Mechanism of Xiaoyao San in treating non-alcoholic fatty liver disease with liver depression and spleen deficiency: based on bioinformatics, metabolomics and in vivo experiments

Abstract Xiaoyao san (XYS) plays an important role in treatment of non-alcoholic fatty liver disease (NAFLD) with liver stagnation and spleen deficiency, but its specific mechanism is still unclear. This study aimed to investigate the material basis and mechanism by means of network pharmacology, metabolomics, systems biology and molecular docking methods. On this basis, NAFLD rat model with liver stagnation and spleen deficiency was constructed and XYS was used to intervene, and liver histopathology, biochemical detection, enzyme-linked immunosorbent assay, quantitative PCR assay and western blotting were used to further verify the mechanism. Through the above research methods, network pharmacology study showed that there were 94 targets in total for XYS in the treatment of NAFLD. Metabolomics study showed that NAFLD with liver depression and spleen deficiency had a total of 73 differential metabolites. Systems biology found that PTGS2 and PPARG were the core targets; Quercetin, kaempferol, naringenin, beta-sitosterol and stigmasterol were the core active components; AA, cAMP were the core metabolites. And molecular docking showed that the core active components can act well on the key targets. Animal experiments showed that XYS could improve liver histopathology, increase 5HT and NA, decrease INS and FBG, improve blood lipids and liver function, decrease AA, increase cAMP, down-regulate PTGS2, up-regulate PPARG, and decrease PGE2 and 15d-PGJ2. In conclusion, XYS might treat NAFLD with liver depression and spleen deficiency by down-regulating PTGS2, up-regulating PPARG, reducing AA content, increasing cAMP, improving insulin resistance, affecting glucose and lipid metabolism, inhibiting oxidative stress and inflammatory response. Communicated by Ramaswamy H. Sarma HIGHLIGHT Network pharmacological results found that XYS might treat NAFLD through multiple targets and multiple pathways. Quercetin and kaempferol were main components of XYS in treatment of NAFLD. System biology research results found that PPARG and PTGS2 were the core targets of XYS in the treatment of NAFLD. Metabolomic results suggest that AA and cAMP were differential metabolites of NAFLD. In vivo animal experiments showed that XYS might treat NAFLD by increasing PPARG and decreasing PTGS2, reducing AA and increasing cAMP to regulate glucose metabolism and lipid metabolism.


Introduction
The syndrome of liver stagnation and spleen deficiency is a common syndrome pattern in clinical practice (Huang et al., 2018).It is usually caused by depression or excessive psychological pressure leading to liver dysfunction in ensuring the smooth flow of Qi and spleen dysfunction in controlling digestion and transportation (Li et al., 2020).Due to people's unhealthy eating habits and long-term emotional stress from life and work in modern society, the symptoms of the liver stagnation and spleen deficiency are very common.In clinical practice, the syndrome of liver stagnation and spleen deficiency has become a high incidence and common syndrome (Zheng et al., 2021).Non-alcoholic fatty liver disease (NAFLD) is a common modern clinical disease with liver stagnation and spleen deficiency syndrome.Asia-Pacific Working Party on Non-alcoholic Fatty Liver Disease Guidelines define this disease as a condition in which there is a large accumulation of lipids in the liver in the absence of evidence of heavy alcohol consumption, viral infection, or other specific causes (Wong et al., 2018).One quarter of the global population is estimated to have NAFLD (Sveinbjornsson et al., 2022).It ranges from simple steatosis to inflammation, liver fibrosis, and even hepatocellular carcinoma (Huby & Gautier, 2022).The pathophysiological mechanism of NAFLD is complex, including lifestyle, nutritional factors, lipogenesis, cell death, insulin resistance, chronic low-grade inflammation and intestinal microbiome changes (Zhao et al., 2023).
However, the world health organization (WHO) has not paid enough attention to NAFLD.In January 2022, the Global multidisciplinary expert Group issued the Global Public Health Consensus Statement on Non-alcoholic Fatty liver Disease, suggesting that WHO and its member states should treat NAFLD as a public health hazard and a chronic noncommunicable epidemic, and formulate corresponding public health countermeasures at the international, national or regional levels (Lazarus et al., 2022).Common therapeutic drugs for NAFLD include regulation of glycolipid metabolism to protect the liver and anti-inflammation.In recent years, therapies for NAFLD based on enterohepatic axis and targeting intestinal flora have gradually emerged, and a variety of new metabolic regulatory drugs are also under clinical development.But the development of related drugs is slow, which has been regarded as a major problem (Rong et al., 2022).So far, no specific drug for NAFLD has been approved by the Food and Drug Administration (FDA).
Traditional Chinese medicine has shown its unique advantages in the treatment of the above-mentioned disease.Xiaoyao San (XYS) is derived from 'TaiPing Huimin Heji Ju Fan', which is a classic prescription for soothing liver and invigorating spleen and treating liver stagnation and spleen deficiency (Meng et al., 2013).Guidelines for diagnosis and treatment of NAFLD with traditional Chinese medicine (grass roots doctors version) state that XYS is an effective formula for patients of NAFLD with liver stagnation and spleen deficiency.Previous clinical studies (Liu et al., 2022) also showed that XYS has excellent clinical efficacy in treatment of NAFLD, can improve the symptoms of liver depression and spleen deficiency of patients, and is worthy of clinical application and promotion.However, the material basis and mechanism of XYS in the treatment of NAFLD have not yet been clarified.
The formula of XYS is complex, including Radix Bupleuri (Bupleurum chinense DC.), Paeoniae Radix Alba (Paeonia lactiflora Pall.), Angelicae Sinensis Radix (Angelica sinensis (Oliv.)Diels), Atractylodes Macrocephala Koidz (Atractylodes lancea (Thunb.)DC.), Poria Cocos (Schw.)Wolf.(Poria cocos (Schw.)Wolf.), Menthae Herba (Mentha haplocalyx Briq.), licorice (Glycyrrhiza uralensis Fisch.),Zingiber Officinale Roscoe (Zingiber officinale Roscoe), the plant name has been checked with http://www.theplantlist.org.It is difficult for traditional pharmacology to explain its mechanism in the treatment of NAFLD with liver depression and spleen deficiency.The emerging network pharmacology at the current stage is in line with the connotation of TCM holism, multi-component and multi-target.Through the discovery of biologically active components and biomarkers, it provides important ideas for revealing the mechanism of action of complex TCM formulas (Wang et al., 2020(Wang et al., , 2021;;You et al., 2020).Metabolomics can systematically monitor the dynamic changes of the body, help us understand the changes of small molecule metabolites in the body under different disease states, and reflect the microscopic performance of the body under macroscopic conditions.It provides new ideas for Chinese medicine research and accelerates the process of Chinese medicine research (Izzi-Engbeaya et al., 2018;Mayneris-Perxachs & Swann, 2019).
Through network pharmacology, metabolomics and systems biology techniques, this study intends to investigate the possible mechanism of XYS in the treatment of NAFLD with liver stagnation and spleen deficiency, and to validate the therapeutic effect and specific mechanism of XYS on NAFLD with liver stagnation and spleen deficiency through animal experiments.

Methods of network pharmacology research
The TCMSP database (Ru et al., 2014) (http://tcmspw.com/tcmsp.php)was utilized in this study to find and collect the ingredients and corresponding targets of XYS.And the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used as previously described (Yuan et al., 2020).To obtain NAFLD disease targets, enter the keyword 'nonalcoholic fatty liver disease' into the GeneCards database (Stelzer et al., 2016) (https://www.genecards.org/) and the OMIM database (Amberger et al., 2019) (https://www.omim.org/).The target genes predicted by XYS active ingredient and the target genes predicted by NAFLD disease were intersected, yielding the target genes of XYS in the treatment of NAFLD.For protein-protein interaction (PPI) analysis, enter the intersection genes into the STRING database (Szklarczyk et al., 2023) (https://cn.stringdb.org/).Then screen core genes with the R3.5.0 software and draw the compounds-targets-disease network diagram with Cytoscape 3.6.1 software (Shannon et al., 2003).Finally, utilize the DAVID database (Dennis et al., 2003) (https://david.ncifcrf.gov/) to conduct enrichment analysis of Gene Ontology (GO) (Milano, 2022) (http://geneontology.org/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa & Goto, 2000) (https://www.kegg.jp/kegg/).The cellular component (CC), biological process (BP) and molecular function (MF) enrichment analyses were displayed as enrichment circles in the GO enrichment analysis, while the KEGG pathway enrichment analysis was displayed as a sankey dot.The detailed operating method of network pharmacology was described previously (Ruan et al., 2020).

Clinical sample collection
The clinical cases were outpatients and inpatients from the department of liver diseases and endocrinology of the Hubei No. 3 People's Hospital of Jianghan University (Ethics Approval Number: (2019) ethics review department) No. ( 25)) from August 2019 to August 2020, included 30 patients diagnosed with NAFLD of liver depression and spleen deficiency and 30 healthy controls.The basic information for NAFLD group and healthy control group were showed in Table S1.All subjects signed informed consent.And the study followed the guidelines of the Declaration of Helsinki for humans.All NAFLD of liver depression and spleen deficiency and normal control group did not receive any drug treatment 1 month before blood collection.After fasting for at least 8 h, venous blood was collected in the morning and stored in a À 80 � C refrigerator (Forma 900 series; Thermo Fisher Scientific) for metabolomics detection.

Metabolites extraction and GC-TOF-MS analysis
Transfer 100 lL sample to EP tube and add 400 lL precooled extract (methanol), with 10 lL internal standard (adonitol, 0.5 mg/mL stock) were added, vortex mixing for 30 s. Ultrasound for 10 min (ice bath).Centrifuge the samples at 4 � C and 12,000 rpm for 15 min.Carefully transfer the 180 lL supernatant into a 1.5 mL EP tube.Take 35 lL of each sample and mix them into QC samples.Dry extract in vacuum concentrator.After evaporation in a vacuum concentrator, 30 lL of Methoxyamination hydrochloride (20 mg/mL in pyridine) was added and then incubated at 80 � C for 30 min, then derivatized by 40 lL of BSTFA regent (1% TMCS, v/v) at 70 � C for 1.5 h.Gradually cooling samples to room temperature, 5 lL of FAMEs (in chloroform) was added to QC sample.All samples were then analyzed by gas chromatograph (7890 A, Agilent, USA) coupled with a time-of-flight mass spectrometer (Pegasus HT, Leco, USA) (GC-TOF-MS).
GC-TOF-MS analysis was performed as previously described (Yang et al., 2019).The mass spectrometry data were acquired in full-scan mode with the m/z range of 50-500 at a rate of 12.5 spectra per second after a solvent delay of 4.9 min.

Data preprocessing, annotation and analysis
Raw data analysis, including peak extraction, baseline adjustment, deconvolution, alignment and integration, was finished with Chroma TOF (V 4.3x, LECO) software, and LECO-Fiehn Rtx5 database was used for metabolite identification by matching the mass spectrum and retention index (Kind et al., 2009).Finally, the peaks detected in less than half of QC samples or RSD > 30% in QC samples was removed (Dunn et al., 2011).
The final dataset containing the information of peak number, sample name and normalized peak area was imported to SIMCA16.0.2 software package (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate analysis, including PCA (principle component analysis, PCA) and OPLS-DA (orthogonal partial least squares discriminant analysis, OPLS-DA).The 95% confidence interval in the PCA and OPLS-DA score plot was used as the threshold to identify potential outliers in the dataset.The metabolites with VIP > 1 and p ＜0.05 (student t test) were considered as significantly changed metabolites.In addition, commercial databases including KEGG and MetaboAnalyst (Chong & Xia, 2020) (http://www.metaboanalyst.ca/)were used for pathway enrichment analysis.

Systems biology analysis and molecular docking validation
Import the core targets screened of network pharmacology and differential metabolites screened by metabolomics into IMPaLA: Integrated Molecular Pathway Level Analysis (Kamburov et al., 2011) (http://impala.molgen.mpg.de/) for integrated analysis, and use Cytoscape software to construct the core compounds-core targets-metabolites network.Then further application of molecular docking technology to preliminary verification of the screened core compounds and core targets.The detailed operating method of molecular docking was described previously (Ruan et al., 2020).

Preparation of drugs
The components of XYS formulation: Radix Bupleuri, Paeoniae Radix Alba, Angelicae Sinensis Radix, Poria Cocos (Schw.)Wolf., Atractylodes Macrocephala Koidz, Menthae Herba, Zingiber Officinale Roscoe, Licorice.The ratio of them was 3:3:3:3:3:1.5:1:1.The above-mentioned herbal medicine was purchased from Anhui Haozhou Yonggang Slice Factory Co., Ltd.It was authenticated by director Juhua Yang of the pharmacy of traditional Chinese medicine of the Hubei No. 3 People's Hospital of Jianghan University as authentic and in compliance with the provisions of the Chinese pharmacopoeia.Soak all herbs (except Menthae Herba) for 20 min, add water to inundate the herbs, burn them in strong fire for 5 min, and then fry them in soft fire for 30 min.Menthae Herba was decocted together for the last 10 min, and finally prepared into a decoction containing crude drug 4.995 g/mL.High-fat feed was purchased from Jiangsu Synergy Medicine Bioengineering Co., Ltd.Fenofibrate capsules (manufacturer: RECIPHARM FONTAINE, France; registration number: H20181239) were dissolved in pure water and prepared into a solution with a concentration of 4 mg/mL.

Animal grouping, model building and group dosing
40 SPF Wistar rats (aged 6-7 wk; body weight 200-220 g), half male and half female (purchased from Hubei Provincial Laboratory Animal Control Center, license number SCXK (E) 2020-0018), approved by the ethics committee (No.: Safety Assessment Center Action (Fu) No. 202120106).The rats were raised in an environment with a humidity of more than 60% and a temperature of 20-25 � C, and the bedding was replaced and disinfected twice a week.After 1 week of adaptive feeding, the rats were randomly divided into a normal control group (8 rats) and a NAFLD model group with liver depression and spleen deficiency (32 rats).A high-fat and high-cholesterol diet (10% lard þ 2% cholesterol þ 88% normal diet) was used to build the NAFLD rat model.Syndrome modeling was performed at the same time as the disease modeling, and the liver stagnation and spleen deficiency syndrome model was constructed according to the modified compound etiology modeling method.The specific operation was to restrain the rats for 3 h every day, then swim in a water bath at (22 ± 1) � C for 10 min, and pinch the tail for 30 min every other day.The above disease models and syndrome models were operated continuously for 10 wk.At the end of the 10th week of modeling, liver color Doppler ultrasound was used to preliminarily verify the success of the model.Two animals in each group were randomly sacrificed to verify the effect of the model.At the 11th week of modeling, the liver depression and spleen deficiency NAFLD model group with liver depression and spleen deficiency was randomly divided into liver depression and spleen deficiency NAFLD low, medium, and high dose groups; western medicine intervention group; and liver depression and spleen deficiency NAFLD model group (6 animals in each group).From the 12th week, the drug intervention group was given 2 mL of gavage per day.The doses of XYS low, medium and high dose groups were 8.33 g/kg/d, 16.65 g/kg/d, and 33.30 g/kg/d, respectively, and the western medicine intervention group was given fenofibrate capsules at 18 mg/kg/d.The model control group was given the same amount of double-distilled water once a day at 9:00 am, and each group was given continuous gavage for 4 wk.The normal control group received no treatment and was fed normally.

Histopathological examination
The liver tissue was made into paraffin-embedded sections and frozen sections, stained with HE and Oil Red O, respectively.Images were taken under an Olympus microscope (BX53, Tokyo, Japan) at 200 � magnification.

Quantitative PCR assay
Total RNA was isolated from liver tissue using Trizol reagent (Aidlab, Lot: 252250AX) according to the manufacturer's instructions.The purity and concentration of RNA were determined with a UV spectrophotometer (JY02S, Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.).PCR was performed using SYBR Green Master Mix (VAZYME, Q111-02) and primers specific for the target gene (Table S2).Gene expression was assessed by the DDCT method, using GAPDH as the housekeeping gene.

Western blotting
Western blotting (WB) was used to detect the expression of PTGS2 (AF7003, Affinity) and PPARG (Ab215191, Wuhan Sanying Biotechnology Co., Ltd.) protein in rat liver tissue.The samples were homogenized using RIPA lysis buffer (Biyuntian, P0013B).The homogenates were subjected to centrifugation (12,000 rpm) at 4 � C for 5 min to obtain proteins.BCA protein concentration assay kit (Biyuntian, P0010) was used to detect protein concentration.Afterward, the protein was isolated using 10% SDS-PAGE, and it was then transferred to a polyvinylidenefluoride (PVDF) membrane (0.45 lm, Millipore, IPVH00010).After reacting with the primary antibody overnight at 4 � C, the membrane was immersed in a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature.Subsequently, the greyscale images were obtained and analyzed with Image J software, and GAPDH (AB-PR 001, Hangzhou Xianzhi Biotechnology Co., Ltd.) was used as an internal reference.

Statistical analysis
The results were expressed as the mean ± SEM.Statistical analysis was performed using one-way analysis of variance (ANOVA).Histograms were generated by GraphPad Prism (San Diego, USA), p < 0.05 was considered to be statistically significant.

Results of network pharmacology
Through TCMSP database search and ADME standard screening, a total of 161 active compounds of XYS and corresponding to 222 targets were obtained.Among them, the number of active compounds corresponding to Radix Bupleuri, Angelicae Sinensis Radix, Paeoniae Radix Alba, Poria Cocos (Schw.)Wolf., Atractylodes Macrocephala Koidz., Menthae Herba, Zingiber Officinale Roscoe and licorice was 17, 2, 13, 15, 7, 10, 5, 92; the number of corresponding targets were 166,40,71,17,15,94,41,199 (Table S3). Figure 1A showed the regulatory network of the active components and targets of traditional Chinese medicine in XYS.From Figure 1A, it can be seen that quercetin, kaempferol, naringenin, beta-sitosterol, stigmasterol, luteolin, isorhamnetin, formononetin and other active ingredients have the largest number of targets, which were the main active ingredients of XYS.Some of the active ingredients and their abbreviations were shown in Table S4.After searching the GeneCards database and the OMIM database, 944 and 398 NAFLD targets were obtained, respectively (Figure 1B), and 1315 NAFLD disease targets were finally obtained by combining deduplication.
Use Cytoscape software to draw a compound-targetdisease network diagram (Figure 1C), sort by degree.layout in the network diagram, and screen the top 10 compounds and the top 8 core targets (Table 1).By taking the intersection of the corresponding targets of the above active ingredients of XYS and NAFLD disease targets, the targets of XYS for the treatment of NAFLD were obtained (Figure 1D).Combining Figure 1A and C, it can be seen that quercetin, kaempferol, naringenin, beta-sitosterol, stigmasterol, luteolin, isorhamnetin and formononetin were not only the main active components of XYS, but also the main active components of XYS in the treatment of NAFLD.
The targets of XYS in the treatment of NAFLD were imported into the STRING database for PPI network (Figure 2A).The top 15 core genes were screened according to the degree.layout of the PPI network (Figure 2B).Furthermore, GO and KEGG enrichment analysis were performed on the targets of XYS in the treatment of NAFLD, and the number of GO and KEGG entries in the enrichment analysis was 2082, 151, and the number of BP, CC, and MF in the GO entries was 1914, 52 and 116, respectively.The top 10 BP, CC and MF entries (Table S5) and the top 20 KEGG entries for the screening ranking were presented in the form of circle plots (Figure 2C) and sankey dot plots (Figure 2D), respectively.

Differential metabolite screening for NAFLD
Metabolomics data is usually multivariate and complex data.
The application of PCA analysis can reduce the dimensionality of the data.OPLS-DA can overcome the shortcomings of PCA unsupervised analysis.The above PCA and OPLS-DA analyses can obtain a score scatter plot of PCA and OPLS-DA models for NAFLD group vs. control group (Figure 3A, B).As can be seen from Figure 3A, B, the NAFLD group and control group were significantly different.In order to prevent overfitting of the OPLS-DA model, a permutation test was further performed on the OPLS-DA model for NAFLD group vs. control group (Figure 3C).As shown in Figure 3C, there was no overfitting phenomenon in OPLS-DA model.PCA and OPLS-DA analysis focus on the relationship between metabolites.
In order to explore the independent changes in metabolite levels, the card value standard used in this study is that the P-value of Student's t-test (P-value) is less than 0.05.At the same time, the variable importance in the projection (VIP) of the first principal component of the OPLS-DA model is greater than 1, and finally, 73 differential metabolites of NAFLD group vs. control group were obtained (Table 2), which were displayed in a volcano diagram (Figure 3D).

Functional analysis of differential metabolites
Hierarchical clustering analysis helps us to group metabolites with the same characteristics into one group, and to discover the changing trends of metabolites in the disease group and the healthy control group.We calculated the Euclidean distance matrix for the quantitative values of the differential metabolites, clustered the differential metabolites with a complete linkage method, and displayed them in a heatmap (Figure 4A).In order to measure the closeness of the correlation between different differential metabolites, we  calculated the correlation coefficient for the quantitative value of the differential metabolite.The value of the correlation coefficient r is between À 1 and 1.The closer the absolute value of r is to 1, the stronger the correlation, the closer the absolute value of r is to 0, the weaker the correlation between the two variables.The calculation method adopts the Pearson method and is displayed in the form of a heat map (Figure 4B).Substance classification and source attribution were performed on the differential metabolites, and the spearman method was used to calculate the differential metabolites, which were finally visualized in a chord diagram (Figure 4C).It can be seen from Figure 4C that the 73 differential metabolites in the NAFLD group relative to the normal control group were mainly concentrated in alkaloids and derivatives, lipids and lipid-like molecules, nucleosides, nucleotides, and alogues, organic acids and derivatives, organoheterocyclic compounds, organonitrogen compounds, organooxygen compounds and so on.

Metabolite pathway analysis of differential metabolites
Through the enrichment analysis and topology analysis of the pathways where the differential metabolites are located, we found that the differential metabolites are mainly concentrated in lipid metabolism, carbohydrate metabolism and amino acid metabolism (Table S6).And the key pathways with the highest correlation with the metabolite differences were found and displayed in a bubble plot (Figure 5A).To further understand how differential metabolites affect NAFLD through key pathways, we displayed the top 5 metabolic pathways and their key metabolites (Figure 5B).

Results of systems biology and molecular docking verification
In order to further explore the mechanism of XYS in the treatment of NAFLD, what specific targets are used by the core active components in XYS, and which metabolites are affected to play a role in the treatment of NAFLD?We integrated the previous network pharmacology and metabolomics analysis, and used Cytoscape software to construct a targets-pathwaysmetabolites regulation network (Figure 6A).Combining the drugs-targets-disease network (Figure 1C), the target PPI diagram (Figure 2A) and the targets-pathways-metabolites network diagram (Figure 6A), it was found that PTGS2 and PPARG were the key targets of XYS in the treatment of NAFLD, and quercetin, kaempferol, naringenin, beta-sitosterol and stigmasterol The abscissa where the bubble is located and the size of the bubble represent the size of the impact factor of the pathway in the topology analysis.The larger the size, the greater the impact factor; the ordinate where the bubble is located and the color of the bubble represent the enrichment analysis.The P value (take the negative natural logarithm, i.e.À lnP-value), the darker the color, the smaller the P value, and the more significant the degree of enrichment.(B) The top 5 metabolic pathways and their key metabolites.
were the core active components in XYS that play a role in the treatment of NAFLD, and the core metabolites affected are mainly AA, cAMP, etc.But can the core active ingredients in XYS act well on the core target?With this question in mind, we conducted molecular docking between the core target and the core compound, and used the binding free energy to evaluate the binding of receptors and ligands (Table 3).The lower the binding free energy, the better the binding between the receptor and the ligand.Generally, a binding energy of less than À 5 kcal/mol is considered to be significant.The results showed that PTGS2, PPARG protein crystal residues and small molecule active components quercetin, kaempferol, naringenin, luteolin, beta-sitosterol, isorhamnetin, formononetin, stigmasterol docking results were good, and the docking scores of each receptor protein and small molecule active components were all less than À 5 kcal/mol.It can be seen from Table 3 that the best binding compounds to PTGS2 (6n4e) are isorhamnetin, kaempferol and luteolin, and the best binding compounds to PPARG (6c5t) are quercetin and  B-E) were the two-dimensional and three-dimensional structural diagrams of the molecular docking of the targets PTGS2 (6n4e) and isorhamnetin, luteolin, PPARG (6c5t) and quercetin, stigmasterol, respectively.Table 3. Docking energy scores for the docking of PTGS2, PPARG and core active ingredient molecules (kcal/mol).

PTGS2(6n4e)
PPARG(6c5t) beta_sitosterol À 7.2 À 7.9 formononetin À 6.9 À 6.9 isorhamnetin À 7.4 stigmasterol.The protein crystal 6n4e corresponding to PTGS2 was used as the receptor, and the core active components isorhamnetin and luteolin were used as ligands; the protein crystal 6c5t corresponding to PPARG was used as the receptor, and the core active components quercetin and stigmasterol were used as ligands for docking.The docking results were displayed using the Discovery Studio 3.5 software to draw the two-dimensional and three-dimensional structural diagrams of the ligand-receptor interaction (Figure 6B-E).

In vivo experiments to verify the efficacy of XYS
The rats in the control group had neat and shiny hair, bright eyes, flexible movements and granular stools.The early stage of the NAFLD-MOL group was an acute stress period, which was manifested as erect hair, eyes wide open, screaming and struggling, irritability, and stool formation, which was in line with the 'liver depression syndrome' stage.The mid-term was the transition period from acute stress to chronic stress, which was characterized by emotional instability, rough hair, less screaming and struggling when restrained, and occasional unformed stools.The latter stage was the chronic stress period.The hair of the rats was dry, yellow, messy and lacklustre, the expression was sluggish, the behaviors such as struggling and screaming were obviously reduced, the fatigue was lessened, the diet was reduced, the rats liked to lie together and lie down, and the stool was loose.At this stage, the liver stagnation was prolonged and the spleen earth was restrained, which was in line with the stage of 'liver stagnation and spleen deficiency syndrome'.After XYS intervention, the symptoms of liver stagnation and spleen deficiency syndrome of the rats in each group were alleviated compared with the NAFLD-MOL group, and the highdose group was the most obvious.HE and Oil red O staining of the liver tissue of the rats in the control group showed the liver cells were clearly structured, neatly arranged, uniform in size, and had no obvious steatosis.In the NAFLD-MOL group, the hepatic lobule structure was disordered, the lobular structure partially disappeared, and fat vacuoles of different sizes appeared in most of the liver cytoplasm, the boundaries between cells were blurred, and there was extensive hepatocyte steatosis, ballooning degeneration and hepatocyte necrosis.After the intervention, the NAFLD þ XYS-L, NAFLD þ XYS-M, NAFLD þ XYS-H groups and Fenofibrate groups had different degrees of improvement in hepatocyte steatosis, ballooning degeneration and hepatocyte necrosis, and NAFLD þ XYS-H group improved significantly (Figure 7A, B).
Compared with the control group, 5-HT and NA in the hippocampus and HDL-C of the NAFLD-MOL group were significantly decreased, while the FBG, INS, TC, TG, LDL-C, ALT, AST and GGT were increased.After intervention, compared with NAFLD-MOL group, 5-HT, NA and HDL-C were increased, while the FBG, INS, TC, TG, LDL-C, ALT, AST and GGT were decreased in NAFLD þ XYS-L, NAFLD þ XYS-M, NAFLD þ XYS-H and NAFLD þ Fenofibrate groups in some degree, and NAFLD þ XYS-H group improved significantly (Figure 7C-F).

Validation of core metabolites and core targets in vivo
Compared with the control group, the levels of AA, PGE2 and 15d-PGJ2, the mRNA and the protein of PTGS2 in the NAFLD-MOL group were increased, while the cAMP, the mRNA and the protein of PPARG were decreased.Compared with the NAFLD-MOL group, the levels of AA, PGE2 and 15d-PGJ2, the mRNA and the protein of PTGS2 decreased, while the cAMP, the mRNA and the protein of PPARG increased in the NAFLD þ XYS-L, NAFLD þ XYS-M, NAFLD þ XYS-H and Fenofibrate groups to some degree, and the NAFLD þ XYS-H group improved significantly (Figure 8A-D).

Discussion
Network pharmacology is one of the most popular research methods in the field of traditional Chinese medicine (Dogan et al., 2023).It not only integrates biological information and systems medicine, but also conforms to the connotation of the holistic theory of traditional Chinese medicine, multicomponent and multi-target.It can analyze the multi-component action mechanism of traditional Chinese medicine compounds as a whole (Liu et al., 2020).Network pharmacology, metabolomics and molecular docking technology play an important role in explaining the specific mechanism of XYS in the treatment of NAFLD with liver stagnation and spleen deficiency.Network pharmacology showed that XYS acted on multiple targets through multiple active components, regulated multiple biological processes, affected multiple signaling pathways in the treatment of NAFLD.
In recent years there has been increased use of Metabolomics to examine the underlying aetiology of disease states and develop biomarkers for diagnosis or prognosis (Wilkinson et al., 2020).Metabolomics research showed that NAFLD with liver depression and spleen deficiency had 73 differential metabolites, and these differential metabolites were closely related to glucose and lipid metabolism, indicating that its pathogenesis was related to abnormal glucose and lipid metabolism at the metabolic level.Systems biology showed that the main active components in XYS, such as quercetin, kaempferol, naringenin, luteolin, beta-sitosterol, isorhamnetin, formononetin and stigmasterol, played a key role in the treatment of NAFLD by acting on the targets PTGS2 and PPARG, and regulating the metabolites AA and cAMP.Molecular docking results further indicated that the active ingredients of XYS could well act on disease targets.Animal experimental study showed that XYS had a significant effect on NAFLD with liver stagnation and spleen deficiency, it can improve liver function and inhibit insulin resistance, lower blood sugar, and improve lipid metabolism.At the same time, it can down-regulate PTGS2, up-regulate PPARG, decrease AA content, increase cAMP, reduce inflammatory indicators (PGE2, 15d-PGJ2).The above results all showed that the NAFLD þ XYS-H group had the best effect.
The pathogenesis of NAFLD is currently recognized as the 'two-hit theory'.The 'initial hit' is associated with lipid accumulation in the liver, which induces lipotoxicity and can promote hepatic steatosis (Rao et al., 2019), an excessive accumulation of lipid droplets in hepatocytes, which is caused by dysfunctional lipid metabolism and insulin resistance (IR) (Liu et al., 2017).The study of Shi et al. (2020) showed that the pathogenesis of NAFLD was usually related to abnormal liver lipid metabolism, gluconeogenesis, insulin resistance, oxidative stress and inflammation.The role of PPARG in NAFLD is mainly reflected in two aspects.On the one hand, PPARG attenuates IR and lipid accumulation.Gallagher and LeRoith (2015) studies have shown that PPARG activation leaded to adipocyte differentiation and decreased production of prostaglandins, TNF-a, IL-6, leptin and resistin, thereby increasing INS sensitivity.Studies have shown that PPARG agonists could reduce liver inflammation and fibrosis, regulate glucose and lipid metabolism in a mouse model of NAFLD, and have shown favorable results in early clinical trials (Hundertmark et al., 2018).On the other hand, PPARG activation can induce the expression of glucose and lipid metabolism-related genes.Wang et al. (2020) showed that in NAFLD liver, increased PPARG expression in adipocytes activates lipid-producing genes, which was beneficial to reducing liver fat accumulation and improving inflammation.
PTGS2, also known as COX-2, is a key enzyme in prostaglandin biosynthesis and is responsible for participating in inflammation and prostaglandin biosynthesis (Balde� on Rojas et al., 2016; Wang et al., 2015), which can convert AA to prostaglandins (Zhu et al., 2017).PTGS2 is involved in lipid metabolism and may be involved in regulating the expression of lipid metabolism-related gene PPARG.PTGS2 mediates 15d-PGJ2, a metabolite of AA, which induces hepatic IR and NAFLD by activating PPARG.PTGS2 may play a role in preventing diet-related obesity NAFLD and insulin resistance development (Tsujimoto et al., 2016).The study of Hsieh et al. (2009) found that in adipose inflammation, PTGS2 activation is important for the development of insulin resistance and fatty liver in high-fat-induced obese rats.
As a x6 long-chain polyunsaturated fatty acid, AA is a metabolite of phospholipase A2.Under external stimulation, due to the action of phospholipase, AA will be detached from the cell membrane and converted into prostaglandins.
Free AA is involved in various life activities in the body, including cell proliferation, differentiation, autophagy, inflammatory response, immune regulation, angiogenesis, recognition of knowing function and so on.AA is involved in the pathogenesis of NAFLD and may be associated with pathological processes such as oxidative stress, inflammatory response, insulin resistance and lipid metabolism disorders.Oxidative stress can promote mitochondrial damage and inflammatory responses that further exacerbate NAFLD.Changes in AA content may be an early indicator of irreversible changes in inflammation and NAFLD progression (Deng et al., 2020;Sztolsztener et al., 2020).PTGS2 is a key enzyme in the metabolism of AA into efficient autocrine and  Lenzen, 2010;Qiu et al., 2017).The homeostasis of glucose and lipid metabolism is the basis of life activities, and the liver is the core organ to maintain its homeostasis.AA plays an important role in regulating lipid metabolism in the body (Zhang et al., 2021), and the main pathways of AA metabolism are closely related to the homeostasis of hepatic glucose and lipid metabolism.cAMP has become a key molecule in signal transduction in all life forms.cAMP is involved in the occurrence and development of NAFLD, and the cAMP signaling pathway is a good pharmacological target for the treatment of liver diseases through its anti-inflammatory, anti-lipogenic and anti-fibrogenic effects.Extensive evidence to date supports a role for cAMP as an active mediator in NAFLD in reducing steatosis and suppressing inflammatory responses.It is known that PKA is involved in hepatic lipid metabolism, and cAMP can promote PKA activation to inhibit glycogen synthesis and fat degradation, and can participate in the transcription of glucose and lipid metabolism genes to regulate glucose and lipid metabolism (Bloyd et al., 2021).In addition, cAMP can also affect autophagy and participate in the pathogenesis of NAFLD.Zhang et al. (2015) showed that resveratrol induced autophagy through the cAMP-PRKA-AMPK-SIRT1 signaling pathway and partially improved hepatic steatosis.Elevating cAMP levels through various pathways can improve lipid metabolism, inflammatory response, oxidative stress and fibrosis in NAFLD.
In conclusion, the results of this study showed that XYS could increase the content of NE and 5-HT in the hippocampus of NAFLD with liver stagnation and spleen deficiency, improve the behavioral manifestations of liver stagnation and spleen deficiency syndrome, relieve liver histopathological damage, improve liver function, and improve glucose and lipid metabolism, indicating that XYS has a good effect in the treatment of NAFLD.XYS can improve insulin resistance, regulate glucose and lipid metabolism, and achieve the purpose of treating NAFLD by regulating the targets PPARG and PTGS2, and the metabolites AA and cAMP (Figure 9).

Conclusion
In this study, network pharmacology, metabolomics, systems biology, the pathogenesis of NAFLD with liver stagnation and spleen deficiency and the intervention mechanism of XYS were preliminarily discussed, and preliminarily verified by in vivo animal experiments.It was found that the main active components in XYS, quercetin, kaempferol, naringenin, luteolin, beta-sitosterol, isorhamnetin, formononetin and stigmasterol may act on PTGS2, PPARG, regulate AA, cAMP, improve insulin resistance, regulate glucose and lipid metabolism, and achieve the purpose of treating NAFLD with liver stagnation and spleen deficiency.Through this study, on the one hand, it provides an important reference for the clinical diagnosis and treatment of NAFLD with liver stagnation and spleen deficiency; on the other hand, it also provides a good research idea for the study of metabolic diseases such as NAFLD and TCM syndrome types such as liver stagnation and spleen deficiency syndrome.However, this study only preliminarily discussed the possible mechanism of XYS in the treatment of NAFLD with liver stagnation and spleen deficiency from the perspective of metabonomics and molecular biology.In the later stage, proteomics, genomics and other technologies should be further used to explore the possible related mechanisms from multiple dimensions, and largescale clinical trials should be conducted to confirm the safety and effectiveness of XYS in the treatment of metabolic diseases such as liver depression and spleen deficiency NAFLD, so as to further expand the clinical application of XYS.

Figure 1 .
Figure 1.Active components of XYS and their corresponding targets and disease targets.(A) The yellow nodes represent the eight traditional Chinese medicines in XYS, the green nodes represent the active compounds, and the red nodes represent the targets corresponding to the compounds.(B) The nodes in the graph represent NAFLD targets in genecards and OMIM, respectively.(C) The middle green nodes in the figure represent targets, and the peripheral red nodes represent active compounds.(D) The red part represents the number of targets corresponding to NAFLD, the green part represents the targets corresponding to the core active compounds in XYS, and the middle intersection part is the target of XYS for the treatment of NAFLD.

Figure 2 .
Figure 2. Core targets and enrichment analysis.(A) The nodes represent proteins, and the connections represent interactions between proteins.The more connections there are, the greater the degree of connection.(B) The top 15 gene names and the gene degree.layoutassociated with them.(C) The outer, middle and inner circle represent the name, the number of all genes, and the number of core genes enriched in the GO entry, respectively.Different colors represent different GO types, yellow represents BP, purple represents CC, and blue represents MF. (D) The left side is a sankey diagram, which represents the genes contained in each pathway, and the right side is a conventional bubble diagram.The size of the bubble represents the number of genes to which the pathway belongs, and the color of the bubble represents the P value.

Figure 3 .
Figure 3. Multivariate statistical analysis.(A, B) The abscissa t[1]P represents the predicted principal component score of the first principal component, showing the difference between sample groups; the ordinate t[1]O represents the orthogonal principal component score, showing the difference within the sample group; each scatter point represents a sample; scatter shapes and colors indicate different experimental groupings.(A) The PCA model's score scatter plot for the NAFLD vs. control groups.It can be seen from the results of the PCA score that the sample was basically within the 95% confidence interval.(B) The score scatter plot of the OPLS-DA model showed that the NAFLD group and control group were significantly different, and the sample was basically within the 95% confidence interval.(C)OPLS-DA model permutation test for the NAFLD vs. control groups.The abscissa represents the permutation retention degree of the permutation test, the ordinate represents the value of R 2 Y or Q 2 , the green dot represents the R 2 Y value, the blue square point represents the Q 2 value, and the two dashed lines represent the regression line for R 2 Y and Q 2 .The Q 2 value of the random model of the permutation test was all smaller than the Q 2 value of the original model; the intercept of the regression line of Q 2 and the vertical axis was less than zero; at the same time, as the permutation retention gradually decreased, the proportion of the permuted Y variable increased, and the Q 2 of the random model gradually decreased.It showed that the original model had good robustness, and that there was no overfitting phenomenon.(D) Volcano plot for the NAFLD group vs. control group.Each point in the volcano plot represents a metabolite, the abscissa represents the fold change of each substance in the group compared with each other (take the logarithm with the base 2), and the ordinate represents the P-value of the student's t-test (take the pair with the base 10).The size of the scatter point represents the VIP value of the OPLS-DA model, and the larger the scatter point, the greater the VIP value.Scattered colors represent the final screening results, with significantly up-regulated metabolites in red, significantly down-regulated metabolites in blue, and non-significantly different metabolites in grey.

Figure 4 .
Figure 4. Functional analysis of differential metabolites.(A) the abscissa represents different experimental groups, the ordinate represents the differential metabolites compared in the group, the color Blocks at different locations represent the relative expression of the metabolites at the corresponding locations, and the red represents the content of the substance at high expression, while the blue indicates low expression of the substance.(B) The horizontal and vertical coordinates in the figure represent differential metabolites; red represents positive correlation, blue represents negative correlation, and the darker the color, the stronger the correlation.Meanwhile, non-significant correlations are marked with a cross.(C) The size of the point represents the value of LOG_FOLDCHANGE.The larger the point, the larger the value of LOG_FOLDCHANGE.The color of the point represents the source classification of the differential metabolites in the group comparison, and the line represents the correlation coefficient value of the metabolite at the corresponding position.

Figure 5 .
Figure 5. Metabolite pathway analysis of differential metabolites.(A) Each bubble in the bubble diagram represents a metabolic pathway.The abscissa where the bubble is located and the size of the bubble represent the size of the impact factor of the pathway in the topology analysis.The larger the size, the greater the impact factor; the ordinate where the bubble is located and the color of the bubble represent the enrichment analysis.The P value (take the negative natural logarithm, i.e.À lnP-value), the darker the color, the smaller the P value, and the more significant the degree of enrichment.(B) The top 5 metabolic pathways and their key metabolites.

Figure 6 .
Figure 6.Targets-pathways-metabolites regulation network diagram and molecular docking structure diagram.(A) The diamond-shaped nodes in the figure represent the target proteins, the rectangular nodes represent the pathways, the arrow nodes represent the metabolites, and the connecting lines represent the associations between nodes.(B-E) were the two-dimensional and three-dimensional structural diagrams of the molecular docking of the targets PTGS2 (6n4e) and isorhamnetin, luteolin, PPARG (6c5t) and quercetin, stigmasterol, respectively.

Figure 7 .
Figure 7. Histopathology and related index detection in rats.(A) HE staining of the liver (�200 times).(B) Oil red O staining of the liver (�200 times).(C) Comparison of 5-HT and NA in each group of rats.(E) Comparison of the insulin and fasting blood glucose in each group of rats.(F) Comparison of blood lipids (TG, TC, HDL-C, LDL-C) in each group of rats.(G) Comparison of liver function (ALT, AST, GGT) in each group of rats.Compared with the normal control group, �� P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01.

Figure 8 .
Figure 8. Validation of core metabolites and core targets in vivo.(A) The contents of AA and cAMP in each group.(B) PTGS2, PPARG mRNA expression levels in each rat group.(C) PTGS2, PPARG protein expression level in each group.(D) The content of PGE2 and 15d-PGJ2 in each group.Compared with the normal control group, �� P < 0.01; compared with the model group, # P < 0.05, ## P < 0.01.

Figure 9 .
Figure9.Mechanism of XYS in the treatment of NAFLD with liver depression and spleen deficiency.After the application of XYS, the amount of AA detached from the cell membrane is decreased.AA is converted into PGs under the action of PTGS2, and the reduction of PTGS2 reduces the increase of PPARG bound to PGJ2.On the one hand, elevated PPARG can directly or through the transcriptional regulation of glucose and lipid metabolism-related genes inhibit insulin secretion by pancreatic islet cells, reduce IR, lower blood sugar, and inhibit lipid accumulation; on the other hand, elevated PPARG can inhibit oxidative stress and inflammatory responses.Decreased PGE2 increases cAMP, which can reduce FBG and lipid accumulation directly or through transcriptional regulation of glucose and lipid metabolism-related genes.

Table 1 .
The core active components and core targets.

Table 2 .
Differential metabolites in NAFLD group vs control group.