2.1. Isolation and extraction compositions from Citrus wilsonii Tanaka fruit
Citrus wilsonii Tanaka fruits were collected from farms in Nanzheng District of Shaanxi Province with the permission of the owner. The fruit samples were pretreated into powders as previously described [8]. Briefly, ten grams of the powders were macerated and extracted in 1000 mL distilled water for 1 h at 95°C. The extracted supernatants were collected after centrifugation at 3000 g for 20 min at 4°C. Then the yielded pellets were re-extracted twice following the same procedure. Supernatants were then combined, freeze-dried, and resulted in the aqueous extract of Citrus wilsonii Tanaka fruit (ZQAE). Naringin was recrystallized from ZQAE by water and deposited in ethanol and identified by Mass Spectrum. Briefly, the supernatants of the aqueous extract liquid were standing for overnight at 4°C and deposited precipitate. The precipitate was collected and preliminary purified by ethanol. The filtrate, on standing at 4°C, and deposited crystals; the precipitate was collected and re-dissolved with water, on standing, and deposited crystals, repeated the steps for twice. The recrystallized precipitate was obtained and freeze-dried.
2.2. Analysis of compositions from Citrus wilsonii Tanaka fruit
ZQAE was profiled as described previously [8]. Briefly, ZQAE was dissolved with distilled water to 1mg/mL, filtered with a 0.22 µM membrane, and subjected to high performance liquid chromatography (HPLC) quantification to measure the contents of naringin, hesperidin, neohesperidin, rhoifolin, naringenin and poncirin. The purified naringin identification was carried out by using UPLC-QTOF- MS/MS. The purity was determined using an HPLC method with an external standard.
2.3. Animals, myocardial ischemia/reperfusion model and experimental design
All protocols were approved by the Institutional Animal Care and Use Committee of Wuhan University (Approval Number: 2015 − 0563) and performed following the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, revised 1996). Male Sprague-Dawley rats weighing 200–250 g were purchased from the Animal Experiment Center of Wuhan University (Wuhan, China).
Myocardial I/R model and experimental design. Rats were anesthetized by 45 mg/kg body weight (BW) sodium pentobarbital (i.p., Sigma, St. Louis, USA). Then, animals were ventilated artificially with a volume-controlled rodent respirator, and an electrocardiogram was monitored with a computer-based EP system (LEAD2000B, Jinjiang Ltd., Chengdu, China). The myocardial I/R rat model was established as previously described [12]. Rats were euthanized with 22.5 mg/kg BW sodium pentobarbital (i.p.), and the blood and heart samples were collected for evaluation of myocardial injury and other biochemical assessments.
Three times animal experiments were carried out. (1) Sprague-Dawley rats were randomly assigned to five groups (n = 6/group) as following: Sham-operated control (Sham): rats were pretreated with 0.9% NaCl (solvent for the ZQAE) by intraperitoneal (i.p.) injection for 5 consecutive days before the surgical manipulation without ligation of the left anterior descending coronary artery (LAD). I/R group: rats were treated with 0.9% NaCl (i.p.) for 5 consecutive days, and then subjected to LAD occlusion for 30 min followed by reperfusion for 4 h. Rats were pretreated with ZQAE at 25, 50, or 100 mg/kg BW, by i.p. injection for 5 consecutive days), and then the LAD was occluded for 30 min followed by reperfusion for 4 h. (2) Rats were randomly assigned to three groups (n = 10/group). Doses of ZQAE at 50 mg/kg were pretreatment for 5 days and then the LAD was occluded for 30 min followed by reperfusion for 4 h. (3) Rats were randomly assigned to four groups (n = 10/group). Doses of naringin at 5 mg/kg and diltiazem at 5mg/kg were pretreatment for 5 days and then the LAD was occluded for 30 min followed by reperfusion for 4 h.
2.4. Histopathological examination
Myocardial tissues were fixed in 4% paraformaldehyde (in PBS) and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E). Five fields at 200× magnification were scored in each group to quantify the histological cardiac damage. The scoring rule was described previously [12].
2.5. Assessment of myocardial injury
Serum levels of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) were measured to assess the myocardial injury. Standard techniques were performed using commercial kits for CK-MB and cTnI (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
2.6. Assessment of infarct size
The infarct size was measured using the 2,3,5-triphenyl tetrazolium chloride staining (TTC, Sigma-Aldrich, St. Louis, USA) as previously described [11]. Briefly, after reperfusion, LAD has occluded again, and the rats were injected with 2 ml of 1% Evans blue dye (Sigma-Aldrich, St. Louis, USA) via the femoral vein. The hearts were removed from the body, frozen at -80°C for 15min and then cut (approximately 2 mm) into five slices from the apex to the base. The slices were immersed and incubated in 1% TTC for 20 min at 37°C to separate the infarct area (white) from the risk area (red) and then fixed in 4% paraformaldehyde. The white tissues and red tissues from each section were determined with image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Silver Spring, USA). Five rats per group were used to measure the myocardial infarct size. The infarct size (%) was expressed as: infarct area / (risk + infarct) area × 100%.
2.7. TUNEL assay
Myocardial apoptosis was analyzed by the TUNEL assay using a commercial kit (Roche Applied Science, Indianapolis, USA) according to the manufacturer’s instructions. The apoptosis index was defined by the percentage of apoptotic cell number/total cell number, as calculated using Image-Pro Plus 6.0 software. Notably, brown staining in the nucleus indicated apoptosis.
2.8. Measurement of oxidative stress and inflammatory cytokines in myocardial tissues
Oxidative stress markers, including malondialdehyde (MDA) contents and antioxidant superoxide dismutase (SOD) activities, were determined spectrophotometrically in myocardial tissues, using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was assessed in ischemic areas of the left ventricle by enzyme-linked immunosorbent assays according to manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
2.9. Western blot
Frozen ischemic areas of left ventricle samples were analyzed by Western blot analysis. The antibody against interleukin-23 (IL-23) (1:800) was purchased from Proteintech Group Inc, USA. Anti-total-Caspase3 (1:1000) was purchased from Abcam (UK). Anti-cleaved-Caspase3 (1:1000), anti-p38 (1:1000), and anti-p-p38 (1:1000) were obtained from Cell Signaling Technology, Danvers, USA. Anti-high mobility group box 1 (HMGB1) (1:300) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000) were received from the Boster, Wuhan, China.
2.10. Statistical analysis
SPSS 19.0 was used for data analysis. All data are expressed as mean ± SD. Student’s t-test was used for comparison between two groups. Multiple comparisons among groups were evaluated by one-way ANOVA or a Welch test. Student-Newman-Keuls or Dunnett’s T3 test was used for post hoc multiple comparisons. P value < 0.05 was considered statistically significant.