Collection and verification of DXXK ingredientsSince DXXK capsules are herbal preparations, we searched for the corresponding ingredients using its most prominent API (D. nipponica Makino) as the keyword. The ingredients of DXXK were mainly obtained from the Encyclopedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (Batman-TCM) databases, both of which contain comprehensive and standardized information on the specific ingredients of Chinese herbs and formulas. To ensure the completeness and accuracy of the ingredients in DXXK, some ingredients were hand-collected from other literature21. Their chemical formulas were determined by Pubchem and stored in 2D.sdf format. The pharmacological properties of each ingredient were evaluated separately in accordance with the Swiss ADME database. We also reviewed the literature on the antidiabetic activity of these ingredients, followed by literature verification of the ingredients excluded according to the Swiss ADME screening criteria. If the pharmacological effects of these ingredients were clearly relevant to the subject of the study, they were included directly in the study.Prediction of DXXK-related targetsEach ingredient of DXXK was individually loaded into the PharmMapper database (a comprehensive pharmacophore matching platform) to find potential targets. Based on the number of targets obtained, genes with a normalized fit (NF) score ≥ 0.3 were screened as DXXK-related targets.Prediction of DM-related targetsTo obtain a comprehensive and reliable list of DM-related targets, the keyword “Diabetes mellitus” was selected to search the GeneCards, OMIM, DisGeNET, and DrugBank databases. All DM-related targets in the OMIM and DrugBank databases were collected. In addition, the median relevance score was used for multiple screening in the GeneCards database, while Score_gda > 0.1 was used as a filtering criterion in the DisGeNET database. Diabetic targets were obtained by removing duplicate values and merging the targets extracted from the above databases.Construction of protein–protein interaction (PPI) networksPredicted targets for DM and DXXK were converted to gene symbols using the UniProt database. These gene symbols were then mapped onto a Venn diagram to identify core therapeutic targets. To further elucidate the interactions between overlapping genes, their PPI networks were generated using the STRING database. The species was set to Homo sapiens with a confidence score of 0.4, and the other settings were default. Moreover, the interaction network of DXXK-related components and corresponding targets was constructed using Cytoscape (version 3.7.1).Enrichment analysisKyoto Encyclopedia of Genes and Genomes (KEGG)29,30,31 and Gene Ontology (GO) enrichment analyses (p ≤ 0.01) were performed on the overlapping targets using the Metascape database. The GO enrichment analyses included biological process (BP), molecular function (MF), and cellular component (CC), respectively. After sorting the enrichment analysis results by p-value, the top 20 pathways were selected for visualization.Molecular dockingThe PDB files of the proteins were downloaded from the RCSB database (PDB IDs: 1PY5-TGF-β1, 5E8Y-TGF-β2, 1KHX-p-Smad2, 1MK2-p-Smad3, 1DEV-Smad2, and 1MJS-Smad3). To eliminate the effect of water of crystallization on ligand-target protein interactions and optimize the docking energy, ligands (ingredients of the DXXK) and protein receptors were saved in PDBQT format in the Autodock Vina software (version 1.1.2) after removing water molecules and adding hydrogen bonds, respectively. The appropriate coordinate positions and grid boxes were set to cover the active sites of the proteins by adjusting the number of points in the x, y, and z dimensions and the spacing (angstroms), and then Vina was run. After docking, the conformations with the highest number of hydrogen bonds were analyzed using PyMOL (version 2.5). Table 1 lists the web links to all databases and software.Table 1 Database and software (URL).Medications and reagentsDXXK was manufactured by Di’ao Pharmaceutical Group Co., Ltd (2,106,036, Chengdu, China). Metformin was produced by Xinyi Tian ping Pharmaceutical Co., Ltd (67,210,972, Shanghai, China). Control samples of dioscin, protodioscin, and pseudoprotodioscin were purchased from Yuanye Biotechnology Co., Ltd (B21176, B21621, B21303, Shanghai, China). High-fat diet (HFD) was purchased from Xietong Pharmaceutical Bio-engineering Co., Ltd (XTHF60, Nanjing, China). Streptozotocin (STZ) was purchased from Aladdin (G2115339, Shanghai, China). Citric acid and sodium citrate were purchased from Macklin (C12022793, C12931024, Shanghai, China). Insulin was purchased from Wan bang Pharmaceutical Technology Co., Ltd (H10890001, Hefei, China). Hematoxylin–eosin (H&E) staining kit, Masson’s trichrome (M-T) staining kit, reverse transcription kit, and SYBR Green qPCR kit were purchased from Biosharp (BL700A, BL1059A, BL699A, BL698A, Hefei, China).Constituent analysisDXXK and control samples were dissolved separately in methanol, vortexed and filtered. A volume of 10 μL was injected into a high-performance liquid chromatography (HPLC) system (Agilent1260, USA) for analysis (203 nm). DXXK was separated on a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) using acetonitrile (A) and water (B) as mobile phases. The detection wavelength was 203 nm and the elution conditions were 0–30 min: 15–35% A, 85–65% B; 30–55 min: 35–68% A, 65–32% B.AnimalEight-week-old male C57BL/6 mice were purchased from Ziyuan Experimental Animal Technology Co., Ltd (license number: SCXK [Zhe] 2019–0004, Hangzhou, China). All mice were housed in an animal room (24 ± 2 °C, 12-h light/dark cycle) with access to water and food. Animal experiments were conducted under the approval of the animal ethics committee of Anhui University of Chinese Medicine (No. AHUCM-mouse-2022092). Throughout the experimental process, we strictly adhere to international and national animal welfare guidelines as well as ARRIVE guidelines.DM modeling and drug therapyAfter 1 week of acclimatization, mice were fasted for 12 h and then injected intraperitoneally with STZ (50 mg/kg/d) for 4 days. In the control group, the corresponding volume of saline was injected intraperitoneally. Metformin was used as a positive control in this study. Following 3 days, mice with FBG ≥ 16.8 mmol/L were randomly divided into DM, low-dose, high-dose of DXXK, and metformin groups (n = 6). The low and high doses of DXXK were 75 mg/kg/d and 150 mg/kg/d, respectively, administered once daily by oral gavage. The intragastric administration of metformin was at a dose of 180 mg/kg/d. The control and model groups received equal volumes of saline by gavage daily. All doses were calculated using the Meeh-Rubner formula, with a conversion factor of 9.1X mg/kg (X is the human dose). Treatment with DXXK and metformin for 10 weeks and the specific animal procedures were shown in Fig. 2.Fig. 2Procedure for the treatment of DM mice with DXXK.DM indicators assessmentDuring the administration of DXXK, the mice were monitored biweekly for body weight, water and food consumption, and FBG levels. In addition, post-treatment oral glucose and insulin tolerance tests (OGTT and ITT) were performed. All mice were fasted for 12 h prior to testing. Blood samples were collected from the tail at 0, 15, 30, 45, 60, 90, and 120 min after oral glucose administration (2 g/kg) and then measured (Roche, Switzerland). Similarly, mice were injected intraperitoneally with insulin (1.5 UI/kg) after an 8-h fast and FBG data were recorded as previously described.Serum biochemical assayAt the completion of medication, mice were anesthetized with 20% urethane and then the eyes were removed for blood collection. Serum was collected by centrifugation at 2500 g for 25 min and then analyzed using an automatic analyzer (Hitachi 7020, Japan).Histopathologic assayFollowing euthanasia, the right kidney and liver were immersed in 4% paraformaldehyde, then dehydrated in graded alcohol, followed by hyalinization with xylene, and finally embedded in paraffin. Paraffin Sects. (4 μM) were stained with H&E and M-T stain, respectively. Moreover, all left kidneys were cryopreserved for subsequent experiments.Quantitative real-time PCRTotal RNA from kidneys was extracted with trizol and quantified by ultraviolet–visible (UV–Vis) spectroscopy (Molecular Devices, USA). After reverse transcription, the cDNA was subjected to qRT-PCR using SYBR Green chemistry in a real-time fluorescence quantitative PCR instrument (Applied Biosystems, USA). Primers were listed in Table 2.Table 2 Primer sequences for qRT-PCR.Western blot analysisProteins were extracted from the kidney with pre-cooled RIPA buffer and quantified with bicinchoninic acid (P0010S, Beyotime, China). Chemically denatured proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Membranes were blocked with TBST which containing 5% skim milk for 2 h at room temperature and then incubated for 12 h at 4 °C with diluted primary antibodies: GAPDH (ab8245, Abcam, UK), TGF-β1 (AF1027, Affinity, China), phospho-Smad2/3 (AF3367, Affinity, China), and Smad2/3 (8685, Cell Signaling Technology, USA). Subsequently, incubation with the secondary antibody was performed for 1 h at room temperature. Between blocking and antibody incubation, the membranes were washed with TBST solution (3 times for 10 min each). After the enhanced chemiluminescence reaction on the membrane, the protein expression levels were detected in an imaging system (GelView 6000Plus, China) and the results were processed using Image J (version 1.8).Statistical analysisAll data were expressed as mean ± standard deviation (SD). Differences between groups were compared by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test. Statistical significance was considered when p < 0.05. Statistical analyses were performed using GraphPad Prism 6.0 software (California, USA).
