Reagents
Vaccarin was purchased from Shifeng Technology (Shanghai). D-glucose was bought from Sigma Chemical Co. (St Louis, MO, USA). Kits for serum creatinine (Scr), blood urea nitrogen (BUN) and urine protein were procured from Jiancheng Bioengineering Institute (Nanjing). Antibodies against β-actin, α-SMA, E-cadherin and P-ERK were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against collagen Ⅰ, TGF-β1, T-EGFR, P-EGFR(Y845), P-EGFR(Y1173) and T-ERK from Abcam (Cambridge, MA. USA).
Experimental animals
Male C57BL/6J mice, aged 6–8 weeks, were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All experiments were approved by Institutional Animals Care and Use Committee at Jiangnan University (document number for animal use approval: JN.No20200710c0600131[174]). The animals were housed in a controlled environment with a 12-hour light-dark cycle, regulated temperature, and humidity. They were provided unrestricted access to both standard chow and tap water.
Mice model establishment
T2DM was experimentally induced in mice via a high-fat diet (HFD)/ streptozocin (STZ) regimen as we previously demonstrated [20, 27]. The normal control group (Ctrl) were given standard diet. Group received vaccarin daily (1 mg/kg, i.p.) for 8 weeks was served as vaccarin control group (VAC). While the other mice were given HFD (21.8 kJ/g, 60% fat, D12492). After feeding for 4 weeks, the HFD mice were received a single dose of STZ intraperitoneally (120 mg/kg, pH 4.0 dissolved in 10 mM citrate buffer). Mice with fasting plasma glucose higher than 11.1 mmol/L were diabetic [29]. Thereafter, T2DM mice were randomly allocated to two groups, model group (DN) and vaccarin-treated DN group (DN + VAC). DN + VAC group was given vaccarin (1mg/kg, i.p.) every day for weeks. The mice in DN group and (DN + VAC) group were maintained on a HFD feeding until sacrificed.
Assessment of blood glucose level, albuminuria, blood urea nitrogen and serum creatinine
Fast blood glucose (FBG) was measured using an AccuChek glucose meter weekly. At the end of the experiments, metabolic cages were employed to gather 24-hour urine samples for albuminuria analysis. Blood samples were obtained to extract serum, and the serum concentrations of blood urea nitrogen (BUN) and creatinine (Scr) were assessed following the guidelines provided by the manufacturer [30].
Sample collection and morphological observations
The kidneys were removed and weighed for renal/body weight index calculation. Kidney was fixed with 4% paraformaldehyde and kidney sections were cut at 5 µm. The renal histological changes were assessed by both hematoxylin and eosin (H&E) staining and periodic acid-schiff (PAS). Masson staining was used to evaluate the renal fibrosis. Images were captured by the Pannoramic SCAN (3DHISTECH Ltd., Budapest, Hungary).
Immunohistochemistry assay (IHC)
Renal sections were de-paraffinized, hydrated and underwent antigen retrieval. Following that, sections underwent hydrogen peroxide treatment (3%) to eliminate endogenous peroxidase activity, after which they were blocked with 5% BSA for 60 minutes. Primary antibodies against collagen Ⅰ, α-SMA, and E-cadherin were then applied and left to incubate overnight at 4℃. Subsequently, the sections were exposed to secondary antibodies coupled with horseradish peroxidase (HRP) for 60 minutes at room temperature. Finally, staining was accomplished using 3, 3’-diaminobenzidine (DAB). The resulting images were examined using the Pannoramic SCAN system.
Quantitative real time-PCR (RT-PCR)
Total RNA was extracted from tissues and cells using Trizol reagent (Cwbio, Beijing, China) in accordance with the provided guidelines. Subsequently, an equal amount of RNA was subjected to reverse transcription using HiScriptQ RT SuperMix (Vazyme, Nanjing, China), followed by quantitative real-time PCR utilizing ChamQTM SYBR®qPCR Master Mix (Vazyme, Nanjing, China). Relative gene expression levels were determined using the 2-ΔΔCT method [31]. The primer sequences were provided in Table S1.
Western blotting
Total protein was extracted using RIPA lysis buffer (CWBIO, Taizhou China), and the protein concentration was determined using a BCA kit (Beyotime Biotechnology, Shanghai, China). Subsequently, 20 µg of proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. Afterward, these membranes were blocked with defatted milk in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 1 hour and incubated overnight at 4°C with primary antibodies. Following TBST washing, the membranes were exposed to a 1:2000 dilution of HRP-conjugated anti-rabbit IgG antibody (CWBIO, Taizhou China). The blots were visualized utilizing a chemiluminescence detection system (Millipore Darmstadt, Germany) and semi-quantified using Image J (National Institutes of Health, Bethesda, MD, USA).
Cell culture
HK-2 cells were cultured in low glucose DMEM medium (5.6 mM glucose, Gibco, Carlsbad, CA, USA) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) in a 5% CO2 incubator. The cells were treated with 5 µM of vaccarin for 12 h before HG (35 mM) for 48 h in the following experiments.
Databases analysis
Genes related to DN and diabetic kidney disease were screened from the DrugBank database (https://go.drugbank.com/) and Genecards database (https://www.genecards.org). The predicting targets of vaccarin were searched from PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/). The DAVID database (https://david.ncifcrf.gov/) was applied to predict the biological processes (BP). High-confidence proteins of the protein-protein interaction (PPI) network was constructed by the STRING database (https://string-db.org/) and visualized by Cytoscape.
Molecular docking of EGFR and vaccarin
The 3D structure of EGFR was downloaded from RCSB Protein Data Bank (PDB) (http://www.rcsb.org/). The molecular structure of vaccarin was provided from PubChem Database (https://pubchem.ncbi.nlm.nih.gov/). AutoDock (http://autodock.scripps.edu/) was used to dock EGFR and vaccarin based on network pharmacology. Binding energy was used as docking score to predict binding strength between the key target and the drug.
Measurement of oxidative stress markers
Cellular superoxide anion production was assessed using dihydroethidium (DHE, 10 µM) or 2,7-dichlorofluorescein diacetate (DCFH-DA, 10 µM) staining, conducted in a dim environment for 30 minutes at 37℃. Fluorescence microscope (Zeiss, Germany) was employed to capture the resulting images. Additionally, malondialdehyde (MDA) levels and glutathione peroxidase (GSH-Px) activity in diabetic kidney tissue were evaluated following the manufacturer's instructions [32].
Statistical analysis
Data were presented as mean ± SEM and subjected to statistical analysis using one-way analysis of variance (one-way ANOVA) through GraphPad Prism 8 software (California, USA). A significance level of P < 0.05 was considered statistically significant.