Elevated expression of periostin in diabetic cardiomyopathy and the effect of valsartan

Induction of diabetes

Male healthy Wister-Albino rats (6-week-old; 180-220 g; n = 60) were purchased from the animal center of Lu-kang corporation. All experimental procedures were performed in accordance with the institutional animal care guidelines. Rats were housed in an air-conditioned room at a constant temperature of 20 ± 2 °C and were fed with a standard diet and water at liberty. Following one week of acclimatization, the rats were assigned randomly to a control group (n = 10) and a diabetic group (n = 50). All rats were weighed, then blood glucose levels were measured initially before the experiment and then weekly once throughout the experiment. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg body weight; Sigma, USA) dissolved in 0.1 mmol/L sodium citrate buffer (pH adjusted to 4.2), whereas rats in control group were injected with 0.1 mmol/L citrate buffer alone. All rats were given the same weight of normal (non-high fat and sugar) diet and the same volume of water. Three weeks after the injection of STZ, blood was collected from tail vein and samples were analyzed for blood glucose using a glucometer. Rats with random fasting blood glucose levels greater than 16.7 mmol/L were considered as the distribution criteria of type 1 diabetic rat. In addition, the eligible diabetic rats were randomly assigned to two groups: diabetes group (n = 21) and diabetes plus valsartan group (n = 20). Rats in diabetes plus valsartan group were given valsartan by gavage using a suitable intubation cannula at 30 mg/kg/day dose levels to soluble in 5 mL distilled water, whereas rats in the diabetes group were administered with the same amount of distilled water each day. No additional treatments were given to control rats.

After 16 weeks, all animals from each group were euthanized using pentobarbital and fresh hearts were harvested immediately. Then, weight of the left ventricle was noted in order to observe the degree of left ventricular hypertrophy. Furthermore, a transversal cross-section block was cut from the left ventricle, soaked in 4 % paraformaldehyde, and remained frozen in liquid nitrogen until mechanical testing.

Hematoxylin eosin (HE) and Masson’s trichrome stain of myocardial tissue

After 48 h of treatment with 4 % paraformaldehyde, the tissues were dehydrated and the transparent tissues were embedded in paraffin. The tissues were cut into 6-μm slices, heated at 60 °C for 3 h, dewaxed, and stained with HE and Masson’s trichrome dyeing kits. Each slice was analyzed under a microscope at 400× magnification. The collagen volume fraction was determined as the ratio of interstitial collagen area to myocardial area.

Immunohistochemistry

For immunohistochemical analysis, the transversal sections of the left ventricle were cut into 5-μm sections and the endogenous peroxidase activity was quenched by incubation with 3 % hydrogen peroxide for 15 min. The sections were then blocked using 5 % bovine serum albumin to prevent the nonspecific staining with the secondary antibodies. After overnight incubation at 4 °C with primary antibodies (anti-periostin, anti-TGF-β1, anti-TGF-β1 RII, and anti-collagen I and III antibodies were used at 1:100 dilutions), the sections were exposed to the secondary antibody conjugated with horseradish peroxidase. The reaction was visualized using a light microscopy with 3, 3’-diamno-benzidine tetrahydrochloride.

Western blot analysis

Protein fractions were isolated in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime, China) and protein concentrations were determined using the enhanced bicinchoninic acid protein assay kit (Beyotime, China). Protein fractions were denatured in a loading buffer, and 100 μg of each sample was loaded into alternating lane of 10 % sodium dodecyl sulfate -polyacrylamide gel. Protein blots were transferred to polyvinylidene fluoride membranes (Milipore, USA). After blocking with 5 % non-fat milk, the blots were washed with a mixture of Tris-buffered saline and Tween 20 (TBST) and incubated overnight at 4 °C with an appropriate primary antibody (anti-β-actin, anti-periostin, anti-TGF-β1, anti-TGF-β1 II R, and anti-collagen I and III antibodies were used at 1:1000, 1:500, 1:400, 1:2000, 1:2000, and 1:5000 dilutions, respectively). Membranes were washed with TBST and then incubated at room temperature for 1 h with an appropriate secondary antibody conjugated to horseradish peroxidase and washed again. Finally, the protein blots were visualized using an enhanced chemiluminescence kit (Millipore, USA). Relative intensities of protein bands were measured using Gel-Pro analyzer. β-actin was used as a control.

Real-time polymerase chain reaction (RT-PCR) analysis

The total RNA was extracted from the frozen myocardial tissues using Trizol reagent (Invitrogen, USA), according to the manufacturer’s instructions. The concentration of extracted total RNA was quantified using spectrophotometry. The reverse transcription of RNA to complementary deoxyribonucleic acid (cDNA) was performed using Sensiscript RT kit (TaKaRa Biotechnology, China). Total cDNA was amplified with LightCycler-FastStart DNA Master SYBR Green I (TaKaRa Biotechnology, China). Primers for rats periostin, TGF-β1, TGF-β1 R II, Type I collagen, Type III collagen, and β-actin were designed by TaKaRa Biotechnology Corporation: Periostin S: 5’-GGCTGAAGACTGCCTTGAATGAC-3’, A: 5’-CGTGGCAGCACCTTCAAAGA-3’; TGF-β1 S: 5’-AGGTAACGCCAGGAATTGTTGCTA-3’, A: 5’-CATTGCTGTCCCGTGCAGA-3’; TGF-β1 R II S: 5’-CTACAAGGCCAAGCTGAAGC-3’, A: 5’-AGCCAATGGAAGTAGACATCCG-3’; collagen I S: 5’-AGGGACCCTTAGGCCATTGTGTA-3’, A: 5’-GACATGTTCAGCTTTGTGGACCTC-3’; collagen III S: 5’-GACAGATCCCGAGTCGCAGA-3’, A: 5’-TTTGGCACAGCAGTCCAATGTA-3’; and β-actin S: 5’-AGACCTTCAACACCCCAG-3’, A: 5’-CACGATTTCCCTCTCAGC-3’. Amplification process included an initial denaturation step of 30s at 95 °C followed by 40 cycles of three-step procedure (denaturation at 95 °C for 5 s, annealing at 60 °C for 30s, and extension at 60 °C for 30s) and then a melting-curve procedure at 60 °C for 1 min. At the end of each cycle, fluorescence emitted by the SYBR Green I dye was measured. For each PCR product, a single narrow peak was obtained through melting curve analysis at a specific melting-curve temperature, indicating specific amplifications. The amount of periostin, TGF-β1, TGF-β1 R II, Type I and Type III collagens, and β-actin transcripts was calculated from their respective standard curves using the Light Cycler software. Samples were tested for three times, and the average values were used for quantification. Ultimately, the relative expression levels of each target gene were normalized to the messenger RNA (mRNA) of the internal standard gene β-actin.

Statistical analysis

All data were expressed as mean ± standard error of the mean. Comparisons between two groups were performed using paired student’s t test. One-way analysis of variance was used to compare more than two groups. A value of P less than 0.05 was considered statistically significant.