The build of diabetes combined SCI rat model
8-week-old female Sprague Dawley (SD) rats (weight: 0.2–0.25 kg) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All experimental protocols and procedures were approved by Animal Care and Use Committee of Wenzhou Medical University, and followed in accordance with the ethical guidelines on animal experimentation of Laboratory Animals of China National Institutes of Health. The rats were maintained under a 12 hours (h) light/12 h dark condition and had free access to water and food. The SD rats were randomly divided into the Sham group, T1D group, SCI group, and diabetes + SCI (DS) group. T1D rat model was induced by intraperitoneal injection of STZ (55 mg/kg). Then, the random blood glucose of rats were monitored every day from 72h after STZ injection. After 2 weeks, the random blood glucose ≥ 16.7 mmol/L was determined as the T1D model. 5% isoflurane was anesthetized via air anesthesia machine, then the rats were pried open the spinal plate of T9-T10 segment, exposed the spinal cord, and sutured the muscles and skin without any treatment in Sham group. For SCI and DS group, the T9-T10 segment in spinal cord of rats were hit with Allnes beater. It can be seen that the lower limbs of rats will twitch, tail swing, hyperemia and swelling at the hit place, which is regarded as the successful SCI modeling. these rats did not receive any drug treatment. The rats in DS + F group were intraperitoneally injected with Ferrostatin-1(Fer-1, 1 mg/kg/d, MCE) from the day before SCI. After treatment, the rats were euthanized at the indicated time points, the tissue and blood sample were collected for further study (Fig. 1a).
Locomotor function assessment
The electrophysiology and footprints were performed to evaluate the locomotor function of hindlimb of rats at 14th day after SCI. During electrophysiology assessment, the electrical stimulation was given to the C2-C3 segment of spinal cord in the rat. The electrical fluctuation received by the gastrocnemius muscle of the hind limb was detected through the injured or uninjured T9-T10 segment, and the incubation period and amplitude were obtained (NeuroExam M-800A, MEDCOM, China). For footprints test, the rats' forelimbs were immersed in blue ink and the hind limbs were immersed in red ink, then the rats crawled on white paper to obtain the footprints of hind limbs in each group.
Tissue preparation
The rats were deeply anesthetized, and ventricular perfusion was performed with normal saline. A total of 8 mm spinal cord tissue from the injured area was taken and frozen to -80℃ immediately for subsequent western blot analysis. For transmission electron microscope (TEM), 1–2 mm3 spinal cord tissues from the injury center of each group were soaked in 2.5% glutaraldehyde for 4℃ overnight. For morphological and immunofluorescence staining, after continuous perfusion with 4% PFA, the 1 cm spinal cord tissues were taken from the injured area and fixed in 4% PFA for 24 h.
Hematoxylin &eosin(H&E) staining, Nissl staining and Prussian blue staining
After fixed in 4% PFA, the tissues were dehydrated, waxed and embed in paraffin. Then, the tissues were cut into 5 µm sections for staining. Before staining, the tissue sections were dewaxed and hydrated, and stained with H&E staining kit (G1120, Solarbio), Nissl staining solution (C0117, Beyotime), and Prussian Blue Staining Kit (G1424, Solarbio) respectively. Prussian Blue Staining Kit was used to assess the content of iron in spinal cord tissue. Finally, the images were observed under microscope (Nikon, Japan).
BSCB permeability assessment
Evans blue (EB) dye was used to evaluate the permeability of BSCB. At 1 day after SCI, the rats were injected with 2% EB solution (4 ml/kg) through tail vein. After 2 h, the eyes and ears of rats turned blue. Then, the rats were anaesthetized and performed ventricular perfusion with normal saline and 4% PFA. After that, the spinal cord tissues were collected and fixed in 4% PFA for 24 h, and embeded with OCT. Then, it was cut into 10 µm frozen sections and captured the Evans blue fluorescence in spinal cord tissue under Nikon ECLIPSE 80i microscope.
Cell culture and treatment
Human umbilical vein endothelial cells (HUVECs) were purchased from the cell storage center of Wuhan University (Wuhan, China). HUVECs were cultured in 4.5 g/L DMEM medium at 37℃, 5% CO2 and 95% air, and supplemented with 10% fetal bovine serum (FBS), 100 U/ml streptomycin and 100 U/ml penicillin. The cells were co-cultured with glucose (final glucose concentration 100 mmol/L) and palmitic acid (PA, 0.1 mmol/L) for 24 h to simulate the environment of high glucose and lipid, and then stimulated with 100 µmol/L H2O2 for 1.5 h to mimic the oxidative damage for ECs after SCI. In order to explore the effect of ferroptosis on high glucose-induced ECs injury, the cells were treated with H2O2 (100 µmol/L) in the presence or absence of Fer-1 (8 µmol/L) for 1.5 h (Fig. S1a). Thus, the cells were divided into Control (Ctrl) group, HG group, H2O2 group, HG + H2O2 group and HG + H2O2 + Fer-1 group.
Western blotting analysis
The cell samples and spinal cord tissues were lysed by protein extraction reagents, and quantified by BCA assay. The protein samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane. After blocking with 5% milk for 1 h, the membranes were incubated with following primary antibodies at 4℃ overnight: xCT(1:1000, Ab175186, Abcam), GPx4(1:1000, Ab125066, Abcam), ACSL4(1:1000, Ab155282, Abcam), 4-HNE(1:500, MAB3249, R&D), COX2(1:1000, 66351-1-Ig, Proteintech), Nrf2(1:1000, 16396-1-AP, Proteintech), HO-1(1:1000, 10701-1-AP, Proteintech), ZO-1(1:1000, AF5145, Affinity), Occludin(1:1000, DF7504, Affinity), β-catenin(1:1000, Ab32572, Abcam), p120(1:1000, 14599-1-AP, Proteintech), Claudin5(1:1000, 34-1600, ThermoFisher) and β-actin(1:1000, 66009-1-Ig, Proteintech). After washing with TBST (TBS with 0.05% tween 20) for 3 times, the membranes were respectively incubated with secondary antibody (1:10000, Liankebio) at room temperature for 1 h. The images were captured by ChemiDicTM XRS+ imaging system (Bio-Rad) with an enhanced chemiluminescence (ECL) kit. Finally, the results were quantitatively analyzed by Image Lab 3.0 software followed by statistical analysis.
RNA sequencing analysis
Whole-transcriptome sequencing (Shanghai OE Biotech CO., LTD) was performed on spinal cord of rats in Sham group, SCI group and DS group, with four independent samples in each group. The spinal cord tissues of rats were taken out at 3 days after SCI and immediately placed at -20℃. After extracting total RNA, the cDNA samples were sequenced using Illumina NovaSeq 6000 system. Through quantification and standardization among samples, GO enrichment analysis and Hierarchical clustering analysis of diabetes related to anti-oxidative stress and ferroptosis gene based on the RNA sequencing analysis were conducted for all differentially expressed genes among sample groups.
Immunofluorescence staining
After dewaxing and hydration, the 5 µm thick sections of spinal cord were incubated with sodium citrate for high-pressure antigen repair, then incubated in 3% H2O2 for 15 minutes(min) at room temperature. Next, the sections were incubated with 5% bovine serum albumin (BSA) for 30 min. The cell climbing tablets with HUVECs were fixed with 4% PFA at 4℃ for 20 min. Then, the sections were incubated with the following primary antibodies for 4℃ overnight: GPx4(1:300, Ab125066, Abcam), RAGE(1:400, Ab216329, Abcam), CD31(1:50, AF3628, R&D) and 4-HNE(1:200, MAB3249, R&D). After that, the sections were incubated with Alexa fluor 488/647 labeled secondary antibody(1:1000, Abcam) in a 37℃ oven for 1 h. Finally, the nuclei were stained with DAPI, and the images were captured under a confocal laser microscope (Nikon, Japan).
Dihydroethidium (DHE) staining
After dewaxing, hydration and high-pressure antigen repair, the sections of spinal cord were incubated with 10 µmol/L dihydroethidium (S0063, Beyotime) solution at room temperature in dark for 10 min. After washing with PBS, the nuclei were stained with DAPI. Then, the images were observed under confocal laser microscope.
Transmission electron microscope (TEM) analysis
The spinal cord tissues of rats were soaked in 2.5% glutaraldehyde, then rinsed and fixed with 1% osmic acid for 2 h. After washing, the tissues were immersed in 1% uranium acetate for 2 h. Then, it was dehydrated by acetone gradient and soaked overnight, lastly embedded and polymerized in pure embedding agent. The ultrathin sections were stained with uranyl acetate for 20 min and lead citrate for 10 min. Finally, the mitochondria were observed and captured under the transmission electron microscope (JEOL, JEM-1200EX, Japan).
Detection of iron, MDA and total GSH in serum
After anaesthetized, the serum of rats was collected for the further detection. According to manufacturer's protocol, the contents of iron, MDA and GSH in serum were determined with the serum iron detection kit (A039-1-1, Nanjing Jiancheng), Lipid oxidation (MDA) detection kit (S0131S, Beyotime) and total glutathione detection kit (S0052, Beyotime) respectively.
Detection of amino acids content in spinal cord
The amino acids content in spinal cord were detected in Qingdao Sci-tech Innovation Quality Testing Co. Ltd. In brief, 0.2 g spinal cord tissues of rats were put into a hydrolysis tube, and add 20 mL HCl, and then hydrolyze it in an electric blast drying oven at 110℃ for 24 h. After cooling, it was transferred into a 25 ml colorimetric tube, take 1ml of liquid from it, dry it in an 85℃ water bath, add 1ml of pure water, and then dry it. Then, it need add 10 ml of 0.02 mol/L HCl, take out 500 µl after mixing, add 0.1 mol/L Phenyl isothiocyanate acetonitrile and 1 mol/L triethylamine acetonitrile, 250 µl respectively, and mix well. After derivatization, it will add 2 ml of N-hexane, shake completely, and stand for 10 min. After layering, the lower layer were take out and filter it with 0.45 µm PTFE membrane. Finally, the sample extracts were analyzed by HPLC (Agilent 1260, USA).
Cell viability assay
HUVECs was inoculated in 96 well plates at the density of 3×103 cells per well and cultured for 24 h. The cells were treated differently according to the experimental design. After washed with PBS, 10 µl CCK-8 solution and 90 µl basic medium were added to each well. After incubated for 2 h, the absorbance of each well at 450 nm was monitored under a microplate reader (MD SpectraMax190, USA).
DCFH-DA staining and MitoSOX staining
For DCFH-DA staining, the treated cells were washed and fixed with 4% PFA for 20 min at 4℃. Next, the treated cells were washed with PBS and incubated with 10 µmol/L DCFH-DA at 37℃ for 20 min (S0033S, Beyotime). Then, the cells were washed with basic medium, and the fluorescence at 525 nm emission wavelength was observed under confocal laser microscope (Nikon, Japan). Using MitoSOX Red mitochondrial superoxide indicator (M36008, ThermoFisher), the cells were incubated with 5 µmol/L of MitoSOX at 37℃ for 10 min. Finally, the nuclei were stained with DAPI and observed under confocal laser microscope.
JC-1 staining
The JC-1 staining was used to evaluate the mitochodrial membrane potential with JC-1 assay kit (C2006, Beyotime). The treated HUVECs were incubated with working solution at 37℃ for 20 min. Then, the HUVECs were washed twice with dyeing buffer and added with basic medium. Lastly, the signaling was immediately captured under confocal laser microscope.
MitoTracker staining
After washed with PBS and fixed with 4% PFA at 37℃ for 15 min, the mitochondria of HUVECs were tracked and stained with MitoTracker™ Deep Red FM Dye (M46753, ThermoFisher) according to the manufacturer's protocol. Then, the HUVECs were incubated with the 100 nmol/L MitoTracker working solution that preheated at 37℃ for 20min, and subsequently labeled the nucleus with DAPI. The signaling was captured under confocal laser microscope (Leica, Germany).
Statistical analysis
All data were expressed as mean ± standard deviation (SD). Each experiment was repeated at least 3 times. Image J and Graphpad prism 8.0 statistical software were used for statistical analysis. The significance of each group was evaluated by one-way analysis of variance (ANOVA), and P < 0.05 indicates that the experimental data is statistically significant.