2.1.Experimental animals and treatments
All adult male Sprague-Dawley (SD) rats (weighing 180-200 g) were acquired from the Animal Center of the Renmin Hospital of Wuhan University. All animal care and experimental procedures were approved by the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (revised 2011) and in compliance with the Animal Care and Use Committee of Renmin Hospital of Wuhan University. All rats were maintained under specific pathogen-free (humidity 50 ± 5%; temperature 20-22 °C) with a 12-hour light/dark cycle.
Rats were subjected to either a ligation of left anterior descending branch to make MI model or a sham operation according with previous method (Yuan et al., 2016a). The rats were randomly divided into three groups: a sham operation group (sham), a MI group (MI+vehicle) and a garcinoic acid treatment (GA)+MI group (GA+MI). We administered GA (1 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) or vehicle (PBS+DMSO) by intraperitoneal (i.p.) injection weekly for 2 weeks (Wallert et al., 2019). All the specimens were collected from the peri-infarcted areas (< 2 mm outside the infarct).
2.2.Echocardiography
Echocardiography was conducted 2 weeks after MI. According to previous study, after anesthetized with 1.5-2% isoflurane, transthoracic echocardiography was performed by a Mylab30CV ultrasound (Esaote SpA, Genoa, Italy) using a 15-MHz imaging transducer (Yuan et al., 2016b). Then, the parameters of cardiac function including left ventricle (LV) end-diastolic diameter (LVEDD), ejection fraction (EF), fractional shortening (FS) were collected.
2.3. Cell culture and treatments
Human umbilical endothelial cells (HUVECs; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were used for all in vitro experiments. HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS,GIBCO,USA) / 1% penicillin-streptomycin (100 U/mL) in a cell incubator with humidified 95% O2 and 5% CO2 atmosphere at 37°C for normoxia condition. Hypoxia condition was performed with hypoxic gas mixture (2% O2, 5% CO2 and 93% N2) from cell incubator. Before hypoxia, HUVECs were pretreated in different concentrations of GA (1μM, 2.5μM and 5μM) or vehicle for 24 hours.
2.4.Cell viability
The cell viability was evaluated by the Cell Counting kit (CCK-8) assay (Dojindo, GB707, Japan). The cells were treated with various concentrations of GA and vascular endothelial growth factor (VEGF) and/or hypoxia and incubated with 10 μL CCK8 solution on a 96-well microplate reader for 2 hours,.The absorbance was detected at 450 nm wavelength by an ELISA reader.
2.5.Tube formation assays
After allowing the BD MatrigelTM Basement Membrane Matrix to settle for 30 min in a 37℃, 5% CO2 incubator, HUVECs from different groups were seeded on the layer of polymerized gel on a 96-well plate, hypoxia+VEGF was used as a positive control. 12 hours after incubation, the formation of a capillary-like structure was recorded using microscope and images taken by light microscopy (ECLIPSE 80i; Nikon, Japan). Tube formation was quantified by using image-analysis software (Image J, version 1.40g, NIH).
2.6.Enzyme linked-immunosorbent assay (ELISA)
Plasma creatinine kinase-MB (CK-MB; Jiancheng Bioengineering Institute, Nanjing, China) and cardiac troponin I (cTnI; Life Diagnostic, Pennsylvania, USA) from jugular vein blood at various time points (0, 7 and 14day) were determined with ELISA detection kits as per manufacturer’s instructions. After treatment for 2 weeks, the serum was collected to detect the concentration of BNP with a BNP ELISA detection kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.
2.7.Masson’s Trichrome
Maximum section of heart was fixed in 4% formaldehyde for 24h, after which it was embedded in paraffin, and cut into 5 µm thick slices. The slices were stained with Masson trichrome staining for evaluation of infarcted size in the heart. The fibrosis areas were observed using a light microscope (Olympus D72, Japan) .The infarcted size was calculated as the percentage of the positively stained area of the total myocardial tissue, and then analyzed by using Image J software.
2.8.Immunofluorescence analysis
The dissected hearts in the peri-infarcteded areas were fixed in 4% phosphate-buffered formalin, and then the samples were embedded in paraffin to cut into 5 μm sections. To evaluate vessel density and proliferation, alpha smooth muscle actin (α-SMA)/CD31 and ki67/CD31 co-immunostaining was performed. After blocking with 10% goat serum, the sections were incubated in α-SMA, ki67 and CD31 primary antibody (Abcam, Cambridge, UK) overnight and subsequently incubated with Alexa-Fluor-coupled secondary antibodies, then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). The images were obtained by using a fluorescence microscope (OLYMPUS, Tokyo, Japan) and then analyzed by using Image J software.
2.9.Western blot
Total proteins from rat hearts or cultured HUVECs were extracted by use of RIPA lysis buffer supplemented with protease/phosphatase inhibitor cocktail (CST, Danvers, MA, USA). Equal amount (50 μg) of protein lysate was subjected to 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated overnight with the following primary antibody: HIF-1α (sc-13515, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), VEGF-A (ab46154, Abcam), basic fibroblast growth factor (bFGF; NB600-1536, Novus Biologicals, CO, USA),phosphorylated VEGF receptor 2 (p-VEGFR2) (SAB4504567, Sigma), VEGFR2 (SAB4501642, Sigma), Ser473 -p-Akt (#4060, CST), p-Akt (#9272, CST), Ser1177- p-eNOS (ab138458, Abcam), eNOS (ab199956, Abcam), GAPDH (ab37168, Abcam). Next, the membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. Proteins were treated with an ECL detection kit and then visualized using chemiluminescence system. The specific protein expression levels were normalized to GAPDH.
2.10.Statistical analysis
The data analyses were performed with the GraphPad Prism 6.0. All data were expressed as mean ± SEM. Comparisons for multiple-group were analyzed using ANOVA with the Tukey’s test. A value of P< 0.05 was deemed statistically significant.