Male Wistar rats, weighing 180-200g, were provided by the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China; certificate number SCXK (Jun) 2012-0004). Wistar male rats were raised in a cage with a constant temperature of 22±2°C, a 12-hour light-dark cycle, free drinking water, and a standard diet. All research procedures conformed to Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86–23, revised 1996), and approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine, China (No. TCM-LAEC2016016).
Animal model of pathological myocardial hypertrophy
The abdominal aorta constriction method was used to establish a pressure overload rat myocardia hypertrophy model . In short, a rat was anesthetized with sodium pentobarbital (45 mg/kg, intraperitoneal injection), then fixed on the operating table supine, and a longitudinal incision in the abdominal cavity was performed under aseptic conditions. The abdominal aorta was exposed and the abdominal aorta above the right renal artery branch was separated. A size 7 needle was placed parallel to the abdominal aorta, the two were ligated with a size 4 surgical suture, the needle was removed, and 200,000 U of penicillin were instilled to prevent infection, and the abdominal cavity was closed. At the same time, a small amount of picric acid solution was applied around the incision to prevent wound infection in rats. After the operation, the rats were fed with sugar-salt solution and continuously injected intramuscularly with 200,000 U/d penicillin for 3 days to prevent infection. Rats in the sham surgery group were operated and the suture line was placed without ligation. The remaining procedure was similar to that in the surgery group.
Groups and dosing
4 weeks after the operations, sham and the aortic constriction surgery rats were randomly divided into the Sham-operated control group(intragastric infusion with equal volume of distilled water)，model group (intragastric infusion with equal volume of distilled water), the valsartan group (intragastric infusion at 7.2 mg/kg; Novartis Pharma Ltd., Beijing, China), and the QSYQ group (intragastric infusion at 135 mg/kg; Tasly Pharmaceutical CO., LTD., Tianjin, China). The interventions were administered at 2 time points (4-week intervention and 8-week intervention), and 8 rats were present in each group at each time point. The rats in each group were dosed once daily in the morning.
Morphological and histological analysis
The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (45 mg/kg) after drug intervention for 4 weeks and 8 weeks. The heart tissue was removed, and the heart mass index (HMI, the ratio of heart weight to body weight) and the left ventricular mass index (LVMI, the ratio of left ventricular weight to body weight) were measured and calculated. Subsequently, the heart was fixed in 4% paraformaldehyde overnight, routinely dehydrated, transparent, embedded, and sectioned, 5 μm thick; deparaffinized, dehydrated using an ethanol gradient, and subjected to hematoxylin-eosin (H&E) staining and Masson staining (all Baihao Biological Technology CO., LTD., Tianjin, China). Dehydrate with ethanol gradient, wash with xylene, fix with neutral resin, and observe under an optical microscope. H&E staining was used to estimate cardiac hypertrophy, Masson staining to measure the area of collagen. The image-Pro Plus image analysis software was used to randomly select 5 fields of view from each slide to measure the area of collagen. The average was calculated as the collagen volume factor (CVF) for this myocardial tissue. CVF = area of myocardial collagen fibers/total area of the image.
Real-time quantitative polymerase chain reaction
Ultrapure RNA extraction kit (CWbio Co. Ltd., Cat#CW0581) was used to extract total RNA from the myocardial tissue. The HiFi-MMLV cDNA first-strand synthesis kit (CWbio Co. Ltd., Cat#CW0744) was used for reverse transcription and the UltraSYBR Mixture with Rox (CWbio Co. Ltd., Cat#CW0956) was used for amplification. The primers for the target genes and household genes were purchased from Guangzhou Fulen Gen Co., Ltd., including TGF-beta 1 (GeneCopoeiaTM; Cat# RQP050181), CTGF (GeneCopoeiaTM, Cat# RQP050397), and glyceraldehyde phosphate dehydrogenase (GAPDH) (GeneCopoeiaTM, Cat# RQP049537). The 2-△△Ct method was used for relative quantitative analysis of the raw data of RT-qPCR determination.
The expression of TGF- beta 1 protein and CTGF protein in rats were measured by immunochemical assay, the slides were routinely dewaxed by xylene, the slides were hydrated by gradient ethanol, and the antigens were repaired by microwave. Then an appropriate amount of hydrogen peroxide was added to block the endogenous peroxidase activity of the protein. A primary antibody (rabbit anti-rat IgG) was added and incubated at 4℃ overnight. Further, the slides were incubated with biotinylated goat anti-rabbit IgG. Finally, DBA was used for color rendering and hematoxylin was used for secondary dyeing (antibody against TGF-beta 1, antibody against CTGF, streptavidin – biotin complex with peroxidase (SABC-POD) (rabbit IgG) ready-to-use kits and DAB Chromogenic Reagent Kit were all from Boster Biological Engineering CO., LTD., Wuhan, China). After dehydration, transparent sealing with neutral resin. Determination of results: Cells with a clearly defined structure, having a significantly greater coloration than the background, and brown-yellow granules at the corresponding area were considered to be positive. The cells that did not develop any color or had the same level of coloration as the background were considered to be negative. We randomly selected 5 fields from each slide under an optical microscope of 40× magnification. The area ratio of the positively stained matter was determined using the Image-Pro Plus imaging analysis software and the average was calculated.
All experimental results were statistically analyzed using SPSS software (v11.5; SPSS Inc., Chicago, Illinois, USA), and expressed as mean ±S.D. One-way analysis of variance was used to compare multiple groups, and then the least significant difference (LSD) test was used for multiple comparisons. The value of P<0.05 was considered statistically significant.