Animals
The 8–12 week C57BL/6 mice weighing 25–35 g and Male Sprague-Dawley (SD) rats weighing 350–400 g were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Bejing, China). All animals are raised in the animal care facility of Hebei Medical University in a temperature-controlled room (23 ℃) with a light-dark cycle of 12:12h. Mice were randomly divided into control, Carotid artery ligation and Carotid artery ligation + BY1 groups. BY1 groups were administered BY1 (50 mg/kg/day) for 14d with intraperitoneal injection.
The adult male SD rats assigned to Balloon injury model procedures were randomly divided into several groups. Control, Balloon injury, Balloon injury + DCB (Drug-Coated Balloon) groups, Balloon injury + Hydrogel groups and Balloon injury + NPs groups. The DCB group is to evenly coat BY1 on the balloon with a drug load of 5 mg. In the hydrogel treatment group, the outer wall of the injured blood vessel was incubated with gauze soaked with BY1 solution (25 mg/ml) for 30 minutes, and then the hydrogel was wrapped. The NPs group was the local administration of BY1-NPs-OPN solution to the carotid artery after the injury. At some pressure, the nanoparticle solution was hit into the damaged blood vessels, incubated for 1 min, paused 10 s, and repeated three times.
Cell culture
The mouse Aortic Smooth Muscle cell (MOVAS) lines (CRL-2797) were obtained from ATCC, and maintained in DMEM (Dulbecco's Modified Eagle Medium), Low Glucose with 10% fetal bovine serum, 100 units/mL penicillin and 100 mg/mL streptomycin comprising 5% CO2 controlling the temperature at 37℃.
Reagents and antibodies
Reagents were obtained from the following sources. Antibodies: FTH1 (CST, #4393), NCOA4 (sc-373739), Akt (CST, #9272), p-Akt (CST, #4060), GPX4 ( proteintech, #67763-1-Ig), FPN (Abcam, ab78066), LC3B (Abcam, ab63817), PDGF-BB (MCE, HY-P7055) were dissolved in double-distilled water, while Erastin (MCE,HY-15763), RSL3 (MCE, HY-100218A), Z-DEVD-FMK (MCE, HY-12466), Necrostatin-1(MCE, HY-15760), Ferrostatin-1 (MCE, HY-100579) and Deferoxamine-1(MCE, HY-D0903) were dissolved in dimethyl sulfoxide (DMSO). FerroOrange (Cat. F374, Dojindo, Japan), C11-BODIPYTM581/591 probe(Cat. L267, Dojindo, Japan). LysoTracker Green (Beyotime Biotechnology, C1047S), ROS Assay Kit (Salaibio, CA1410), Masson's Trichrome Stain Kit (Solaibio,G1340), Prussian Blue Iron Stain Kit (With Nuclear Fast Red) (Solaibio,G1422).
Synthesis of BY1, BY3, BY4 and BY7
![](https://myfiles.space/user_files/58853_cdc0f79cc190fa60/58853_custom_files/img1696447688.png)
General procedure A for the synthesis of compound 6a-b
Dissolve the carboxylic acid 5a-b (0.013 mol) in 60 ml acetonitrile and add the compound 4 (4.76 g, 0.013 mol) and NMI (3.52 g, 0.043 mol). To the mixture above, add TCFH (3.37 g, 0.012 mol) and stir for 24 h. Remove the solvent by evaporating and load the residue on a silica Flash column for further purification to give 6a-b as powder or oil.
General procedure B for the synthesis of compound BY1, BY3, BY4 and BY7
Dissolve the compound 6a-b/3a-b (2.0 mmol) in 40 ml 0.4 M EDTA and 40 ml acetone. Add NaHCO3 (0.64 g, 7.6 mmol) and OXONE (3.04 g, 4.9 mmol) per 15 min for about 3 h. The mixture was filtered and the filtrate was evaporated to acetone free and extracted by ethyl acetate 3*40 ml, dried, concentrated to oil, purified by silica Flash column and further purified by prep-HPLC to give BY1, BY3, BY4 and BY7 as colorless to yellow oil.
General procedure C for the synthesis of compound 3a-b
To a solution of 1 (166.9 mg, 6 mmol) and 2a/b(6 mmol) in 40 ml DMF and 5 ml water, add CuSO4 (958 mg, 6 mmol) and DIPEA (1551 mg, 12 mmol). Equip the vessel with N2 protection and then add L- sodium ascorbate aqueous (1189 mg, 6 mmol/L- sodium ascorbate in 5 ml water). Stir the mixture at 45℃ for 5 h. Then remove the solvent under vacuo, add 40 ml water, fully dissolve the mixture with ultrasound, then add 40 ml ethyl acetate, fully dissolve under ultrasound, separate the aqueous and organic phase 1. Extract the aqueous with ethyl acetate 3*40 ml, combine all ethyl acetate solution and organic phase 1, concentrate the mixture and purify the residue on C-18 flash column to give 3a/b as colorless oil.
Synthesis of MPDA nanoparticles
The MPDA nanoparticles were synthesized by one-pot method. 70 mLH2O and 65 mL ethanol were mixed into a 250 mL round-bottled flask, 360 mg F127 and 500 µL TMB were added to the above solution, and dissolved by stirring for 30 min. Then add 90 mg TRIS solution (9 mL H2O) and 60 mg dopamine hydrochloride solution successively. The mixture was reacted at RT for 18 h. The precipitation was collected by centrifugation at 11000 rpm and 4℃ for 15 min, and then re-suspended with acetone/ethanol (1:2, v/v) and ultrasonic precipitation for 30 min. The supernatant was centrifuged and repeated three times. The synthesized MPDA nanoparticles was collected by high-speed centrifugation, and the collected precipitation was dried in an oven at 37 ℃.
MPDA drug loading
2.5 mg MPDA and 2.5 mg polypeptide drug were dispersed in 1 mL H2O and stirred for 24 h. Drugs are gradually enriched in MPDA mesoporous by π-π-accumulation and hydrophilic hydrophobic interaction. The drug-bearing peptide-MPDA nanoparticles were collected by centrifugation at 11000 rpm at 4 ℃. The collected MPDA NPs were then characterized by transmission electron microscope (TEM) and scanning electron microscopy (SEM).
Particle size
1 mg peptide-MPDA nanoparticles were dispersed in 5mL H2O, and the liquid was transferred to a colorimetric dish. The particle size was measured by Marvin particle size analyzer.
Drug release
The 2 mg drug-loaded MPDA NPs were weighed and dispersed in 2 mL of release medium (PBS containing 0.5% Tween 80) with different pH (5.5, 7.4), then bathed in water at 37 ℃ and centrifuged (5000 rpm,5 min) at different time points to obtain 1 mL of liquid supernatant. The liquid was filtered for HPLC analysis and immediately supplemented with 1mL of fresh media. The release of the drug was calculated according to the standard curve.
Hydrogel
Methacrylic anhydride gelatin (GelMA) is a photosensitive biohydrogel material prepared from methacrylic anhydride (MA) and gelatin (Gelatin). The material has excellent biocompatibility and can be stimulated by ultraviolet or visible light to form a three-dimensional structure suitable for cell growth and differentiation with a certain strength. A LAP initiator is added to the hydrogel and then excited with 405 nm blue light with a curing time of 15 s. The concentration of the hydrogel is 10% and the substitution rate is 60%.
Cell viability assay
The cell viability of VSMC with the BY1 treatments was determined using CCK8 (MCE, HY-K0301). About 5×103 cells per well were seeded into 96-well plates, and then treated with various concentrations of BY1 with or without inhibitor or inducer for 24 h. After treatment, 100 mL per well DMEM including 10 percentage CCK-8 was added in wells and incubated at 37 ℃ for 1 h and then measured at 450 nm wavelength.
Real-time quantitative PCR (qRT-PCR)
About 3×105 cells were added in 60 mm dishes and given different treatments. RNA was extracted (Trizol, Ambion) and reversed (Vazyme Biotech, Cat.: R323-01) to cDNA. RNA extracted from the treated cells was prepared for RNA-seq experiments, as described previously. qPCR reactions were performed (Vazyme, Biotech, Cat. Q711) and the program of qPCR was followed by instruction.
Mitochondria mass assessment
The mitochondria mass of MVSMC cells were measured by Mito-Tracker Green (Beyotime Biotechnology, Cat.: C1048). And fluorescence intensity and quantity reflect the number and mass of mitochondria. The cells were added in 6-well plates and given different treatments. After 24 h, cells were dyed with 100 nmol/L Mito-Tracker Green for 1 h. The results were analyzed by a positive fluorescence microscope.
MDA
Samples were collected 24 h after SAH and LPO was measured based on the MDA content using an MDA assay kit (Nanjing Jian Cheng, A0003-1) according to the manufacturer’s instructions. The relative concentration of MDA in cell after treated 24 h was assessed with a Lipid Peroxidation (MDA) Assay Kit (ab118970; Abcam) according to the manufacturer’s instructions. This assay measures the reaction of MDA with thiobarbituric acid (TBA), which generates an MDA-TBA adduct. The MDA-TBA adduct can be quantified colorimetrically (OD = 532 nm).
Measurement/Imaging of Lipid ROS
MVSMCs were plated in 24-well plates at a density of 4–5×104 cells/well. After treatment, cells were washed twice with 1×PBS and then stained with DCFH-DA dye (diluted with serum-free culture at 1:1000 to a final concentration of 10 µmol/L) or BODIPY-C11 dye (50 µmol/L) for 20 min at 37°C. Following staining, cells were washed with PBS, trypsinized, and collected in 1 ml PBS. Then detected by Flow cytometry or Fluorescence microscopy.
Localization of Intracellular Fe2+
Cells were seeded in glass bottom 96-well plates and grew overnight. After treated for 24 h, cells were stained or co-stained with FerroOrange (1 µM) and LysoTracker Green (10 nM) at 37 ℃for 30 min. Digital images were recorded using laser scanning confocal microscope.
Glutathione Measurements
MVSMC cells were seeded into 12-well plates at a density of 5×104 cells/well. After treated as indicated for 24 h, cells were collected and prepared for measurement of Reduced Glutathione (GSH) using the assay kit (Beyotime) according to the manufacturer's instructions. Three independent biological replicates were performed for the experiments.
Western blotting analysis
About 6×105 cells were added in 100 mm dishes. After attachment, the cells were given different treatments for 24 h. cells were lysed in RIPA (Beyotime Biotechnology, Cat.: P0013B) for 30 min on ice. After centrifuged at 8,000 rpm for 10 min, the protein concentrations of supernatant was collected and quantified by the BCA Protein Assay Kit (Beyotime Biotechnology, Cat.: P0010). Equal amounts (30 mg) of total protein were resolved in 10–12% SDS-PAGE and then transferred to PVDF membranes (Milli-pore, Cat.: ISEQ00010). Membranes with proteins were conducted to blocking, washing, incubation with antibodies, and finally detection with enhanced chemiluminescence.
H&E Staining
Vascular tissues were fixed in 4% paraformaldehyde for 24–48 h, embedded in paraffin, and serially sectioned, deparaffinized, and hydrated. The sections were stained with Hematoxylin and Eosin (H&E) for routine histological examination with a light microscope.
Perls and Masson trichrome staining
Vascular tissues were fixed in paraformaldehyde, embedded in paraffin, sectioned at 4 µm, and stained with Perls (G1424, Solarbio, Beijing, China) and Masson trichrome(G1343, Solarbio) according to the manufacturer’s instructions for evaluation of fibrosis and the extent of extracellular matrix or Fe3+ localization. The stained sections were examined microscopically and multiple fields were evaluated.
Immunofluorescence
Vascular tissues sections (4 µm) from mouse or rats were incubated with the following primary antibodies overnight at 4°C for immunofluorescence staining using the methods described in our previous study: anti-FTH1 (1:50), anti-NCOA4 (1:50), anti-LC3B (1:100) and anti-GPX4 (1:100). After washing with PBST, sections were incubated with the following fluorescent secondary antibodies at 37°C for 2 h: Alexa Fluor 555 (1:50, Abcam, ab150078) and Alexa Fluor 488 (1:50, Abcam, ab150113). Digital images were recorded using laser scanning confocal microscope.
Transfection
Small interfering (si)-FTH1 and a negative control siRNA (si-NC) were synthesized by Sangon Biotechnology (Beijing, China). The pcDNA3.1-FTH1 overexpression plasmid, and a negative control overexpression plasmid (pcDNA3.1) were synthesized by GenePharma Biotechnology (Shanghai,China). VSMCs at 60–70% confluency was transfected with 100 nM siRNA or 2 ug of plasmids in the presence of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham,MA, USA) in 6-well plates according to the manufacturer’s instructions.
Immunohistochemistry
Vascular tissues were cut into 4 µm thick slices. Deparaffinization and rehydration of tissue slices were done using a graduated alcohol series. Antigen repair was achieved by microwave treatment of the sections in 10 mM sodium citrate (pH 6.0) for 3 min. The slices were then treated for 10 min at room temperature with 3% H2O2 to inhibit endogenous peroxidase activity. Next, the slices were incubated with rabbit monoclonal antibody against FTH1 (1:100, CST, #4393) and rabbit polyclonal antibody against FPN (1:100, Abcam, ab78066) overnight at 4 ℃. Afterward, RT peroxidase was treated with horseradish coupled secondary antibody for 30 min. Finally, the immune complexes were visualized using diaminobenzidine. These images were taken on an OLYMPUS microscope. The intensity of positive staining in each sample was measured using Image J software. Briefly, three random fields from three different experiments were quantified per sample.
Statistical analysis
Unless otherwise stated, three times at least in all studies were performed. All statistics were calculated with GraphPad Prism (v8.3). Data are expressed as the mean ± SD. Differences between groups were determined by Student’s t tests or one-way analysis of variance (ANOVA). P values of < 0.05 were considered statistically significant.