Eighty-five male Wistar rats (5 weeks old; weight, 200 ± 20 g) were provided by the Experimental Animal Center of the Chinese PLA Military Academy of Medical Sciences [License No.: SCXK-(Army) 2007-004]. The rats were housed in the experimental animal center at the Institute of Basic Theory, CACMS. There were five rats per cage, with a total of 17 cages. All rats were provided free access to water and were maintained on a 12-h light/dark cycle, with a temperature of 20 ± 2 °C and humidity of 50 ± 5 %. The animals were cared for in accordance with the laboratory animal care protocols of the Institute of Basic Theory of Chinese Medicine, CACMS. All animal studies conducted here were approved by the medical ethics committee of the Institute of Basic Theory of Chinese Medicine, CACMS.
The component herbs of WXD included the following: Ginseng Radix et Rhizoma (10 g), Cinnamomi Ramulus (10 g), Allii Macrostemonis Bulbus (15 g), Trichosanthis Fructus (15 g), Pinelliae Rhizoma Praeparatum Cum Alumine (10 g), Paeoniae Radix Rubra (15 g), Ophiopogonis Radix (15 g), and Coptidis Rhizoma (10 g). These were purchased from Beijing Tong-Ren-Tang Pharmacy and were certified as authentic by the Institute of Chinese Meteria Medica, CACMS. They also fulfilled the standard requirements of the 2010 version of the Chinese Pharmacopoeia. The traditional decocting method is described as follows. First, 1000 ml water is added for impregnation, the extract is heated for 30 min, and the liquid is leached. Then, 500 ml water is added to the residue, the extract is heated for 30 min, and the liquid is leached. The two portions of leached liquid are merged for liquid static sedimentation, filtration, and concentration. Finally, the derived product is packed for use. For controlled administration, we prepared WXD as a dry extract according to the guidelines of the School of Chinese Materia Medica, Beijing University of Chinese Medicine. To prepare the dry extract, the herbs were immersed, boiled, and filtered. The filtrates were combined and concentrated in a constant volume, steamed in a water bath until nearly dry, placed in an oven at 105 °C for 3–4 h, and cooled in a dryer for 0.5 h. concentrate leaching into the powder preparation. The extract concentration was 30.58 %, which was equivalent to 3.27 g of the original medicines per 1 g of dry extract. We used a certain amount of distilled water to dissolve the dry extract. The dry extract was prepared in strict compliance with the Chinese Pharmacopoeia ((see Appendix IO) and CGEP (GMP herbal extracts) in order to ensure the quality of medicines.
The other commercial products used in this study were as follows: atorvastatin calcium (Pfizer, Lipitor; code number approved by SFDA: J20070061), vitamin D3 (Shanghai General Pharmaceutical Co., Ltd.; code number approved by SFDA: H31021404; dose: 600,000 IU, 1 ml), AngII-ELISA kit (BG; E02A0204), ET-1ELISA kit (BG; E02E0040), NO-ELISA kit (BG; E02N0041), real-time quantitative polymerase chain reaction (real-time PCR) kit (SYBR Green PCR Mixture, CWbio. Co., Ltd., CW0957), rabbit anti-PI3K P85 (4292S; CST; dilution ratio 1:1000), rabbit anti-p-eNOS (9570; CST; dilution ratio 1:1000), rabbit anti-eNOS (ab11627; Abcam; dilution ratio1:300), rabbit anti-P-AKT (ab38449; Abcam; dilution ratio 1:500), rabbit anti-AKT (ab8805; Abcam; dilution ratio 1:500), rabbit anti-iNOS (ab15323; Abcam; dilution ratio 1:250), and a high-fat diet (4 % cholesterol, 10 % lard, 5 % sucrose, 81 % diet).
Preparation of animal models
After 1 week of acclimatization, the Wistar rats were randomly divided into a normal group (n = 10) and a model group (n = 75). The total time taken for model preparation was 5 months. During the first 3 months, the rats received a high-fat diet combined with intraperitoneal injections of vitamin D3 (150,000 U/kg, once a month). For the next 2 months, they received the high-fat diet alone.
According to the AS index (AI), the 75 model rats were randomly divided into five groups of 15 each: model, atorvastatin, high-dose WXD, medium-dose WXD, and low-dose WXD groups. Each group received continuous drug (suspended liquid gavage) or saline administration for 30 days as follows: normal and model groups, saline (10 ml/kg/d); atorvastatin group, atorvastatin (4.8 mg/kg/d, equivalent to five times the adult human dose); WXD high-dose group, 9 g/kg/d WXD (equivalent to two times the human dose); WXD medium-dose group, 4.5 g/kg/d (equivalent to one time the human dose); and WXD low-dose group, 2.25 g/kg/d (equivalent to half the human dose). All animals were sacrificed after 30 days of drug or saline administration, with no food intake before sacrifice.
On day 180, rats were anesthetized by intraperitoneal injection of 5 % urethane (1000 mg/kg), following which blood samples were collected from the abdominal aorta and the full-length aorta (from the aortic arch to the iliac artery bifurcation) was harvested. Blood samples from the abdominal aorta were drawn (10 ml), and the serum was collected and stored at −20 °C. Serum levels of cholesterol (CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an Automatic Biochemistry Analyzer (HICATHI) according to the manufacturer’s instructions. After removal of the epicardial adipose tissue, the aortic arch and 1 cm of the lower end of the thoracic aorta were harvested and placed in liquid nitrogen for real-time PCR and Western blot analysis. The coronary artery (1 mm3) was fixed in glutaraldehyde for observation under a transmission electron microscope. Part of rat aorta was examined by hematoxylin and eosin staining for histopathological changes.
Total RNA was extracted by an ultrapure RNA extraction kit. In total, 8-μl RNA samples were used for 1 % agarose gel electrophoresis. Reverse transcription was performed using the HiFi-MMLV first strand cDNA synthesis kit (CWbio. Co., Ltd.). RNA templates (2 μl) were added to a 20-μl reaction system to achieve a final concentration of 10 ng. Real-time PCR was performed using the Bioer line-gene quantitative PCR device, and the products were amplified using the SYBR PCR Mixture according to the manufacturer’s instructions. The primers for AKT, eNOS, PI3K, actin, and iNOS were as follows: AKT, 5-AGC ATG GAG TGT GTG GAC AG-3 (forward) and 5-GAT GAT CCA TGC GGG GCT T-3 (reverse); eNOS, 5-AAG GCA AAC CAC CCT CTC TG-3 (forward) and 5-TTA GGC TGA CTC CCT CCC AG-3 (reverse); PI3K, 5-GCT CTT TCC CCA GCT GAA CT-3 (forward) and 5-ACA CAG TGT CGC TGG TTT GA-3 (reverse); actin, 5-CCC ATC TAT GAG GGT TAC GC-3 (forward) and 5-TTT AAT GTC ACG CAC GAT TTC-3 (reverse); and iNOS, 5-ACACAG TGT CGC TGG TTT GA-3 (forward) and 5-AAC TCT GCT GTT CTC CGT GG-3 (reverse). The reaction system was as follows: SYBR PCR Mixture (2×), 10 μl; forward primer (10 uM), 1.5 μl; reverse primer (10 uM), 1.5 μl; and template, 3 μl. All reactions were performed in triplicate. Gene expression was assessed using the 2−△△CT method as follows: △△CT = △CT (drug treatment group) − △CT (normal group) and △CT = CT (target gene) − CT (reference gene).
Western blot analysis
Total protein was isolated using ice-cold RIPA and protease inhibitor cocktail (Roche); phosphorylated protein extraction required phosphatase inhibitors. In total, 50 mg of tissues were homogenized using 500 μl of RIPA lysate buffer, incubated on ice for 20 min, and centrifuged at 13,000 rpm (4 °C) for 20 min. The supernatant was collected and stored at −80 °C. The protein concentration was determined using the BCA Protein Assay Kit according to the manufacturer’s instructions. Protein samples (30 μg) were added for 12 % polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5 % skimmed milk at room temperature for 2 h, incubated with the primary antibody for 1 h at room temperature, and maintained at 4 °C overnight. The following day, the membrane was washed with TBST (six times, 5 min each) and incubated with the secondary antibody at room temperature for 1 h. After washing with TBST (six times, 5 min each), the membrane was developed in a darkroom. The gray scale of the protein bands was analyzed using ImageJ software.
All data are expressed as means ± standard deviations (means ± SDs). Statistically significant differences between groups were determined using one-way ANOVA and Dunnett’s t-tests. The results of real-time PCR and Western blot analysis were analyzed using independent samples two-tailed t-tests. All data were analyzed using SPSS 16.0 software. A p-value of <0.05 was considered statistically significant.