Drugs and Reagents
r-Hirudin was provided by Shanghai Yiyan Biotechnology Co., Ltd., (China, batch number: 8001-27-2). p38 MAPK pathway inhibitor (SB203580) was provided by Shanghai Jizhi Biochemical Technology Co., Ltd., (China, batch number: 124-20-9). Simvastatin tablets were provided by Hangzhou MSD Pharmaceutical Co., Ltd. (China, GYZZ J20130068). Low-molecular-weight heparin sodium (Jipalin) was obtained from Hangzhou Jiuyuan Genetic Engineering Co., Ltd. (China, approval number: GYZZ H19990035). Penicillin sodium was provided by North China Pharmaceutical Co., Ltd., (China, batch number: D0801320).
Total cholesterol (TC) (Nanjing Jiancheng Bioengineering Institute, China, batch number: 20210405), triglyceride (TG) (Nanjing Jiancheng Bioengineering Institute, China, batch number: 20210408), low-density lipoprotein-cholesterol (LDL-C) (Nanjing Jiancheng Bioengineering Institute, China, batch number: 20210407) and high-density lipoprotein-cholesterol (HDL-C) kits (Nanjing Jiancheng Bioengineering Institute, China, batch number: 20210407); β-actin (Proteintech, USA, batch number: 66009-1-Ig), p38 MAPK (Proteintech, USA, batch number: 14064-1-AP), p65NF-κB (Proteintech, USA, batch number: 10745-1-AP), Caspase-9 (Proteintech, USA, batch number: 10380-1-AP), and Caspase-3 (Proteintech, USA, batch number: 19677-1-AP) antibodies; ox-LDL, TNF-α, IL-1β, IL-6, ET-1 and NO enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Optimal Biotechnology Co., Ltd., China, batch number: 202104); a high-purity total RNA rapid extraction kit (Beijing BioTeke Biotechnology Co., Ltd., China, batch number: B027009019); and a BioTeke Super RT kit (Beijing BioTeke Biotechnology Co., Ltd., China, batch number: B020009018) were used. Primers were synthesized by Shanghai Sangon Biological Engineering Co., Ltd.
Animals and Treatments
Seventy healthy male purebred Sprague-Dawley (SD) rats weighing 280~300 g were provided by Liaoning Changsheng Biotechnology Co., Ltd. (SCXK: (Liao) 2015-0001). The experimental rats were housed in the animal room of Jilin University School of Basic Medicine, which was a well-ventilated, quiet room with a controlled temperature of 18 ~ 22°C and a controlled humidity of 50%~70%. The animals were housed on a 12 h light/dark cycle and given ad libitum access to water and chow pellets for adaptive feeding for 1 week.
After 1 week of feeding, the rats were randomly divided into seven groups: the control group, AS model group, simvastatin group, low-dose r-hirudin group, medium-dose r-hirudin group, high-dose r-hirudin group, and pathway inhibitor group (10 rats in each group). The common carotid artery was injured with a balloon in all groups except for the control group, in which the external jugular artery was ligated but balloon injury was not induced. After surgery, the animals were given ad libitum access to a normal diet and water for 1 week. Beginning 2 weeks after the operation, the rats in all groups except the sham operation group, which were still given a normal diet, were fed a high-fat diet. At 6 weeks after operation, the low-, medium- and high-dose r-hirudin groups were given the drug at doses of 0.05 mg/kg/d, 0.1 mg/kg/d and 0.2 mg/kg/d, respectively; the simvastatin group was given 1 mg/kg/d simvastatin tablets; and the pathway inhibitor group was given the p38 MAPK pathway inhibitor SB203580 at a concentration of 100 mg/kg/d by gavage. The sham operation group was given an equal volume of normal saline for 8 weeks. At the end of the experiment, the animals were euthanized with carbon dioxide, and samples were taken for appropriate tests.
Specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with an ultramicrotome at a thickness of 5 µm. After sectioning, hematoxylin and eosin (HE) and Masson staining were performed, and histomorphological changes were observed under a light microscope.
Determination of Blood Lipid Levels
Serum samples were placed in a refrigerator at 4°C for 30 minutes and then incubated at room temperature for 30 minutes. After the serum was fully thawed, TC, TG, LDL-C and HDL-C levels were determined using kits and a multifunctional microplate reader according to the manufacturer’s instructions.
The serum levels of ox-LDL, TNF-α, IL-1β, IL-6, ET-1 and NO were measured by ELISA. The ELISA kits were removed from the refrigerator and warmed at room temperature for approximately 30 minutes. The instructions of the kits were followed precisely.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from the rat common carotid artery using a high-purity total RNA rapid extraction kit. Then, total RNA was reverse transcribed into cDNA by qPCR using the BioTeke Super RT Kit. qRT-PCR was performed using Roche Fast Start Universal SYBR Green Master Mix (Rox). The above operations were performed in strict accordance with the manufacturer’s instructions. β-Actin was used as an internal reference gene, and the relative expression of the target gene in each group was measured for comparison. The PCR conditions were as follows: activation at 50°C for 2 minutes and 95°C for 10 minutes followed by denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 minute. A total of 40 cycles were performed. The specific primer sequences were as follows: p38 MAPK: F, 5'-TTGGTCTGTTGGATGTGTTTAC-3', and R, 5'-TGGATTATGTCAGCCGAGTG-3'; NF-κB: F, 5'-TTGCTGGTCCCACATAGTTG-3', and R, 5'-ATGTATGTGAAGGCCCATCC-3'; caspase-3: F, 5'-GGACTGCGGTATTGAGA-3', and R, 5 '-GGTGCGGTAGAGTAAGC-3'; caspase-9: F, 5'-GTGAAGAACGACCTGACT-3', and R, 5'-AGGATGACCACACACACACACCA-3'; β-actin: F, 5 '-CAAGCTTAATCAGG-3', and R, 5 '-ACATGAAGAGAGAGAGAGAGAGGC-3'.
Western Blot Analysis
The rat common carotid artery was collected, rinsed several times in precooled normal saline, weighed and placed in a tissue homogenizer. Protein was extracted according to the instructions of a tissue cell lysate kit. The protein concentration was determined by the Coomassie brilliant blue method. SDS-PAGE was performed (150 V, 50 min), and the proteins were transferred to a membrane for 1 h at 100 V. After transfer, the membrane was washed with TBST 3 times, blocked with skimmed milk powder for 2 h, washed with TBST, incubated with primary antibody overnight at 4°C, washed with TBST 3 times, incubated with secondary antibody at room temperature for 1 h, and washed with TBST 3 times. Color development was performed with a high-sensitivity ECL chromogenic luminescence kit according to the manufacturer’s instructions. β-Actin was used as an internal reference protein, gray values were analyzed and determined using a gel imaging system, and the relative expression of each protein was calculated as the gray value of the sample band/the gray value of the β-actin band for quantitative analysis. Each experiment was repeated three times.
Statistical analysis was performed with SPSS 21.0 and GraphPad Prism 6.0 software. All data are presented as the mean±standard deviation. One-way analysis of variance and the least significant post hoc difference test were used to evaluate the significance of differences. P<0.05 was considered statistically significant.