Hydrogen Sulfide Promotes Angiogenesis in Diabetic Mice

Animals and experimental protocolAll experimental procedure obeyed the Guide for the Care and Use of Laboratory Animals of the Chinese National Institutes of Health. Type 2 diabetic db/db mice were obtained from Changzhou Cavens Labobratory Animal CO LTD (Changzhou, China), and bred in standard facility with 22 ^°C room temperature, and a 12-hour day/night alternate. Animals were assigned to four groups (8 mice per group): ischemia control (I-C), ischemia treatment (I-T), nonischemia control (N-C), and nonischemia treatment (N-T). All mice had access to food and water ad libitum.
Preparation of animal model and treatmentMice were anesthetized via intraperitoneal injection with 10% chloral hydrate (300 mg/Kg). After shaved, left hind limb was made an incision for exposure of the femoral artery. The femoral artery was carefully separated from the femoral nerve and vein, and ligated for preparation of lower limb ischemia model in the mice from the I-C and the I-T, while not ligated in the N-C and the N-T mice. The I-T and the N-T mice were treated with sodium bisulfide (hydrogen sulfide donor) (0.1 ml, 30 mmol·l^-1) by injection of quadriceps and gastrocnemius. A round incision (6 mm in diameter) was prepared on the dorsal skin of the left hind limb. The wounds from the I-T and the N-T mice were treated with 3% sodium bisulfide plaster. The wound closure was acquired with a digital camera.
Determination of VEGF and PDGFSerum levels of VEGF and PDGF were measured with enzyme linked immunosorbent assay (ELISA) kits (Hefei Bomei Biotechnology CO, LTD, China) by colorimetric methods according to manufacturer’s protocol. The optical density (OD) values of each well were recorded, and the standard curve was plotted for determination of VEGF and PDGF contents.
Morphological analysesAt the end of experiment, the wound tissues and adductor muscles were collected, and fixed in 4% formalin. After embedded in paraffin, fixed samples were cut 5-μm-thickness sections. The sections were deparaffinized, and stained with hematoxylin-eosin (HE) for histological examinations. To measure granulation tissue thickness in the wounds, the sections were stained with Massone trichrome.
Analyses of immunohistochemistryDeparaffinized 5-μm-thickness sections were treated with sodium citrate buffer for antigenic retrieval. To inhibit endogenous peroxidase, sections were incubated with 3% hydrogen peroxide in phosphate buffer solution for 15 min, and followed by treatment with phosphate buffer solution containing bovine serum albumin for blockage of nonspecific sites. Then sections were incubated with PDGF, CD34 primary antibody (Santa Cruz Biotechnology, USA) overnight at 4 ^°C. After rinsed with phosphate buffer solution, the sections were treated with biotin-conjugated secondary antibody for 15 min. The sections were washed with phosphate buffer solution, and hatched in streptavidin-horseradish peroxidase. 3, 3’-diaminobenzidine (DAB) was used to detect antigens by visualization.
Real-time polymerase chain reaction (RT-PCR)RT-PCR was used to assess mRNA expressions of VEGF, PDGF, HF-1α, FGF2, and eNOS. Total RNA of adductor muscles was extracted with Trizol Reagent (Invitrogen, USA). Genomic DNA contamination was removed from the RNA with Dnase. RNA concentration was determined via detecting the absorbance at 260 nm wavelength. RNA was utilized to synthetize cDNA by reverse transcriptase kit. PCR primers and probes are listed in Table 1. β-actin mRNA was used to measure the relative expressions of target genes. The relative levels of gene were expressed as 2^-ΔΔCT.
Western blottingAdductor muscles (50 mg) were collected and lysed in precooled lysis buffer (1% Triton-X 100, 50mM HEPES, 10mM Na₃VO₄, 100 mM NaF, 100 mM Na₄P₂O₇, 10 μg/l leupeptin and aprotinin, 2mmol/l phenylmethanesulfonyl fluoride) on ice for 20 min, and subsequently centrifuged at 12,000 g for 15 min at 4 ^°C. Amount of protein in supernatant was determined with a BCA kit (Bio-Rad). Proteins in the supernatant were electrophoretically separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membranes were immersed in 5% nonfat milk containing primary antibodies VEGF, VEGFR, ERK, p-ERK, PDGF, PDGFR, Akt, p-Akt, and β-actin (1 : 500, Abcam, Cambridge, UK) overnight at 4 ^°C, respectively. After rinsed with PBS, membranes were incubated with a peroxidase-conjugated secondary antibody (1:10000, Sigma, USA) for 2 hr. Target proteins were determined with visualization by DAB. β-actin as an internal control protein was used in the experiment.
Statistical The data were presented as mean ± standard deviation (SD). Statistical differences between two groups were analyzed with one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc analysis. GraphPad Prism (Version 7) was used for statistical analyses. A value of P<0.05 was considered to be statistically significant.