All the experiments were approved by the Animal Care Committee of Harbin Medical University (Heilongjiang, China).
HRS production
Hydrogen-rich saline was prepared as previous description [9]. Briefly, H2 was dissolved in 0.9% saline for 6 h under high pressure (0.4 MPa) to a supersaturated level using a HRS-producing apparatus. The saturated HRS was stored at 4 °C in an aluminum bag without dead volume and sterilized by gamma radiation. To confirm the concentration of hydrogen in the saline, gas chromatography was performed. HRS was freshly prepared every week to ensure a concentration of more than 0.6 mmol/L.
Cardiac hypertrophy model and animal grouping
Male Sprague–Dawley rats, weight 200–220 g, were provided from the Experimental Animal Center of The First Affiliated Hospital of Harbin Medical University (Harbin, China). Rats were housed with free access to food and water under a natural day/night cycle. Rats were acclimated for 7 days before any experimental procedures. All rats were cared according to Laboratory Animal Administration Rules (China, 2013).
Sixty rats were randomly divided into 4 groups: 1:sham-operated (sham); 2: AAC-model;3:AAC + Low HRS of 3 ml/kg(LHRS); and 4:AAC + High HRS of 6 ml/kg (HHRS) groups with 15 rats for each group. HRS was injected into the peritoneal cavityonce a day after surgery. The model of cardiac hypertrophy was established with the constriction of abdominal aorta as described previously [10]. Briefly, after the rat was anesthetized, and placed in supine position, a 2-cm incision along the midline of the abdomen was made. After the abdominal aorta was identified, an 8-cm length of 4–0 silk suture was passed underneath the abdominal aorta between the origins of the right and left renal arteries, and a knot was made around the aorta and a 22G needle, and then the needle was removed immediately to achieve a 0.7-mm diameter constriction. Sham-operated rats received a same surgical procedures without constriction performed.
At 6-week after AAC and HRS treatments, rats were weighed and then killed with a quick decollation. The hearts were soon removed, and rinsed in ice-cold PBS. The body weight, heart weight and the left ventricular weight were determined.
Histological analysis
The left ventricles were dissected and immersed in 4% paraformaldehyde overnight. Then the left ventricles were paraffin-embedded, and sectioned at a thickness of 4 μm. Masson staining were performed for light microscopy. The collagen volume fraction (CVF) was calculated as collagen area /total tissue area in a field.
Western blot analysis
The left ventricles were harvested and Western blot analysis was carried out as described previously [11], total protein concentration was determined by Bradford reagent (Bio-Rad), and the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in left ventricles were determined with western blot. Briefly, about 50 μg of sample was separated by electrophoresis on a 4–12% SDS-polyacrylamide NuPAGE gradient gel (Invitrogen) and transferred to a PVDF membrane followed by probing with primary antibodies and followed by secondary antibodies. The protein bands were visualized using chemiluminescent substrates (Pierce). ANP and BNP antibodies (1/1000, respectively) were from Millipore (Temecula, CA) and antibodies of IL-6,JAK, STAT3 and p-STAT3were from Abcam (Cambridge, MA). Densitometry analysis of the bands was conducted with Scion Image Beta 4.02 software. The quantitative measurements of IL-6, JAK and STAT3 expression in the heart tissue were assessed with commercial ELISA kits following the manufacturer’s instructions. Absorbance was read on a microplate reader, and the concentrations were determined based on its standard curve. Native proteins from the left ventricles were isolated using Total Protein Extraction Kit (Biochain Institute, Inc.).
Apoptosis analysis
The apoptotic cells were detected byterminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay (Promega) according to manufacturer’s instructions. The apoptotic index’s quantification was conducted at 400 X. In all groups, 20 fields were randomly picked and the apoptotic index of each field was estimated as the percent of TUNEL-positive cells.
Statistics analysis
The data are expressed as mean ± S.D. Statistical analysis was performed with SPSS 11.5 (SPSS Inc., Chicago, IL, USA). Statistical comparisons were conducted through one-way analysis of variance with post hoc test of Student–Newman–Keuls. AP value of less than 0.05 was considered statistically significant.