Screening candidate compounds and targets of mSMG

As shown in Table 1, mSMG is a TCM formula consisting of five herbs: Atractylodis Rhizoma (AR, Pinyin name: Cangzhu, Plant source: Atractylodes lancea (Thunb.) DC.), Phellodendri Chinensis Cortex (PCC, Pinyin name: Huangbo, Plant source: Phellodendron chinense C.K.Schneid.), Coicis Semen (CS, Pinyin name: Yiyiren, Plant source: Coix lacryma-jobi var. ma-yuen (Rom.Caill.) Stapf), Artemisiae Scopariae Herba (ASH, Pinyin name: Yinchen, Plant source: Artemisia capillaris Thunb.), and Plantaginis Herba (PH, Pinyin name: Cheqiancao, Plant source: Plantago asiatica L.). The herbal name referred to the Chinese Pharmacopoeia 2020. Each herb corresponded to its plant source in the Chinese Pharmacopoeia 2020 and the latest revision of Medicinal Plant Names Services (MPNS, Royal Botanic Gardens, Kew, Version 12, accessed on September 13, 2023, http://mpns.kew.org). We screened the compounds of each herb in the Traditional Chinese Medicine Systems Pharmacology database (TCMSP, http://tcmspw.com/tcmsp.php) and Traditional Chinese Medicine integrative database (TCMID, http://119.3.41.228:8000/tcmid/) by entering the Pinyin names of herbs and filtered them with the screening criteria: oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The compounds from the TCMID database were imported into the TCMSP database due to no OB and DL parameters in the TCMID database, and only the compounds that met the screening criteria were included. The duplications of compounds were removed. Next, by entering the names of the screened compounds, corresponding targets were derived from TCMSP, Search Tool for Interacting Chemicals (STITCH, http://stitch.embl.de/), and DrugBank (https://www.drugbank.com/) databases. After deleting duplicate targets, their full names were input into the Uniprot database (https://www.uniprot.org/) and the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) for conversion into gene symbols.

Table 1 Information of mSMGScreening genes related to IR in T2DM

The genes related to T2DM were collected by searching “Type 2 diabetes” or “Type 2 diabetes mellitus” or “Diabetes Mellitus, Non-Insulin-Dependent” or “T2DM” in the GeneCards (https://www.genecards.org/), OMIM (https://omim.org/) and DisGeNET (https://www.disgenet.org/) databases. Download files and delete duplicate genes. Meanwhile, the IR-associated genes were retrieved by searching “insulin resistance” in the three databases. Then, using the Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/), the overlaps between the genes related to T2DM and the IR-associated genes were filtered out, which were considered as the IR-associated genes in T2DM.

Potential action targets of mSMG against IR in T2DM and compound-target network construction

In order to obtain the potential action targets, we used the candidate targets of mSMG to intersect the IR-associated genes in T2DM using the Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/). These overlapping targets and related candidate compounds were regarded as potential action targets and compounds of mSMG against IR in T2DM. Correspondingly, candidate compounds acting on these targets were also considered as potential compounds of mSMG against IR in T2DM. Subsequently, these potential compounds and action targets were used to construct the compound-target network and visualized by Cytoscape 3.7.1 software.

Construction of PPI network and identification of hub targets

To explore the interaction among the potential targets of mSMG, we imported the potential action targets of mSMG against IR in T2DM into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://cn.string-db.org/), selected “Homo sapiens” as the organisms, set high confidence (0.700) as a minimum required interaction score, and hided disconnected nodes in the network, then a protein–protein interaction (PPI) network was established. TSV format of the updated results were downloaded. Visualization and analysis were performed using Cytoscape 3.7.1. Firstly, node1, node2, and combined scores were extracted and imported into the Cytoscape software to create a PPI network, and the network was analyzed as follows: Step 1: analyze the topology properties of the network: Tools→Network Analyzer→Network Analysis→Analyze Network, save the CSV format of the network result and extract the Degree value; Step 2: create a network map according to the Degree value: Tools→Network Analyzer→Network Analysis→Generate Style from Statistics→Map Node Size to Degree (Low value to small sizes) →Map Node Color to Degree (Low value to dark colors), and save the PPI network map. Subsequently, we used 11 topological algorithms of plug-in cytoHubba [18] in Cytoscape 3.7.1 to identify the hub targets in the PPI network. Among the 11 methods, Degree, Closeness, and Betweenness are the most used to measure the importance of a node in a network. Degree refers to the number of nodes connected by a node; Closeness is used to measure the average distance between a node and other nodes; Betweenness is an indicator of node importance characterized by the number of shortest paths passing through a node [19]. The detailed operation process was as follows: Step 1, install cytoHubba: Apps→App Manager→Search cytoHubba→Install; Step 2: analyze the topological algorithms: cytoHubba→Calculate, save the CSV format of the calculation result and extract the Degree, Closeness, and Betweenness value. Afterwards, the top 15 targets respectively ranked by the 3 topological algorithms were intersected, and the overlapping targets were filtered out as the hub targets of mSMG against IR in T2DM.

GO function and KEGG pathway enrichment analyses

To characterize the function and biological implications behind the potential action targets of mSMG, enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were carried out in the KEGG Orthology Based Annotation System database (KOBAS, http://kobas.cbi.pku.edu.cn/). We input gene symbols, selected “Homo sapiens (human)” as species, and set corrected P-value < 0.05 as statistical significance. Then, re-render the key KEGG pathway map using the “Pathview R” package. Based on the input action targets, add green color to the boxes corresponding to the relevant genes in KEGG pathway map to integrate information of KEGG pathway and action targets of mSMG against IR in T2DM for intuitive viewing.

Reagents

Metformin (B25331) was purchased from Sino-US Shanghai Squibb Pharmaceutical Co., Ltd (Shanghai, China) and berberine (S9046) was purchased from Selleck (USA). TNF-α (11948), insulin receptor substrate 2 (IRS-2, 3089), Akt (4691), p-Akt (Ser473) (4060), glycogen synthase kinase-3β (GSK-3β, 12456), p-GSK-3β (Ser9) (5558), β-actin (8457), anti-rabbit IgG (7074), and anti-mouse IgG (7076) antibody were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). JNK1 (ab199380), p-JNK1 (T183) (ab47337), and glucose transporter 2 (GLUT2, ab54460) were purchased from Abcam (USA). p-IRS-2 (Ser388) (07-1517) was purchased from Sigma Aldrich Inc. Glucose kit (glucose oxidase method, A154-1-1), liver glycogen assay kit (A043-1-1), and enzyme linked immunosorbent assay (ELISA) kits for glycated hemoglobin (HbA1c, H464), insulin (H203), and TNF-α (H052) were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Glycogen synthase activity assay kit (YB-GCS-1) was purchased from Shanghai Yubo Biotechnology Co., Ltd (Shanghai, China).

Preparation and quality control of mSMG

All the herbs were processed into boil-free granules, provided by Sichuan Neo-Green Pharmaceutical Technology Development Co., Ltd (Sichuan, China) and quality controlled. Table 1 lists the amount of granular extracts of each herb in 1 unit of modified Si-Miao formula. They were mixed and brewed with boiling water like coffee based on Chinese Pharmacopoeia 2020. The main chemical components in mSMG were identified by Ultra-high-performance Liquid Chromatography-Quadrupole-Orbitrap Mass Spectrometry (UPLC-Q-Orbitrap-MS) as our previous description [20]. In brief, 1000 μL 80% methanol was added into 200 μL mSMG extract. Then it was swirled for 10 min, centrifuged at 4 °C for 10 min with centrifugal force of 20,000×g. Supernatant was filtered for UPLC-Q-Orbitrap-MS analysis. Mass spectrometric detection was performed on Q Exactive high resolution mass spectrometer (Thermo Fisher Scientific, USA). Mass spectrum condition: ion source: electric spray ionization source (ESI); scanning method: positive and negative ion switching scanning; detection method: full mass/dd-MS2; resolution ratio: 70,000 (full mass), 17,500 (dd-MS2); scan range: 100.0–1500.0 m/z; spary voltage: 3.2 kV(Positive, Negative); capillary temperature: 300 °C; collision gas: high purity argon (purity ≥ 99.999%); collision energy (N) CE: 30; sheath gas: nitrogen (purity ≥ 99.999%), 40 Arb; auxiliary gas: nitrogen (purity ≥ 99.999%), 15 Arb, 350 °C; data collection time: 30.0 min. Chromatographic analysis was performed on UltiMate 3000 RS (Thermo Fisher Scientific, USA). Chromatographic condition: chromatographic column: AQ-C18, 150 × 2.1 mm, 1.8 μm, Welch; flow rate: 0.30 mL/min; aqueous phase: 0.1% formic acid/aqueous solution; organic phase: methanol; column temperature: 35 °C automatic injector temperature: 10.0 °C; automatic injector volume: 5.00 μL. The data were preliminarily collated by CD2.1 (Thermo Fisher) and then retrieved and compared in mzCloud database.

Animals and experimental design

Male C57BL/6J mice and KK-Ay mice aged 8 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd (Permission No: SCXK 2019–0008). All mice were individually housed in a specific pathogen-free (SPF) condition with free access to sterile water and chow diet. The housing ambient temperature was 22 ± 2 °C with a humidity of 55 ± 15% and a 12-h light/12-h dark cycle. All mice were allowed to acclimatize for 1 week. C57LB/6J mice fed with normal diet were taken as a normal control (Normal) group. KK-Ay mice were fed with high-fat diet (HFD; 4.73 kcal/g, 20% kcal from protein, 35% kcal from carbohydrates and 45% kcal from fat; H10045, Beijing Huafukang Biotechnology Co., Ltd, China). After 4 weeks, KK-Ay mice with fasting blood glucose (FBG) level more than 11.1 mmol/L were classified as T2DM [21], and then these mice were randomly divided into four groups (n = 6 per group): T2DM model group (Model), metformin treatment group (Metformin, 0.308 g/kg), low dose mSMG treatment group (mSMG-L, 1.665 g/kg), and high dose mSMG treatment group (mSMG-H, 3.330 g/kg). Dosage conversion between humans and mice was determined according to Pharmacological Experiment Methodology (4rd version) [22]. The dosages of metformin and mSMG in mice were respectively calculated by multiplying 0.025 g/kg (1.5 g metformin/60 kg adults/day) and 0.135 g/kg (8.1 g mSMG/60 kg adults/day) by 12.33 (conversion coefficient), resulting in an approximate dosage of 0.308 g/kg for metformin and 1.665 g/kg for the low dose mSMG, while the high dose was twice of that amount. mSMG suspension or metformin was administered to mice by oral gavage. Mice in Normal group and Model group were intragastric administered with the same solvent distilled water. Once a day. The adverse reactions were observed and recorded during the experiment. After 12 weeks of treatment, mice were euthanized, and blood and liver samples were collected. The animal protocols were strictly implemented following the Guidelines of the National Institutes of Health on Animal Care and Ethics and approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (Approval Number: 20220512003).

Oral glucose tolerance test

To detect the ability of the glycemic regulatory in mice, the oral glucose tolerance test (OGTT) was performed. After fasting for 15 h, glucose (1 g/kg) was administered by gavage, and blood glucose levels were measured using an automated analyzer (Roche Diagnostics, Germany) at 0, 30, 60, and 120 min, respectively. Then, the area under the curve (AUC) of blood glucose (AUC of OGTT) was calculated as following: AUC of OGTT = (G0 + G120)/4 + (G30 + G60)/4 + (G60 + G120)/2 [23].

Serum biochemistry, HOMA-IR and TNF-α measurements

Blood samples were collected from the medial canthus vein and centrifuged (3000 r/min) for 15 min at 4 °C. FBG, total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate transaminase (AST), serum creatinine (Scr), and blood urea nitrogen (BUN) were examined by an automatic biochemistry analyzer (Rayto Technologies, China). HbA1c, fasting insulin (FINS), and TNF-α in serum were measured using ELISA kits according to the manufacturer’s protocol. Homeostasis model assessment-IR (HOMA-IR) was used to evaluate the IR in T2DM, which was calculated as described previously [23]: HOMA-IR = FBG (mmol/L) × FINS (μU/mL)/22.5.

Hematoxylin and eosin staining

Briefly, after fixation in 4% paraformaldehyde for 24 h, liver tissues were isolated, dehydrated in graded volumes of ethanol alcohols, and embedded into paraffin. Then, the sections (3.5 μm) of liver tissues were stained by hematoxylin and eosin (H&E), and the pathology was observed under a microscope [21]. The H&E staining images were evaluated using the NAFLD activity score (NAS) system as previously reported [24].

Oil Red O staining analysis

Frozen sectios (5 μm) of liver tissue were washed with phosphate buffered saline (PBS) twice. Then they were fixed in 4% paraformaldehyde for 40 min, washed twice in 60% isopropyl alcohol, and stained using an Oil Red O stain kit (P0047, Wuhan Pinofi Biotechnology Co., Ltd.) following the manufacturer’s instructions. Next, Positive cells were observed using an optical microscope [25].

Molecular docking

To detect the binding capacity between compounds derived from the UPLC-Q-Orbitrap-MS analysis of mSMG and the hub target TNF identified by network pharmacology, we conducted molecular docking. Compounds of mSMG were selected as the ligands and the hub gene TNF was selected as the receptor. The molecule structures of ligands (compounds) were downloaded from the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/). Based on the screening criteria: (1) protein source organism: Homo sapiens; (2) refinement resolution < 2.5 Å; (3) complete protein structure with corresponding ligand; and (4) pH within the physiological range of the human body, the molecule structures of receptor (2AZ5) were downloaded from the Protein Data Bank Database (www.rcsb.org). Then, we preprocessed the ligand using ChemBio3D software and automatically optimized its 3D conformation using the default system settings. PyMOL 1.7.2.1 software was utilized to remove water molecules and impurity from the receptor protein, followed by the separation of the original ligand to obtain a standardized receptor. Next, the ligand and receptor were imported into AutoDockTools 1.5.6 software. The receptor underwent the addition of polar hydrogen atom and Gasteiger charge. Subsequently, the Grid tool was utilized to automatically identify the Grid Box for the docking. The Grid Box parameters were set as follows: (1) Energy range and num modes were set as 5 and 20, respectively; (2) Spacing (angstrom) was 1; (3) The center of Grid Box was coincided with the center of the receptor; and (4) the dimension of Grid Box was manually adjusted based on the protein volume until the receptor was completely enveloped. Finally, molecular docking was performed using AutoDock Vina 4.2.6 software, and the docking results were visualized and assessed for hydrogen bond formation using PyMOL 1.7.2.1 software. Usually, binding energy lower than − 7.0 kcal/mol indicates excellent binding activity between receptor and ligand [15, 26].

Preparation of mSMG-mediated serum (mSMG-MS)

Ten New Zealand rabbits were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine and kept in a clean condition. The preparation and identification methods were the same as we described before [20]. In brief, the rabbits were randomly divided into the normal (n = 4) group and mSMG (n = 6) group. Rabbits in the mSMG group were administered mSMG 1.247 g/kg (three times the clinical equivalent dose) once a day, whereas animals in the normal group were intragastrically administered equal volume of distilled water. After continuous gavage for 6 days, the rabbits were fasted for 12 h. Then they were anaesthetized at 1 h after the last administration, and carotid blood samples were collected and centrifuged at 4000 r/min for 15 min. The supernatant was inactivated in a 56 °C water bath for 30 min and removed bacteria with a 0.22 μm microporous filter. Finally, we obtained the mSMG-mediated serum (mSMG-MS) and the normal serum (NS) and stored them at − 80 °C.

Cell culture, TNF-α-induced IR model and treatment

The human hepatoblastoma cell line HepG2 was purchased from China Type Center for Type Culture Collection (CCTCC) (Wuhan, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. According to the previous description [27, 28], IR in HepG2 cells (IR-HepG2 cells) was induced by 5 µM insulin and 30 ng/mL TNF-α for 4 h, and we treated the IR-HepG2 cells with corresponding drugs. Specifically, HepG2 cells were seeded in 6-well plates at a density of 1 × 104 cells/well and grown for 48 h to 80–100% confluence. The cells were then incubated for 4 h in medium containing blank (Control group), or 5 µM insulin and 30 ng/mL TNF-α (IR group), or 5 µM insulin and 30 ng/mL TNF-α added with 20%NS (NS group) or 20% mSMG-MS (MS group) or 50 µM berberine (BBR group).

Glucose consumption assay

Cellular glucose uptake was assayed in the medium with a glucose assay kit. After the treatment, culture medium was collected and separated at 1000 rpm for 10 min, and the supernatant was used to detect glucose content according to the manufacturer’s instructions. After incubation at 37 °C for 10 min, the absorbance was measured at 505 nm by a microplate reader (BioTek, Waltham, MA, USA) and the glucose content was calculated. Glucose consumption (mmol/L) = glucose content of blank well − glucose content of treatment well.

Cell counting kit-8 (CCK-8) assay

The HepG2 cells (5 × 104/well) were seeded in 96-well plates overnight. Then, the cells were incubated in medium with 5 μM insulin and 30 ng/ml TNF-α. Meanwhile, they were treated with mSMG-MS (0–40%) or berberine (0–250 µM) for 4 h. Next, 10 µL CCK-8 (C0038, Beyotime, China) was added to each well, and the cells were incubated on the condition of 37 °C, 5% CO2 for 2 h. Eventually, the absorbance was measured at 450 nm by a microplate reader, and cell viability (%) = optical density (OD) value of experimental group/OD value of control group × 100% [20].

Glycogen content and glycogen synthase activity assay

After the treatment, glycogen was detected utilizing a glycogen assay kit according to the manufacturer’s instructions, and the absorbance was measured at 570 nm by a microplate reader. Additionally, the protein content was quantified by BCA assay kit (P0010, Beyotime, China), and the glycogen content was calculated as a ratio of glycogen (mg)/protein (g) [29]. Glycogen synthase (GS) is a key rate-limiting enzyme in glycogen synthesis. We measured it by ELISA method with a glycogen synthase activity assay kit according to the manufacturer’s instructions.

Western blotting

According to our previously described method [20], the liver tissues or HepG2 cells were lysed with RIPA lysis buffer (P0013B, Beyotime, China) containing 1% protease inhibitor (5871, CST, USA) and 1% phosphatase inhibitor (5872, CST, USA). After quantification by BCA assay kit, the protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane. The PVDF membrane was sealed with 5% bovine serum albumin (BSA) on a shaker at room temperature for 1 h. Then, according to the molecular weight of the target protein, the PVDF membrane was incubated with TNF-α, JNK1, p-JNK1, IRS-2, p-IRS-2 (Ser388), Akt, p-Akt (Ser473), GSK-3β, p-GSK-3β (Ser9), GLUT2, or β-actin antibody on a shaker at 4 °C overnight. The next day, the membranes were incubated with species-related horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) at room temperature for 2 h. Finally, Ultra-sensitive enhanced chemiluminescent (ECL) (SI254786A, Thermo, USA) was employed and ChemiDoc™ XRS + System (Bio-Rad, USA) was used to display protein bands. Image J software (version 1.8.0, NIH, USA) was used to analyze the gray values.

Statistical analysis

SPSS statistical program (version 25.0, IBM Corp, Armonk, USA) and GraphPad prism (version 8.0 La Jolla, CA, USA) were performed for statistical analyses and scientific graphing. When the data conformed to the normal distribution and the variance was homogeneous, the one-way Analysis of Variance (ANOVA) was used. When the one-way ANOVA had statistical differences, the Tukey test is used for multiple comparisons, and the data was described by the mean ± standard deviation (\(\overline\)±s). When the data did not conform to the normal distribution or the variance is uneven, Nonparametric Kruskal–Wallis H test was used, and the data was described by M (P25-P75). All statistics were conducted by double-sided test. The test level (α) was 0.05, and the difference was statistically significant with P < 0.05.