Establishment of the HF Model in Rats and Grouping
The HF model was established by abdominal aortic constriction. After feeding adult Wister male rats for 1 week, pentobarbital sodium anesthetized rats, and then the abdominal aorta was separated 1cm above the left renal artery through a median incision. Abdominal aorta was sutured with no. 22 needle 4-0 silk thread to form abdominal aortic stenosis (about 50% ~ 60%). Abdominal dissection was performed. At the same time, 1×105 U penicillin was intraperitoneally injected to prevent infection. The rats were monitored daily after surgery, and after 10 weeks, AsC medication interfered with HF (2.5 mg/kg/day) for 4 weeks.
The rats were divided into four groups (sham group, HF model, HF+PBS and HF+ AsC group) and five of each group. Sham group rats received DMEM 70μl for control.
All rats were used for subsequent experiments in accordance with the Laboratory Animal Management Regulations and Animal Ethical Requirements before modeling. Animal experimental protocols were approved by the Ethics Committee of the Inner Mongolia People’s Hospital and the study was carried out in compliance with the ARRIVE guidelines. Rats were sacrificed and anesthetized by intraperitoneal injection of pentobarbital sodium solution (1%) at a dose of 50mg/kg. We tried our best to reduce the number of animals that are used and reduce their suffering.
Detection of myocardial function
Ten weeks after the last administration, the rats in each group were anesthetized. After removing the chest hair and applying the coupling agent, the VisualsonicVevo 2100 imaging system was used to evaluate the preoperative and postoperative cardiac function through echocardiography. Mice were anesthetized with 2.5l /min isoflurane before evaluation. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular fraction shortening (LVFS) and left ventricular ejection fraction (LVEF) were calculated with Vevo analysis software25.
Histological examination in rats
The rat heart was dissected, fixed with 4% formalin for 24 hours, embedded in paraffin, and cut into 5μm-thick slices. Then the slices were stained with Masson's trichrome staining (Solarbio, Beijing, China) and hematoxylin-eosin (HE) staining to observe heart tissue morphology. Image analysis software (Image-Pro Plus v4.0, Media Cybernetics, USA) was used to calculate the area occupied by collagen
Analysis of serum biochemical indexes
The contents of creatine kinase (CK), creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST),lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) in plasma were determined by automatic chemical analyzer (Labospect-008, Hitachi High-Tech Diagnostics (Shanghai) Ltd., Japan)
ELISA Analysis
The levels of BNP, TNF-α, AngII, IL-6, IL-4, IL-1b in plasma were quantified using Glucagon Quantikine ELISA Kit (Elabscience Biotechnology Co., Ltd, Wuhan, China). Analyze according to manufacturer's instructions.
Apoptosis Assay
According to the manufacturer's instructions, cardiomyocyte apoptosis was detected by TUNEL (EMD Millipore, Billerica, MA). The apoptosis of cardiomyocytes was brown. The average percentage of apoptotic cells in 5 randomly selected fields (under magnification ×40) was calculated by Olympus microscope.
Western blotting (WB)
Total proteins were extracted from cardiac tissue and the levels of cardiac proteins P/t-PI3K, P/t-AKt, Bcl-2, Bax, Caspase-3 and cytochrome C were detected by WB.
Statistical analysis
All statistical analyses were performed using GraphPadPrism7.0 software. The differences between two groups and multiple groups were analyzed using t-test and one-way ANOVA, respectively. P < 0.05 was considered a statistically significant difference.