participants (9). The JTTZ consists of eight herbs, including Radix Salviae, Zingiberis Rhizoma, red yeast rice, Coptidis Rhizoma, Momordica charantia, Aloe vera, Schisandrae Chinensis Fructus and Anemarrhenae Rhizoma, which is derived from a classic formula, Dahuang Huanglian Xiexin Decoction. These herbs achieves effects on improvement of glucose and lipid metabolic disorder by interaction of different biological functions. Although we had con rmed the signi cant therapeutic effect of JTTZ against T2D with obesity and hyperlipidemia by clinical trials (9), its action mechanism and molecular targets is still unclear, which would limit it as a potential drug to be widely used in clinical practice.
The network pharmacology adopt a network perspective to describe the complex interactions among genes related to diseases and drugs, which is in accord with the holistic theory of traditional Chinese medicine (TCM) (10)(11)(12). There had been so many studies for revealing the potential mechanism and action targets of classic formula by applying TCM network pharmacology methods (13)(14)(15). It is appropriate for the TCM network pharmacology methods to reveal drug-gene-disease associations, so we explored the possible mechanism and potential therapeutic targets of JTTZ against T2D with obesity and hyperlipidemia through TCM network pharmacology. The owchart of the experimental procedures of our study is shown in Fig. 1 (16). TCMID is a comprehensive database to provide information and bridge the gap between Traditional Chinese Medicine and modern life sciences (17). TCM Database@Taiwan is currently the largest non-commercial TCM database for drug screening (18). We set the indexes of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as screening criteria. The candidate ingredients which satis es the selective threshold of OB and DL were regarded as the potential component.

Network Construction
The compounds-targets network was constructed and visualized in the Cytoscape software (version 3.7.2, Boston, MA, USA, https://cytoscape.org/) (24), which showed the relationship between the possible targets of JTTZ and the known targets of T2D with obesity and hyperlipidemia. The node shape, color and size were set in the Cytoscape software by generate style from statistic tool. The different node shape and color re ected different categories, such as targets, compounds and pathways, et al. The size of node depends on the magnitude of the degree centrality (DC) and betweenness centrality (BC), which re ects the number of direct links to nodes and describe the topological importance of targets in networks. The protein-protein interaction (PPI) network carried out by the string software (version 11.0, https://string-db.org/) (25), with species limited to "Homo sapiens", and results were saved in PNG and TSV format. The PNG les were showed as gure and the TSV les were imported into Cytoscape software to analyze and screen hub genes according to DC and BC parameter.

GO and KEGG Pathway Enrichment Performance
Gene Ontology (GO) analysis with the biological process (BP), cellular component (CC) and molecular function (MF) was carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.7, https://david.ncifcrf.gov/) (26). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed using ClueGO plugin in Cytoscape software, with species limited to "Homo sapiens" and P value ≤ 0.01 (27). Then, the ClueGO results about KEGG pathway enrichment in TSV format were downloaded, and import into Cytoscape software to construct pathway-targets network.

Targets Screening of JTTZ and T2D with Obesity and Hyperlipidemia
We obtained a total of 133 chemical ingredients of the eight herbal medicines in JTTZ from TCMSP, TCMID and TCM database@Taiwan. 110 compounds were nally selected after removing 23 compounds which did not link to any targets (Table 1)

Compound-Targets Network Analysis
Compound-targets network of JTTZ against T2D with obesity and hyperlipidemia was consisted of 276 nodes (110 compounds in JTTZ and 166 candidate targets) and 1423 edges which represented the interaction of compounds and targets (Fig. 2). We analyzed the network according to topological parameter including DC and BC, which indicated the compounds and targets with more DC and BC is more important on the effect of JTTZ against T2D with obesity and hyperlipidemia. The results showed that the top ve compounds included quercetin, arachidonic acid, kaempferol, luteolin and stigmasterol acted on 48, 38, 37, 35 and 33 candidate targets respectively. The OB represents the radio of the amount and speed absorbed into system circulation to achieve biological functions of oval drugs. The OB of quercetin, arachidonic acid, kaempferol, luteolin and stigmasterol is 46.43, 45.57, 41.88, 36.16 and 48.38, respectively, which means they might be the crucial active compounds of JTTZ by reason of their considerable positioning in the network.

PPI Networks Analysis
The PPI network of JTTZ against T2D with obesity and hyperlipidemia-related candidate targets contained 164 nodes and 1735 edges, which illustrated 164 interacting proteins and 1735 interactions (Fig. 3A). The protein tends to form macromolecular complexes by interaction to accomplish biological functions, so the PPI network play a vital role in mechanism analysis of biological processes. In order to select the more important interactive proteins, we set indexes of con dence score ≥ 0.9 and DC ≥ 8.294 (the average value of DC) to re-constructed the PPI network containing 59 nodes and 375 edges (Fig. 3B). The targets were further screened and 19 targets with BC ≥ 0.034 (the median values of BC), which were involved in various pathogenic processes of T2D with obesity and hyperlipidemia (Fig. 3C).

GO and KEGG Pathway Enrichment Analysis
GO of 166 candidate targets was analyzed based on BP, CC and MF, and the top 20 was screened out, respectively (Fig. 4). The results indicated that BP of these targets protein related to in ammatory response, steroid and bile acid metabolism, cellular proliferation and apoptotic, insulin secretion, vasoconstriction, response to hypoxia, and oxidation-reduction process, such as in ammatory response referring to G-protein coupled receptor signaling pathway and response to lipopolysaccharide. CC is mainly distributed in integral component of plasm, integral and partial component of membrane, nuclear chromatin, endoplasmic reticulum body and membrane, neuronal cell body, projection and dendrite, receptor complex, endocytic vesicle and extracellular exosome. And MF contains molecular activities like G-protein coupled serotonin receptor, steroid hormone receptor, and molecular bind, such as steroid, drug, enzyme, ligand-activated sequence-speci c DNA, coupled to transmembrane movement of substances, coupled via Gs.
KEGG pathway analysis was performed to show that these targets were mainly enriched in 13 pathway by ClueGO plugin (Fig. 5)

Identi ed Candidate Targets by Analysis of Pathway-Targets Network
To further explore the mechanism of JTTZ in treatment of T2D with obesity and hyperlipidemia. The pathway-targets network is constructed based on the signi cantly enriched pathways and genes regulating these pathways (Fig. 6). The network contains 148 nodes (13 in pathways and 135 in related-genes) and 337 edges. The topological analysis results showed that adipocytokine signaling pathway has the most maximum BC with 48 directed edges and the main 10 targets included AKT2, PRKCB, RELA, NFKB1, GSK3B, BRAF, EDNRA, PTGS2, SLC2A1, PDGFRB and HDAC2.

Discussion
In our previous studies, we discovered that JTTZ could potentially be developed as an alternative medicine because it appeared to alleviate symptoms of T2D with obesity and hyperlipidemia. However, the molecule mechanism and action targets is still unclear. Therefore, we explored possible mechanism of JTTZ against T2D with obesity and hyperlipidemia by the TCM network pharmacology.
The JTTZ formula consists of eight herbs to be used for stomach heat syndrome in clinical application. In the present study, a compound-targets network of JTTZ was constructed containing 110 compounds and 166 targets. The results showed that the most compounds acted on multiple targets, such as quercetin, arachidonic acid, kaempferol, luteolin and stigmasterol effecting 48, 38, 37, 35 and 33 candidate targets respectively, which means they might be the crucial active compounds of JTTZ by reason of their considerable positioning in the network. Quercetin is a polyphenol, and have signi cant effects on anti-in ammation, anti-obesity and anti-diabetes through enhancing antioxidative activities, regulating MAPK and NF-κB/JNK pathway, reducing the proin ammation enzymes activity and cytokine levels (28). Arachidonic acid is a strong inducer of insulin secretion and its metabolism is involved in free fatty acids and hyperglycemia mediated in ammation base on lipid and immune pathway (29). Kaempferol is a kind of natural avonoids and has been exerted extensive biological activities, for example, anti-oxidation, anti-in ammation, antibacterial and antitumor activities, moreover, it also show a notable inhibition activity on a-glucosidase (30). Luteolin also is a represent avonoid and it could alleviate brosis, hypertrophy and apoptosis, and inhibit in ammation and oxidative stress through inhibition of NF-κB and induction of the Nrf2 pathway (31). Stigmasterol is a minor phytosterol and showed reducing plasm cholesterol by decreasing cholesterol absorption, suppressing hepatic bile acid and cholesterol synthesis, and stimulating transintestinal cholesterol excretion mediated by liver X receptor activation (32). Although the biological functions of single herbal compounds is different, due to overlapping targets in network, the multiple compounds of JTTZ would act on same targets and pathways to achieve synergistic effects.
The main pathological mechanism of T2D with obesity and hyperlipidemia refers to variety organs, such as pancreas, liver, adipose tissue, skeletal muscle, kidney, brain and small intestine, and is characterized by decreased insulin sensitivity, common insulin resistance, pancreatic β-cell failure, adipose tissue dysfunction, sterol and bile acids metabolic disorders and chronic systematic in ammation (2,5). By the PPI, GO and KEGG pathway enrichment analysis, biological process of JTTZ related to in ammatory response, steroid and bile acid metabolism, cellular proliferation and apoptotic, insulin secretion, response to hypoxia, vasoconstriction, and oxidation-reduction process, and major pathways contained adipocytokine signaling pathway, AMPK signaling pathway, cAMP signaling pathway and cGMP-PKG signaling pathway, NF-κB and MAPK signaling pathway, we summarized several potential action mechanisms and therapeutic targets, including adipose tissue dysfunction, in ammation response, and insulin resistance (Fig. 7).

Adipose tissue dysfunction
Adipose tissue, which main included white adipose tissue (WAT) and brown adipose tissue (BAT), regulates multiple physiological processes and its dysfunction is associated with imbalance of metabolic homeostasis, insulin resistance, adiposity and T2D through dysregulated adipokine secretion, disrupted mitochondrial function, in ammation, brosis, and hypoxia (33,34). On the one hand, WAT play an important role in energy storage in the form of free fatty acids (FFA), which would maintain lipid metabolic homeostasis based on triacylglycerol (TAG) hydrolysis by lipoprotein lipase (LPL) activity to move non-esteri ed fatty acids (NEFA) into adipocytes and triacylglycerols (TAGs) lipolysis via cAMP activates protein kinases A (PKA) and cGMP activates protein kinases G (PKG) leading to phosphorylation of perilipin and hormone-sensitive lipase (HSL) (35,36). In addition, the activated AMPK exerts the antilipolytic effects on adipocytes by regulating HSL controlled by PPARγ (37,38). PPARγ is key regulator of adipocyte differentiation and glucose homeostasis, whose agonist commonly used to treat diabetes (39). And BAT are important in thermogenesis via generating ATP within a large number of mitochondria for energy production (35). GO and KEGG pathway enrichment analysis also showed JTTZ could ameliorate T2D patients with obesity and hyperlipidemia via AMPK signaling pathway, cAMP signaling pathway and cGMP-PKG signaling pathway. On the other hand, adipose tissue is a remarkably complex endocrine organ to secret a number of adipocyte-derived factors called as adipokines, such as leptin and adiponectin derived from WAT, insulin-like growth factor 1 (IGF-1) derived from BAT, and a variety of proin ammatory cytokines like tumor necrosis factor (TNFα), interleukin-1β (IL-1β) and interleukin-6(IL-6)(34). Our present study showed adipocytokine signaling pathway had the most maximum BC and occupied an important position in pathway-targets network. The past studies revealed that both leptin and adiponectin could promote increased insulin sensitivity and glucose tolerance via activities of AMPK and exerting anti-in ammatory effects through IGF-1 and the inducement of M2 macrophages, respectively (40)(41)(42). Moreover, expanded adipose tissue synthesizes and secretes cytokines including TNFα, IL-1β, IL-6, which cause adipose tissue in ammation that may be a driving factor in the development of systemic IR and T2D in obesity (34,43). Therefore, the regulation of dysfunctional adipose tissue may be potential therapeutic strategies target for JTTZ formula against T2D with obesity and hyperlipidemia. 1β; IL-6, interleukin-6; TNFR, TNF receptor; ILR, interleukin receptor; TRAF, TNF receptor-associated factor; IRAK, interleukin receptor-associated kinase; IKKs, Ikappa-B kinases; IκB, I-kappa-B; NFκB, nuclear factor-κB; MAPK, mitogen-activated protein kinase; JNK, jun N-terminal kinase; IRS, insulin receptor substrates; PI3K, phosphatidylinositol-3-kinase; AKT2, serine/threonine protein kinase B; GSK3, glycogen synthase kinase-3; GS, glycogen synthesis.

Systematic in ammation
The results of GO and KEGG pathway analysis showed in ammation referred to response to lipopolysaccharide and NF-κB and MAPK signaling pathway.
Obesity-induced diabetes is accompanied with a chronic low grade in ammation, which may in part be mediated by endotoxins (especially lipopolysaccharide, LPS) derived from gram-negative bacterial species (44,45). LPS toxicity is triggered by lipid A component via recognition of Toll-like receptors (TLR), but LPS has a short half-life, so the complex of LPS-lipopolysaccharide binding protein (LBP) to cluster of differentiation (CD14) leads to in ammatory responses via activation of TLR4 (45,46). The activation of TLR4 interacting with LPS could trigger various signaling transduction, mainly including NF-κB and MAPK (47). NF-κB is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events, which is a homo-or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, et al (48,49). Our pathway-targets network indicates that RELA and NFKB1 may be therapeutic targets of JTTZ formula against T2D with obesity and hyperlipidemia. NF-κB is in an inactive state because of its combination with I-kappa-B (IκB). The interaction with LPS and TLR4 leads to the phosphorylation of I-kappa-B kinases (IKKs) complex via myeloid differentiation primary response gene (MyD88), IL-1 receptor-associated kinase (IRAK), TNF receptor-associated factor (TRAF), NF-κB inducing kinase (NIK), which causes that IκB is phosphorylated to degrade NF-κB complex for translocation to the nucleus and subsequent expression of pro-in ammatory genes (47,49). MAPK represent a family of kinases for phosphorylation of serine and threonine, whose signaling pathway includes the extracellular signal-regulated kinases such as JNK, p38 MAPK and extracellular signal-related kinase (50). The activation of TLR4 stimulates JNK and p38 MAPK signaling pathway (51). JNK exerts direct inhibitory phosphorylation of insulin receptor substrates (IRS) 1 and 2 leading to decrease recruitment of the phosphatidylinositol-3-kinase (PI3K)-serine/threonine protein kinase B (AKT2) signaling pathway in response to insulin, otherwise, JNK play a major role in obesity-induced macrophage activation leading to increase levels of M1 macrophages to induce in ammatory cytokines driving insulin resistance (50,52). p38 MAPK activation reduces the expression of IRS1 and IRS2 contributing to insulin resistance, and accelerate biosynthesis of in ammatory cytokines such as TNFα, IL-1β and IL-6 in LPS-stimulated human monocytes (53,54).

Insulin resistance
The pathological state of insulin resistance is associated with obesity-induced T2D, moreover, our the analysis of network pharmacology revealed that JTTZ formula may be act on insulin signaling pathway especially AKT2 and glycogen synthase kinase-3 (GSK3) gene targets to treat T2D with obesity and hyperlipidemia (55). The classic insulin signaling starts when it binds to IRS1 and IRS2, which the phosphorylation of these proteins enable trigger PI3K/AKT2 pathway to regulate insulin metabolic actions (47,55). AKT2 is responsible of the regulation of glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface, and is contributed to the storage of glucose in the form of glycogen by phosphorylating GSK3 (56,57).The phosphorylating of GSK3 is inactive action to eliminate the activation of glycogen synthesis (GS) and glucose uptake (58). GSK3 is a multi-functional serine/threonine kinase which is increased expression in the skeletal muscle of patients withT2D, and GSK3 inhibitors have been regarded as an anti-diabetic therapeutic target(59, 60).

Conclusion
Our study systematically explored the potential action mechanism and therapeutic targets of JTTZ formula against obesity and hyperlipidemia used network pharmacologic method. Although JTTZ formula have variety components involved in multiple targets, different compounds could act on same targets via multiple biological process and signaling pathway. The results indicate that JTTZ formula could achieve effects on anti-diabetes, anti-obesity and anti-hyperlipidemia through regulation and improvement of adipose tissue dysfunction, systematic in ammation and insulin resistance, which may be mediated by multiple targets, such as RELA, NFKB, AKT2, GSK3, et al. However, there is still some limitation in this study due to network research and machine algorithms, therefore, it is necessary to further validate mechanisms of JTTZ formula by experiments.

Availability of data and materials
The data used to support the results of this study can be obtained from the corresponding author.

Competing interests
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.  Compound-targets network of JTTZ.

Figure 3
The PPI network of candidate targets for JTTZ against T2D with obesity and hyperlipidemia. KEGG pathway enrichment of candidate targets of JTTZ against T2D with obesity and hyperlipidemia.

Figure 6
Targets gene-pathway network of JTTZ against T2D with obesity and hyperlipidemia.