Animals and groups
Healthy male Sprague-Dawley (SD) rats (license number: SCXK2009-001) weighing 220–280 g were provided by the Laboratory Animal Center of Shanxi Medical University. The animals were housed in separate cages at a room temperature of 20–25°C and relative humidity of 50–65% under a 12-h light-dark cycle and given free access to water and food. In total, 50 SD rats were divided into control (n = 14) and model groups (n = 36); seven rats died during the modeling process. After successful modeling, rats were randomly divided into the T1DM group (n = 14) and Hst group (n = 15). All animal experimental procedures complied with the Experimental Animal Ethics Committee of Shanxi Medical University.
Drug treatment
After fasting for 12 h, all rats, except those in the control group, were intraperitoneally injected with 1% streptozotocin (STZ; Sigma, USA) dissolved in citrate buffer (pH 4.2–4.5) at a dose of 65 mg/kg. Control rats were injected with the same volume of citrate buffer. After 3 days, blood glucose was measured via the tail vein, and rats with a blood glucose level exceeding 16.7 mM for more than 1 week were considered as having T1DM. Rats in the Hst group were then intragastrically administered 100 mg/kg/day Hst (Sigma) dispersed in 0.1% sodium carboxymethyl cellulose for 4 weeks, starting at 2 weeks after the STZ injection. The control and T1DM groups were intragastrically administered an equal amount of 0.1% sodium carboxymethylcellulose.
Hematoxylin and eosin (HE) staining
At the end of the experiment, rats in each group were anesthetized, their chest was opened, and the heart was removed and placed into a beaker containing PSS (pH 7.4) solution in an environment saturated with a 95% O2 and 5% CO2 mixture at 4°C. After routine treatment, the hearts of three rats in each group were stained using HE dye solution (Servicebio, China), observed using microscopy (DM2500; Leica, Germany), and photographed using Leica Application Suite X software (LAS X).
Preparation of proteomic vascular samples
The hearts were removed from the beaker and fixed in a Petri dish with pins. After finding the coronary arteries using an anatomical microscope (Phenix, China), they were separated by carefully removing the muscle tissue around the blood vessels using surgical microscissors and forceps. The vascular specimens were placed in a cryotube, rapidly frozen in liquid nitrogen, and stored in a freezer at -80°C.
TMT-labeled quantitative proteomics
Protein sample preparation
Vascular samples were ground into a powder in liquid nitrogen and transferred to a 5-mL centrifuge tube. Then, four volumes of lysis buffer (8 M urea, 1% protease inhibitor, 3 μM TSA, 50 mM NAM) was added to the cell powder, followed by sonication three times on ice using a high-intensity ultrasonic processor (Scientz, China). After centrifugation at 4°C and 12000 × g for 10 min, the supernatants were collected, and the protein concentration was determined using a BCA kit (Beyotime, China).
Trypsin digestion
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at 25±2 ℃in the dark. The urea concentration of the protein sample was diluted to less than 2 M. Trypsin was added at a mass ratio of 1:50 and 1:100 (trypsin:protein) for the first digestion overnight at 37°C and the second 4-h digestion, respectively.
TMT labeling
After trypsin digestion, peptides were desalted using a StrataXC18 SPE column (Phenomenex) and freeze-dried in a vacuum. Peptides were dissolved in 0.5 M TEAB and labeled with a TMT kit (Thermo, USA) according to the manufacturer’s instructions. Briefly, the labeled reagent was thawed and dissolved in acetonitrile, mixed with peptides, and incubated at room temperature for 2 h. The peptide mixtures were pooled, desalted, and freeze-dried in vacuum. Three technical replicates were carried out per sample, and the samples were labeled as follows: control group, 126, 127C, 128N; T1DM group, 128C, 129N, 129C; Hst group, 130N, 130C, 131.
Fractionation using high-performance liquid chromatography (HPLC)
The peptides were fractionated using high pH reverse-phase HPLC using an Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated using a gradient of 8–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into nine fractions and freeze-dried in vacuum.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
The peptides were dissolved with LC mobile phase A (0.1% formic acid and 2% acetonitrile) and directly loaded onto a reversed-phase column (15 cm length, 75 μm ID). The gradient comprised an increase from 8% to 20% mobile phase B (0.1% formic acid and 90% acetonitrile) over 50 min, 20% to 35% in 35 min, and increasing to 80% in 3 min, then held at 80% for the last 3 min, all at a constant flow rate of 400 nL/min using an EASY-nLC 1000 UPLC system.
After separation using an ultra-high-performance liquid phase system, the peptides were injected into the NSI ion source for ionization and then analyzed using Orbitrap Fusion Lumos MS. The applied ion source voltage was 2.0 kV, and the scan range of the first-level MS was set to m/z 350–1550 with 60,000 resolution; the second-level MS was set to m/z 100 with 30,000 resolution. To improve effective utilization of the mass spectrum, the automatic gain control was set at 5E4, the signal threshold was 50000 ions/s, the maximum injection time was 70 ms, and the dynamic exclusion time of the tandem MS scan was 30 s to avoid repeated scanning of the peptide precursors.
Database search
LC-MS/MS raw data were processed from the UniProt Rattus database concatenated with a reverse decoy database and a common pollution database using Maxquant (v.1.5.2.8). The parameters were set as follows: algorithm, trypsin; maximum missed cleavages, 2; minimum peptide length, 7 amino acid residues; maximum modification number, 5. The mass error tolerance values of the precursors in the first and main search were 20 and 5 ppm, respectively, and the second fragment tolerance was 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification. Variable modifications were oxidation on Met and N-terminal acetylation of protein. The quantification type was TMT-10plex. The false discovery rate was set to 1%.
Bioinformatics methods
Gene Ontology (GO) annotations
The GO annotation proteome was derived from the UniProt-GOA database (https://www.ebi.ac.uk/GOA/). First, we converted the identified protein IDs to UniProt IDs and then mapped the proteins to GO IDs. If some identified proteins were not annotated by the UniProt-GOA database, InterProScan software was used to annotate the functions of the proteins based on the protein sequence alignment method. Then, proteins were classified into biological process, cellular component, and molecular function categories. For each category, two-tailed Fisher’s exact tests were used to evaluate the enrichment of differentially expressed proteins. GO categories with corrected p values < 0.05 were considered significant.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation
The KEGG (https://www.genome.jp/kegg/pathway.html) database was used to annotate protein pathways. First, the KEGG online service tool KAAS was used to annotate protein, followed by mapping the annotation result of the KEGG pathway database using KEGG online service tools and KEGG mapper. Wolfpsort (https://www.genscript.com/psort/wolf_psort.html) was used to predict subcellular localization. For domain annotation, the identified protein domain functional descriptions were annotated using InterProScan and the InterPro domain database (http://www.ebi.ac.uk/interpro/), which integrates diverse information about protein families, domains, and functional sites. Lastly, the KEGG database was used to identify enriched pathways using two-tailed Fisher’s exact tests. KEGG pathways with corrected p values of less than 0.05 were considered significant.
Protein-protein interaction network
All differentially expressed protein database accessions or sequences were searched against the STRING database (v. 11.0) for protein-protein interactions.
Western blot analysis
Coronary artery tissues of rats were ground in RIPA lysis buffer and protease inhibitor (phenylmethylsulfonyl fluoride) using an MP FastPrep-24 5G rapid sample preparation instrument (MP, USA). The precipitate was gently shaken for 30 min on a shaker and removed by centrifugation for 20 min at 4°C and 13000 × g, and the protein concentration was determined using a BCA kit (Boster, China). The samples were diluted to the same concentration, mixed with 5× loading buffer, boiled at 100°C for 5 min, cooled to room temperature, and stored at -20°C until use. The proteins were then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked at room temperature for 2 h with 5% skim milk dissolved in TBST, washed with TBST three times for 10 min each, and incubated with the following antibodies overnight on a shaker at 4℃: anti-β-actin (1:5000 dilution; Bioworld, China), anti-Kng1 (1:1000 dilution; ABclonal, China), anti-S100A9 (1:1000 dilution; ABclonal), and anti-S100A8 (1:1000 dilution; ABclonal). The membranes were washed three times with TBST for 10 min each and incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:10000 dilution; Boster) on a shaker at room temperature for 1 h. After washing the membranes with TBST, an appropriate amount of ultra-sensitive ECL Ready-to-use Substrate (Boster) was added, and the membranes were exposed to a ChemiDoc MP Imaging System (Bio-Rad Laboratories, USA).
Statistical analysis
Statistical analysis was performed using SPSS software (version 26.0). The results were compared using a one-way analysis of variance followed by least significant difference post-hoc tests. All data are expressed as means ± standard deviations and P < 0.05 was considered statistically significant.