Investigation of the potential mechanism of the Shugan Xiaozhi decoction for the treatment of nonalcoholic fatty liver disease based on network pharmacology and molecular docking

Background: Nonalcoholic fatty liver disease (NAFLD) is a metabolic-related disease with its incidence increasing annually. Shugan Xiaozhi (SGXZ) decoction, a composite traditional Chinese medicinal prescription, was demonstrated to exert a therapeutic effect on NAFLD. However, the underlying mechanisms were incompletely elucidated. In this study, the potential bioactive ingredients and mechanism of the SGXZ decoction against NAFLD were explored via network pharmacology and molecular docking. Methods: Compounds in SGXZ decoction were identi�ed and collected from literature studies, and the corresponding targets were predicted through SEA database. Potential targets related to NAFLD were overlapped by using DisGeNET and GeneCards database. The compound-target-disease and PPI network were then constructed based on the putative intersection targets of SGXZ decoction and NAFLD to recognize the key compounds and targets. Functional enrichment analysis was performed to elucidate the biological process and signaling pathway that SGXZ decoction treated NAFLD. Finally, molecular docking combined with homology modeling was applied to further verify the binding between key active compounds and targets. Results: A total of 31 active compounds and 220 corresponding targets in SGXZ decoction were collected. Moreover, 1544 overlapped targets of NAFLD were obtained, of which 78 common targets were intersected with targets of SGXZ decoction. Key compounds and targets were recognized from compound-target-disease and PPI network. Functional enrichment analysis revealed that multiple biological pathways were annotated including insulin resistance, HIF-1, PI3K-Akt, and MAPK signaling pathways. Molecular docking con�rmed that gallic acid, chlorogenic acid and isochlorogenic acid A could combine �rmly with all of the key targets. Conclusion: SGXZ decoction could produce a promising effect for its multi-component, multi-target and multi-biological process in the treatment of NAFLD. The present study provides the novel insight into a comprehensive understanding for further study.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a progressive condition ranging from simple NAFL to nonalcoholic steatohepatitis (NASH), liver brosis, liver cirrhosis and even worse, the hepatocellular carcinoma (HCC) [1][2]. It is a metabolic disease representing the hepatic manifestation of a systemic metabolic disorder [3], and is demonstrated to be associated with obesity-related disorders and diabetes [4][5]. With the increase epidemic of obesity and metabolic-related comorbidities, global incidence of NAFLD is estimated to be 25% and keeps rising continually [6]. NAFLD has been one of the most leading courses of chronic hepatic disease, affecting approximately 1.7 billion individuals worldwide [7].
Moreover, it is one of the most common indication for liver transplantation and NAFLD-HCC is now the fastest growing demand of liver transplantation in the USA [8 -9]. Lifestyle modi cation including healthy diet and increased physical activity is recommended as the rst-line treatment in NAFLD management [10]. Nevertheless, their effectiveness is limited in NAFLD patients due to the low readiness to change lifestyle, especially in terms of increasing physical activity [11]. Another challenge for the lifestyle intervention has been the occurrence of weight regain [12][13]. Up to date, there are no FDA-approved pharma therapies for the treatment of NAFLD at present [14]. Insulin sensitizer (rosiglitazone, pioglitazone and metformin), antioxidants (vitamin E), anti-in ammatory and lipid lowering drugs (atorvastatin and simvastatin) have been in practice for the treatment of NAFLD [15][16]. However, usage of rosiglitazone could increase the risk of cardiovascular causes [17], while long-term use of vitamin E [18][19] was reported to increase the risk of prostate cancer and might increase all-cause mortality.
Moreover, the adverse outcome of pioglitazone [20][21], atorvastatin [22] and simvastatin [23] including weight gain, edema, heart failure, cytotoxicity and hepatotoxicity, should not be neglected. Therefore, it is an urgent need to develop pharmacological strategies for the treatment of NAFLD.
Based on the integration of system biology, bioinformatic and pharmacology [34][35][36], network pharmacology is currently used to explore the potential pharmacological effect and underlying mechanisms of a drug on a disease [37][38]. In particular, it concentrates on the elucidation of complex biological relationship among the drugs, targets, pathways and diseases from a systemic and holistic perspective [39]. The wholeness, relevance and systematic nature of network pharmacology are in line with the concept and treatment theory of TCM, providing a novel method and powerful tool to decipher the therapeutic mechanisms of TCM [40]. In addition, molecular docking is a computational method based on the analysis of the binding pose and a nity between small molecule and macromolecular target, which is widely utilized to predict and identify the potentially active compounds [41][42]. In our study, network pharmacology combined with molecular docking was applied to explore the pharmacological and molecular mechanisms involved in the treatment of SGXZ decoction on NAFLD.
First, chemical compounds in SGXZ decoction were collected and recognized through literature study and their corresponding targets were then obtained from Similarity ensemble approach (SEA) database.
Second, the potential targets involved in the pathology of NAFLD were predicted from DisGeNET and GeneCards, and compared with the predicted targets of SGXZ decoction to intersect and identify the common targets. Subsequently, the network of "compound-target-disease" and protein-protein interaction (PPI) network were constructed to identify the potentially active ingredients and key targets. Furthermore, functional enrichment including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was analyzed via DAVID to explore the potential mechanism of SGXZ decoction for the treatment of NAFLD. Last, active ingredients were screened against the key targets by performing molecular docking to identify the compounds treating NAFLD. The overall owchart of this study is described in

Collection of compounds and the putative targets of SGXZ decoction
The active compounds in SGXZ decoction were identi ed and collected through literature studies. Then, 2D or 3D conformations of the compounds were downloaded either from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) or through sketching in ChemDraw software. Moreover, the simpli ed molecular input line entry system (SMILS) numbers (Supplementary Table 1) of them were also acquired from PubChem or the conversion by ChemDraw. To predict the putative gene target of SGXZ decoction, the Simpli ed molecular input line entry system (SMILES) of each chemical compound was submitted into Similarity ensemble approach (SEA) database (https://sea.bkslab.org/), which relates proteins based on the set-wise chemical similarity among their ligands [43]. The predicted protein targets of SGXZ decoction were listed in Supplementary File 1.

Identi cation of potential targets of NAFLD
The "Non-alcohol Fatty Liver Disease" or the "Non-alcoholic Fatty Liver Disease" was used as the key words to predict the potential NAFLD-related gene targets by means of GeneCards (https://www.genecards.org/) and DisGeNET (https://www. disgenet.org/home/) disease database. DisGeNET is a discovery platform containing one of the largest publicly available collections of genes associated to human diseases [44]. Based on all annotated and predicted human genes, GeneCards provides comprehensive, user-friendly information via searchable, integrative database [45]. After removing the repetitive targets from the two databases, the NAFLD-related protein targets were retained for further study and the detailed information of the targets were provided in Supplementary File 2.

Outcome of PPI network
The PPI network was constructed from the common 78 targets (Fig. 4). Due to the lack of any interactions with other targets, Chymotrypsin-C (CTRC) and Alpha-ketoglutarate-dependent dioxygenase (FTO) were removed from the network. Therefore, the network contained 76 nodes and 376 edges with the average degree value of 9.64. The target proteins with higher degree value than average degree value were Interleukin-6 (IL-6), Vascular endothelial growth factor A (VEGFA), Estrogen receptor (ESR1), Hypoxia-inducible factor 1-alpha (HIF1A), Matrix metalloproteinase-9 (MMP9), Amyloid-beta precursor protein (APP), cAMP responsive element binding protein 1 (CREB1), Heat shock protein family A member 5 (HSPA5), Transcription factor p65 (RELA) and Interleukin-2 (IL-2). The 10 targets were indispensable for constructing interaction between protein and protein in the network, and were considered as the putative key target of SGXZ decoction for the treatment of NAFLD.

GO enrichment analysis
Gene ontology (GO) functionally annotated targets in PPI network into three main aspects containing molecular function (MF), cellular component (CC) and biological process (BP). In total, 185 GO entries including 45 of MF , 25 of CC and 115 of BP were acquired based on the P value (P 0.05). For BP, the targets were mainly concentrated on the oxidation-reduction process, positive regulation of angiogenesis, negative regulation of apoptotic process, regulation of insulin-like factor receptor signaling pathway and so on. For CC, the targets were mainly responsible for the extracellular space, extracellular exosome, cell surface, extracellular region, mitochondrion, extracellular matrix and so on. In regard to MF, the intersection targets were distributed in enzyme binding, insulin-like growth factor binding, electron carrier activity, oxidoreductase activity, receptor binding, protein heterodimerization activity, iron ion binding, glycosphingolipid binding and protein binding. The bubble plot and histogram of the most signi cant enriched GO terms were exhibited in Figure 5.

KEGG enrichment analysis
The 43 annotated pathways were totally obtained based on the targets in PPI network, and the top 20 of them with smallest signi cance value were shown in Figure. 6A and Table 3. The bubble plot of the pathways suggested a concentration on the insulin resistance, amino acid metabolism, cancer-related pathways, in ammation-related pathways including MAPK signaling pathway, HIF-1 signaling pathway, TNF signaling pathway and PI3K-Akt pathway. Moreover, the common targets also concentrated on the endocrine participated biological process such as estrone signaling pathway and immunological pathway of T cell receptor. To reveal the network interaction of the pathways and the involved targets, target-pathway network was constructed and analyzed which consisted of 58 nodes and 130 edges as shown in Figure 6B, indicating a complicated interaction among them. Pathways with largest degree including pathways in cancer, PI3K-Akt, MAPK signaling pathway, proteoglycan in cancer and HIF-1 pathway, suggesting their signi cant role in the treatment of SGXZ decoction on NAFLD.  Figure 1). However, the crystal structure of CREB1 could be obtained neither from the PDB database nor through the homology modeling since none of templates with good quality existed. Besides, 3 duplicated key targets between compound-target-disease network and PPI network were removed. Thus, a total of 10 key compounds were docked into 16 key targets. After docking, 160 pair of compound-target complexes were retained according to their best binding a nity, and the heat map of their docking energy was shown in Figure 7. Moreover, the binding energy of each pair of compound-target complex was provided in Supplementary Table 3. Binding energy with negative value indicated that the ligand molecule was able to combine with the receptor target proteins. The lower and more negative binding energy suggested a better binding a nity of active compounds with target proteins. Almost half of the key compound could combine with most of the targets. Among them, gallic acid (SX01), chlorogenic acid (SX07) and isochlorogenic acid A (SX13) could bind into all the key targets with the lowest binding energy. The accurate values of binding energy between these three key active compounds and key targets were shown in Table 4 Table 4. Moreover, the interaction between the best poses of three compounds and targets were displayed in Figure. 8. The results of molecular docking revealed that gallic acid, chlorogenic acid and isochlorogenic acid A might be the most potentially active ingredients of SGXZ decoction treating NAFLD. Table 4 Binding energy between key active compounds and targets (kcal/mol) 4. Discussion NAFLD is a metabolic related syndrome characterized as dysfunctional hepatic lipid metabolism and insulin resistance [52][53], which has been considered the fastest growing cause of HCC [54] and strongly associated with the increasing risks of type 2 diabetes, cardiovascular disease, chronic kidney disease and hypertension [55][56][57]. Currently, no pharmacological therapies were approved by FDA in the treatment of NAFLD and seeking an effective agent is urgently desiderated. TCM has been proved to treat In the present study, active compounds of SGXZ decoction with corresponding targets were rstly identi ed, and the overlapping targets between SGXZ decoction and NAFLD were considered to be the main targets these compounds acted on. Moreover, compounds including series of phenolic acids and avonoids were selected as the key bioactive ingredients for the treatment of SGXZ decoction on NAFLD based on their contributions in the compound-target-disease network (Figure2, Table 1). Previous study indicated that the intake of phenolic acids alleviated hepatic steatosis, reduced brosis and the insulin resistance in NAFLD patients [64]. Gallic acid, a simple polyphenol, was reported to reduce lipid accumulation that is related to β-oxidation and ketogenesis [65]. Speci cally, the hepatoprotective effect of gallic acid attributed to the repression of in ammatory signaling pathways including nuclear factor-κB and IL-6 in liver, which was associated with regulation of gut microbiota and an increase of Glucagon-like peptide-1 secretion [70][71]. Isochlorogenic acid A was suggested to possess properties of hepatoprotective and anti-hepatitis B through suppressing oxidation [72]. Moreover, it was indicated that isochlorogenic acid A exerted a protective effect on liver brosis through inhibiting in ammation via HMGB1/TLR4/NF-kB signaling pathways [73]. In addition, avonoids are natural products widely distributed in plants, exhibiting not only antidiabetic and hypoglycemic activities but also antiin ammatory and antioxidant properties, and were recognized to have protective effect in the treatment of NAFLD [74][75][76]. As a kind of avonoid, liquiritin could ameliorate cyclophosphamide-induced liver injury and in ammation by inhibiting the elevated MMP-9 expression, hepatic in ltration of neutrophils, myeloperoxidase activity, IL-6 mRNA expression and NF-κB activation [77]. Naringin is a avanone glycoside isolated from Aurantii Fructus Immaturus (Zhi-Shi) [78] and has been proved to improve lipid metabolism disorders through reducing hepatic lipid accumulation in tissue-engineered NAFLD model [79]. Neohesperidin could induce the PGC-1α expression through activating AMP-activated protein kinase (AMPK), increasing hepatic mitochondrial biogenesis and fatty acid oxidation in NAFLD mice [80]. To sum up, three phenolic acids (gallic acid, chlorogenic acid, isochlorogenic acid A) and three avonoids (liquiritin, naringin, neohesperidin) with hepatoprotective effect could serve as the potentially main active ingredients of SGXZ decoction for the treatment of NAFLD.
Together with key targets in compound-target-disease network ( Figure 3, Table 2), 10 targets with highest degree in the PPI network were as well recognized as core targets of SGXZ decoction treating NAFLD promoted liver brosis in NAFLD by activating PTEN/ NF-κB p65 signaling pathway [91]. RELA, also known as p65, is one of the ve members in NF-κB family and is a pivotal transcription factor regulating in ammatory molecules [92]. Inhibition of NF-κB signaling alleviated hepatic lipid accumulation and hepatic in ammation in NAFLD [93][94]. Moreover, abnormal lipid deposition as well as insulin resistance in NAFLD often leads to endoplasmic reticulum (ER) stress, which further triggers the unfolded protein response and thereby causes in ammation in hepatocytes [95]. GRP78 (HSPA5) is a chaperone heat shock protein playing the central role in maintaining ER proteostasis under excessive stress [96].
LGALS3 has been shown to participate in glucose intolerance and lipid metabolism disorders, which is an essential regulator of insulin resistance, brosis and in ammation cytokines including TNF-α, IL-6 and IL-1β [100][101]. FGF1 exerts a protective role in series of metabolic disorders [102]. Investigations revealed that FGF1 could reduce blood glucose and ameliorate hepatic steatosis, in ammation and brosis through modulation of oxidative stress and ER stress [103][104]. The onset of hepatic in ammation caused the brogenesis in NAFLD, which was manifested by deposited extracellular matrix (ECM) proteins including collagens, elastin and bronectin [105][106]. MMP-9 performs the vital role in modulating and degrading gelatins, collagens and other ECM compounds [107][108]. A decreased MMP-9 was associated with more advanced brosis and serum liver injury indices (AST, GGT) in NAFLD patients, while increased MMP-9 activity could precede the clearance of brotic matrix [109][110]. SLC5A1 encodes the sodium glucose cotransporter 1 (SGLT1), and inhibition of SGLT1 not only contributed to regulate hepatic glucose metabolism but also mitigate development and progression of NAFLD [111][112]. To conclude, it was assumed that SGXZ decoction might perform comprehensive regulations of anti-in ammation, anti-lipid deposition, anti-insulin resistance, anti-brosis, and anti-ER stress on NAFLD through these key targets.
After that, GO functional enrichment analysis revealed that the targets in PPI network mainly involved oxidation, positive regulation of angiogenesis, metabolic process, hypoxia, extracellular matrix and insulin-like factor binding, and other biological processes ( Figure 5). The results of GO analysis coincided with the indispensable contribution of aforementioned key targets among all targets in PPI network. In addition, KEGG enrichment analyses indicated that the action pathway mainly included insulin resistance, amino acid metabolism, cancer-related pathways, in ammation-related pathways, estrone signaling pathway and T cell receptor pathway ( Figure 6A, Table 3). As shown in Figure. 6B, the 'target-pathway' interaction network suggested that bioactive compounds in SGXZ decoction performed therapeutic role in regulation of NAFLD through multi-targets and multi-pathways. Through literature retrieval, six signaling pathways including PI3K-Akt, MAPK, insulin resistance, HIF-1, Tryptophan metabolism and Estrogen signaling pathway were recognized as core pathways involved in the treatment of SGXZ decoction on NAFLD. The six core pathways and their interaction were cited from KEGG database and displayed in Figure 9. Among the core pathways, PI3K-Akt, MAPK and HIF-1 signaling pathway were associated with in ammation that is the driving force for the development and evolution of NAFLD [113][114].
Tryptophan is a kind of amino acid that could produce indoles by bacterial enzyme trytophanase A in intestinal epithelial cell. Indoles promote intestinal barrier function and could translocate from the intestinal to the liver to modulate hepatic lipid metabolism and in ammation to protect against NAFLD [124]. Moreover, the hepatic metabolism and in ammation in the onset and progression of NAFLD showed a sex difference, which was potentially due to different estrogen signaling activity [125][126]. Estrogen de ciency increased risk for liver brosis, and postmenopausal women were more likely to develop NAFLD than men [127][128]. In general, the above six core pathways and their interaction were correlated to NAFLD and might be acted on by SGXZ decoction in the treatment of NAFLD.
In the end, molecular docking (Table 4, Figure 8) showed that most of the key compounds could be docked into the key protein targets. Moreover, bioactive compounds of isochlorogenic acid A, chlorogenic acid and gallic acid could bind best with all of the key targets, especially with HSPA5, RELA, MMP9, SLC5A1, HIF1A and FGF1. The results of molecular docking simulations provided a putative pharmacological activity for the treatment of SGXZ decoction on NAFLD. Our present study applied network pharmacology combined with molecular docking to predict the key active ingredients and pathways of SGXZ decoction in NAFLD treatment, which still need to be veri ed by future experimental validations.

Conclusions
In this study, network pharmacology approach combined with molecular docking method were performed to elucidate the underlying mechanisms, including the putative active ingredients, key targets and pivotal signaling pathways, of SGXZ decoction in the treatment of NAFLD. Among the predicted key compounds, three bioactive ingredients of SGXZ, isochlorogenic acid A, chlorogenic acid and gallic acid might exert the essential pharmacological effect. Furthermore, VEGFA, IL-6, IL-2, HSPA5, RELA, MMP9, SLC5A1, HIF1A and FGF1 were assumed to be the key targets acted on by those bioactive ingredients. In addition, signaling pathways of in ammation, oxidative stress, insulin resistance, intestinal barrier function, and lipid metabolism were mainly involved. The results provided an insight into the therapeutic strategies of NAFLD and evidence for future research. Nonetheless, more experimental approaches should be carried out to validate the ndings. Ethical approval and consent to participate Not applicable.

Consent for publication
Not applicable. Figure 1 Schematic diagram of the whole study design.      Heat map of the docking energy (kcal/mol) between key active ingredients and key targets. The redder the color, the lower the binding energy and the stronger the binding ability.

Figure 8
Interaction mode between SX01, SX07, SX13 and targets. Target proteins were displayed as solid ribbon and surface mode, and SX01, SX07 and SX13 were displayed in sticks and colored in yellow, pink and blue respectively. The residues interacting with the compounds were shown as sticks with their name labeled in red.

Figure 9
The predicted six core pathways and their interaction involved in the therapeutics mechanisms of SGXZ