Materials
Caffeic acid (CA, 99%), ellagic acid (EA, 99.5%) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All chemicals used in measurements were of the highest purity commercially available.
Animals
Male Balb/c mice, 3–4 wk old, were obtained from National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light:dark schedule; water and mouse standard diet were consumed ad libitum. The use of mice was reviewed and approved by China Medical University animal care committee (CMU-97-22-N). To induce diabetes, mice with body weights of 22.9 ± 0.8 g were treated with a single i.v. dose (50 mg/kg) of streptozotocin dissolved in citrate buffer (pH 4.5) into the tail vein of 12-h fasted mice. The blood glucose level was monitored on d 5 and 10 from the tail vein using a one-touch blood glucose meter (Lifescan, Inc. Milpitas, CA, USA). Mice with fasting blood glucose levels ≥ 14.0 mmol/l were used for this study.
Experimental design
CA or EA at 2 g was mixed with 98 g power diet containing (g/100 g): 64 starch, 23 protein, 3.5 fat, 5 fiber, 1 vitamin mixture and 3 salt mixture (PMI Nutrition International LLC, Brentwood, MO, USA). After diabetes was induced, mice were divided into three groups (15 mice per group): diabetic mice with normal diet, diabetic mice with 2% CA treatment, and diabetic mice with 2% EA treatment. One group of non-diabetic mice with normal diet was used for comparison. All mice had free access to food and water at all times. Body weight, consumed water volume and food were recorded. After 12 wk supplementation, mice were sacrificed with carbon dioxide. Blood was collected, and plasma was separated from erythrocytes immediately. Cardiac tissue was removed and perfused for 2 min by phosphate buffer saline (PBS, pH 7.2) to remove the remaining blood. Cardiac tissue at 0.2 g was homogenized on ice in 2 ml PBS, and the filtrate was collected. The protein concentration of plasma or cardiac tissue filtrate was determined by the method of Lowry et al. [15] using bovine serum albumin as a standard. In all experiments, the sample was diluted to a final concentration of 1 g protein/l using PBS, pH 7.2.
Content of CA or EA in cardiac tissue
An HPLC method described in Yamada et al. [12] was used to analyze the cardiac content of intact form of CA or EA, in which an octadecylsilica column (4.6 × 250 mm, Wakopak, Wako Pure Chemical Industry, Tokyo, Japan), and a mobile phase consisting of 95.6% H2O, 4.1% ethyl acetate and 0.3% acetic acid were used at 30°C with a flow rate of 0.8 ml/min.
Assay of glucose, insulin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities
Plasma glucose level (mmol/l) was measured by a glucose kit (Sigma Chemical Co., St. Louis, MO, USA). Plasma insulin level (nmol/l) was measured by using a rat insulin radioimmunoassay kit (SRI-13K, Linco Research Inc., St. Charles, MO, USA). For glucose and insulin, the intra coefficients of variation (CVs) were 7.1 and 5.4%; and the inter CVs were 6.3 and 5.8%. Serum activity of ALT and AST were determined by using commercial assay kits (Randox Laboratories Ltd., Crumlin, UK), their intra and inter CVs were in the range of 2.5–4.6%.
Lipid analyses
Triglyceride (TG) and total cholesterol (TC) levels (g/l) in plasma were determined by triglycerides/GB kit and cholesterol/HP kit (Boehringer Mannheim, Germany), respectively. For TG and TC, their intra CVs were 4.3 and 5.0%; and their inter CVs were 3.7 and 4.8%. Total lipids were extracted from cardiac tissue, TG concentration (mg/g wet tissue) was quantified by a colorimetric assay [16], and total cholesterol (mg/g wet tissue) was measured using o-phthalaldehyde [17].
Measurement of blood coagulation and anticoagulation factors
Blood samples were anticoagulated using sodium citrate according to the protocols provided by the manufacturers of the kits used. Plasma fibrinogen level (g/l) was measured based on the principle of salting out using a commercial kit (Iatroset Fbg, Iatron Laboratory, Tokyo, Japan). Plasminogen activator inhibitor-1 (PAI-1) activity (kU/l) was assayed by a commercial kit (Trinity Biotech plc, Co. Wicklow, Ireland). For fibrinogen and PAI-1, the intra and inter CVs ranged from 5.4 to 6.7%. The activity (%) of AT-III and protein C in plasma was determined by chromogenic assays according to the manufacturer'sinstruction s using commercial AT-III and protein C kits (Sigma Chemical Co., St. Louis, MO, USA), and was shown as ratio of those in normal human plasma. For AT-III and protein C, the intra CVs were 3.4 and 4.7%; and the inter CVs were 6.0 and 7.2%.
Determination of oxidative and anti-oxidative status
Glutathione (GSH) and oxidized glutathione (GSSG) concentrations (nmol/mg protein) in cardiac tissue were determined by commercial colorimetric GSH and GSSG assay kits (OxisResearch, Portland, OR, USA). For GSH and GSSG, the intra CVs were 5.0 and 5.8%; and the inter CVs were 4.2 and 6.0%. Glutathione peroxidase (GPX), catalase and superoxide dismutase (SOD) activities (U/mg protein) in cardiac tissue were determined by commercial assay kits (Calbiochem Inc., San Diego, CA, USA). Their intra CVs were in the range of 6.3–7.5%; and inter CVs were in the range of 5.9–7.8%. Lipid oxidation in cardiac tissue was determined by measuring the level of malondialdehyde (MDA, μmol/mg protein) via an HPLC method [18]. The method described in Privratsky et al. [19] was used to determine the amount of ROS in cardiac tissue. Briefly, 10 mg cardiac tissue was homogenized in 1 ml of ice cold 40 mM Tris-HCl buffer (pH 7.4), and further diluted to 0.25% with the same buffer. Then, samples were loaded with 10 μmol/l 2', 7'-dichlorofluorescin at 37°C for 30 min. After rinsing, and the fluorescence intensity was measured using a fluorescent microplate reader with excitation wavelength at 480 nm and emission wavelength at 530 nm. Untreated samples were used to determine background fluorescence.
Cardiac inflammatory factors analyses
Cardiac tissue was homogenized in 10 mM Tris-HCl buffered solution (pH 7.4) containing
2 M NaCl, 1 mM ethylenediaminetetraacetic acid, 0.01% Tween 80, 1 mM phenylmethylsulfonyl fluoride, and centrifuged at 9000 × g for 30 min at 4°C. The resultant supernatant was used for cytokine determination. The levels of IL-1beta, IL-6, TNF-alpha, IL-4, IL-10 and MCP-1 were measured by ELISA using cytoscreen immunoassay kits (BioSource International, Camarillo, CA, USA). Samples were assayed in duplicates according to manufacturer's instructions. The sensitivity of the assay, i.e., the lower limit of detection, was 5 nmol/l for IL-1beta, IL-4, IL-6, IL-10 and 10 nmol/l for TNF-alpha and MCP-1.
Real-time polymerase chain reaction (RT-PCR) for mRNA expression
RT-PCR was used to examine the cardiac mRNA expression of catalase, GPX1, SOD, IL-1beta, IL-6, TNF-alpha and MCP-1. Cardiac tissue was homogenized in guanidinethiocyanate, and RNA was extracted using TRIizol reagent and further digested with DNase. Two μg of total RNA was used to generate cDNA. Reverse transcription was performed in a one-step protocol using the iScript cDNA Synthesis Kit (Bio-Rad Co., Hercules, CA, USA) according to the manufacturer's instructions. The primers were as follows. Catalase: forward, 5'-TTC AGA AGA AAG CGG TCA AGA AT-3', reverse, 5'-GAT GCG GGC CCC ATA GTC-3'; GPX1: forward, 5'-CCC CAC TGC GCT CATGA-3', reverse, 5'-GGC ACA CCG GAG ACC AAA-3'; Cu-Zn SOD: forward, 5'-TGG GTT CCA CGT CCA TCA GTA-3', reverse,5'-ACC GTC CTT TCC AGC AGT CA-3'; IL-1beta: forward,5'-TGT GGC TGT GGA GAA GCT GT-3', reverse, 5'-CAG CTC ATA TGG GTC CGA GA-3'; IL-6: forward, 5'-CAC GGC CTT CCC TAC TTC AC-3', reverse, TGC AAG TGC ATC ATC GTT GT-3'; TNF-alpha: forward, 5'-ACT CAA CAA ACT GCC CTT CTG AG-3', reverse, 5'-TTA CAG CTG GTT TCG ATC CAT TT-3'; MCP-1: forward, 5'-CAG GTC CCT GTC ATG CTT CT-3', reverse, 5'-CAC TGT CAC ACT GGT CAC T-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 5'-TGA TGA CAT CAA GAA GGT GGT GAA G-3', reverse,5'-CCT TGG AGG CCATGT AGG CCA T-3'. The target concentration was expressed relative to the concentration of a reference housekeeping gene, GAPDH. PCR was conducted using the following parameters: 50°C for 2 min, 95°C for 10 min and 40 cycles at 94°C for 20 s and 60°C for 1 min. Generated fluorescence from each cycle was quantitatively analyzed by using the Taqman system based on real-time sequence detection system (ABI Prism 7700; Perkin-Elmer Inc., Foster City, CA, USA). In this present study, the mRNA level of the control group (without diabetes and with normal diet) was defined as 100%; then, mRNA level of other groups were calculated as percentage of the control group.
Statistical analysis
All data were expressed as mean ± standard deviation (SD). A statistical software package, SAS program (Version 5.1), was used to perform statistical analysis. One-way analysis of variance (ANOVA), followed by Dunnett's t-test was used to assess the significance of any change between groups. Statistical significance is defined as p < 0.05.
