A total of twenty 8-week-old male Wistar rats were selected and provided by the Animal Model Center of Nanjing University, with the animal qualification number SCXK (Su) 2015-0001. They were raised in an environment of 22 ± 2°C, freely feeding and drinking water, and were adaptive fed for 1 week before the experiment. Streptozotocin (STZ) (Sigma, St. Louis, MO, USA), Sacubitril valsartan tablets (Novartis, Switzerland), HE kit, and Masson kit were provided by Jiangsu KeyGEN Biotech, NanJing, China. Rat IL-6 ELISA Kit (ab100772), Rat TNF- ELISA Kit (ab100785), Anti-p38 antibody (ab31828), and Anti-phos-p38 antibody (Thr180/Tyr182) (ab4822) were purchased from Abcam, Cambridge, UK.
1.2 Model Establishment
At a dose of 70mg/kg, 1% STZ (pH = 4.5, compound sodium citrate buffer 4 ℃, instantly make immediate use) was injected into the abdominal cavity of rats to destroy the function of islet cells, tail venous blood of rats was extracted 3 days and 1 week after injection of STZ to detect fasting blood glucose (fasting for 8h), When blood glucose ≥16.7mmol/L and polydipsia, polyphagia and polyuria occurred in the rats, the diabetic cardiomyopathy model of rats was considered to be established.
1.3 Group setting
Rats were divided into the following four groups. Firstly, normal rats (NOR) were given normal saline (1ml/100g/d) by gavage for 8 weeks without modeling. Secondly, DCM model group, after modeling, normal saline (1ml/100g/d) was given by gavage for 8 weeks. Thirdly, DCM+ Sac/Val group (Sac/Val)，after modeling, Sac/Val (60mg/kg/d) was given orally for 8 weeks. Fourthly, DCM+ Perindopril group (PER), PER (2mg/kg/d) was given orally for 8 weeks after modeling.
1.4 Left ventricular/body mass index (LVW/BW)
After measured BW, the rats were killed immediately, and then the heart was taken out immediately and washed repeatedly in 0.9% sodium chloride injection. After removing the residual blood, the atrial, great vessels and epicardial adipose tissue were removed, and the left ventricle was dissociated and dried by filter paper. Finally, we use an electronic balance to weigh the lvw. LVW / BW (mg/g) = left ventricular weight (mg) / body weight (g).
1.5 Myocardial histology
We prepared paraffin sections of heart tissue for routine xylene dewaxing, followed by gradient ethanol hydration, distilled water washing, and then stained with HE to evaluate the myocardial arrangement. The paraffin sections of heart tissue were placed in xylene Ⅰ and xylene Ⅱ for 10 min. After dewaxing, they were washed with water, stained with hematoxylin for 10 min, and then washed with water until they turned blue. Then they were stained with Ponceau and fuchsin for 5 min, 1% molybdic acid solution for 10 min, aniline blue solution for 15 min, 0.2% glacial acetic acid solution for 5 min, and 0.2% glacial acetic acid solution for 5 min. Finally, the myocardial histological changes were evaluated by Masson's trichrome staining.
We quickly extracted the hearts of the rats immediately after they were killed and removed about 100mg of myocardial tissue, then rinsed them with PBS buffer. The tissue homogenate was centrifuged at 12,000 g for 10 minutes, and the contents of IL-6 and TNF-α in the supernatant were determined with ELISA kit.
1.7 Western Blot
We isolated cardiac tissue and cut about 100 mg tissue into a precooled EP tube. Then we added 400 μl RIPA lysate into a homogenizer for homogenized the tissue, crushed the tissue as much as possible, put it on ice for 30 min, then transferred the lysate to 1.5 ml Eppendorf tube with a pipette, centrifuged for 5 min, and part of the supernatant was put into 200 μl Eppendorf tube, and the total protein concentration was determined according to the instructions of BCA kit. The SDS-PAGE gel was prepared. The size of the gel was sheared PVDF film and activated in methanol for 1 min, and then immersed in the membrane buffer. The filter paper was placed in the transfer film buffer to soak for 15 min. According to the principle of PVDF membrane≥ gel≥ filter paper, we make transfer sandwich to ensure constant pressure transfer after bubble removal. Add the TBST diluted to the appropriate concentration of a single antibody, close the bag mouth, stay overnight at 4 ℃, add appropriate amount of two resistance, close the bag mouth, and incubate 1 h at room temperature. Tanon 6600 luminous imaging workstation was used for image acquisition. Finally, we detected the expression levels of p38 and phos-p38 in myocardium tissue of rats in each group by Western blot.
1.8 Statistical analysis
We used Single factor analysis of variance (ANOVA) to compare the mean of multiple independent samples. Kruskal-wallis test is used for analysis if there are other conditions such as uneven variance or non-normal distribution. The significance level was bilateral α = 0.05. All data were analyzed using SPSS 20.0.