ABC | Volume 111, Nº4, Octuber 2018

Original Article Yang et al ApoJ in neointimal hyperplasia Arq Bras Cardiol. 2018; 111(4):562-568 (n = 20) and intervention (statin) group (n = 32). All animals were then randomly divided into 4 groups – to be evaluated at 1, 2, 3 or 4 weeks after balloon injury. The study was approved by the Ethics Committee of Tianjin Chest Hospital. Balloon injury The rats wereweighed on the day of operation, and randomly divided into three groups. The rats in the intervention groupwere given 10 mg/kg dose of rosuvastatin. A 2F Fogarty catheter was introduced to induce vascular injury as previously reported. 13 Briefly, the rats were anesthetized after intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 mL/100 g body weight. A 2F balloon catheter was inserted into aortic outlet of carotid artery. The balloon was then inflated and pulled back 3 times to denude the endothelium. At 1, 2, 3 and 4 weeks after surgery, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3 ml/100 g body weight. Then, the animals were sacrificed by intravenous administration of 2-3 mL of potassium chloride solution via subclavian vein; 0.3 cm of the right carotid artery was fixed in 10% neutral formalin for pathological examination, and the other part was frozen immediately in liquid nitrogen and stored at -80°C for further use. Hematoxylin-eosin (HE) staining Vascular specimens were fixed in 10% formaldehyde solution for 3-4h. Routine dehydration and paraffin embedding were performed. The sections were cut evenly and the thickness was 4 μm. The injury of blood vessels was observed under light microscope. Immunohistochemistry (IHC) assay The level of Apo J was assessed by IHC in rat carotid artery. The primary antibody (polyclonal rabbit anti-human Apo J IgG) was purchased from Santa Cruz, Inc. (Cat No. sc-8354). The secondary antibody (labeled goat anti-rat/rabbit IgG polymer) was purchased fromMaixin BioTech (Fuzhou, China). All photos were captured and saved using the ISCapture system, and data collection and analysis are performed using the Image Pro Plus 6 image processing software. Enzyme-linked immunosorbent assay (ELISA) Venous blood was collected and centrifugated at 3000r/min for 10 min. The supernatant was collected using a micropipette and stored in the refrigerator at -20 o C for use. The samples were then thawed at roomtemperature for ELISA. ELISAwas performed using a commercial kit (Rat Competitive ELISA for Apolipoprotein J A 252 SC), following the manufacturers’ instructions. Real-time polymerase chain reaction (PCR) The mRNA level of Apo J was detected by real-time PCR in rat carotid artery. RNA was extracted by Trizol one-step extraction method, and reverse transcription was performed. Primers used for amplification for Apo J were as follows: Forward, TAA GGA GAT TCA GAA CGC CG; reverse, ATC CCT GGT GTC ATC TAG AG. Primers for the control GAPDH were as follows: Forward, GTG ATG CTG GTG CCG AGT AG; reverse, GGT GGC AGT GAT GGC GTG C. Real‑time PCR reactions were prepared following the instructions of SYBR ® Premix Ex Taq ™ system (Perfect Real Time). The mRNA levels in each sample were calculated by 2 -ΔΔCt . Western blotting Proteins were extracted from 30 mg of rat carotid artery. Briefly, proteins were separated using SDS-PAGE with 10% separation gel and 5% concentrated gel. Then the separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes. The membranes were blocked and incubated with antibodies. Relative levels of Apo J were analyzed using Image Lab analysis software. β -actin was used as inner control. Bands were quantified using QUANTITY ONE software (Bio‑Rad, Hercules, CA, USA). Statistical analysis Statistical analysis was performed using SPSS 20.0. Quantitative data were expressed bymean± standard deviation (SD). The difference between two groups was compared using independent-samples t test; comparisons between three groups were analyzed using one-way ANOVA (analysis of variance). P < 0.05 was considered statistically significant. Results Survival and success rates of rat carotid artery model Among the 52 rats of the model group and the intervention group, 2 died during operation by suffocation, and 2 died for arterial hemorrhage 12h after operation. Therefore, 47 rats survived with approximately 90% survival rate. The pathological examination showed intimal hyperplasia and thickening in the experimental group, suggesting that the model was successfully constructed. The mean operation time was 34.19 ± 6.09 min. The feasibility and success rate of this model can be highly reproducible if surgical procedures are properly performed. Level of serum Apo J There was no significant difference in serum Apo J level before and after operation in the intervention group (Table 1). There was no significant difference in the level of serum Apo J at 1, 2, 3 and 4 weeks before ( F = 1.002, p = 0.408) of after ( F = 0.189, p = 0.903) operation. Statin intervention inhibited intimal hyperplasia Results of pathological examination showed that no intimal hyperplasia was observed in the control group (Figure 1). In the model group and intervention group, the intima was slightly thickened 1 week after operation, and further thickened 2 weeks after operation. During 3 weeks after operation, the degree of intimal hyperplasia was further aggravated in the model group; however, this change was not as marked as at 2 weeks after operation, and the cells gradually became paralleled. During 4 weeks after operation, the degree of intimal hyperplasia was further aggravated in the model group, but no significant changes were observed regarding the degree of intimal hyperplasia as compared with week 3 (Figure 2 and 3). 563