Male Sprague-Dawley (SD) rats (n=18; 8-week-old) were purchased from the Animal Center of Zhengzhou University (Zhengzhou, China) and fed in an SPF laboratory. The rats were fed under controlled temperature (23±2°C) and humidity (55±5%) with an artificial 12-h light/dark cycle, and were given free access to food and tap water. The rats were allowed to acclimate to the environment for 1 week. All experimental procedures were performed in accordance with the guidelines on Animal Care of the Fifth Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Rats weighing 200–220 g were used for the experiments in the present study, which were randomized into three groups: i) control group (n=6); ii) diabetic rats with carotid artery injury (model group, n=6); and iii) diabetic rats with carotid artery injury + oleanolic acid (HPLC ≥98%; Aladdin Chemical Co., Ltd., Shanghai, China) treatment (oleanolic acid group, n=6). The establishment of a streptozotocin (STZ)-induced diabetic rat model with carotid artery injury was performed as described previously (3). All other chemicals were of analytical grade and purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

The normal or injured carotid arteries from the rats were fixed in 4% paraformaldehyde for 1 week and then embedded in paraffin. Sections measuring 5 µm were cut and stained with hematoxylin & eosin (H&E). For morphologic analysis of neointimal formation, Image-Pro Plus 5.0 image analysis software (Media Cybernetics Inc., Rockville, MD, USA) was used. The medial and intimal cross-sectional areas were measured, and the intima/media ratios were calculated.

Following sacrifice of the rats, blood was collected using Vacutainer tubes (BD Biosciences, Franklin Lakes, NJ, USA) and centrifuged immediately at 3,000 × g for 10 min at 4°C. The supernatant was then collected and stored at −80°C until used for the subsequent assay. The levels of TNF-α, IL-1β, IL-6 and IL-18 were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (Biosource, Camarillo, CA, USA) according to the manufacturer's instructions. ELISA kits were used to measure the levels of endothelin 1 (ET-1; cat. no. E-EL-R0167; Elabscience Biotechnology Co., Ltd., Wuhan, China), nitric oxide (NO; cat. no. A012-1; Nanjing Jiancheng Biology Engineering Institute, Nanjing, China) and von Willebrand factor (vWF; cat. no. E-EL-R1079; Elabscience Biotechnology) in the supernatant on an ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA) according to the manufacturers' instructions.

Aortic blood vessel leakage was quantitated using Evans blue dye, which binds non-covalently to plasma albumin in the blood stream, as described previously (18). Aortic blood vessels were visualized under a microscope (Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany).

RNA was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The synthesis of cDNA was performed with 2 µg total RNA using Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) and oligo(dT)15 primers (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The qPCR was performed using an Applied Biosystems 7300 Real-Time PCR system (Thermo Fisher Scientific, Inc.). Reaction mixtures (25 µl) were prepared as follows: 12.5 µl SYBR-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 1 µl cDNA, 300 nM of each primer, and DEPC H₂O to a final volume of 25 µl. Thermocycling procedure was performed as follows: 94°C for 1 min, 40 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 30 sec. The quantification cycle fluorescence value (Cq) was calculated using SDS software, version 2.1 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the relative mRNA expression levels of RANK were calculated using the 2^−ΔΔCq method (19) and normalized to the internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primer sequences were used: ET-1 forward, 5′-AAGCGCTGTTCCTGTTCTTCA-3′ and reverse, 5′-CTTGATGCTATTGCTGATGG-3′; NLRP3 forward, 5′-AAAGCCAAGAATCCACAGTGTAAC-3′ and reverse, 5′-TTGCCTCGCAGGTAAAGGT-3′; capsase-1 forward, 5′-AGGCATGACAATGCTGCTACAA-3′ and reverse, 5′-TGTGCAAATGCCTCCAGCTC-3′; IL-1β forward, 5′-TCGCCAGTGAAATGATGGCTTA-3′ and reverse, 5′-GTCCATGGCCACAACAACTGA-3′; GAPDH forward, 5′-ACAGGGGAGGTGATAGCATT-3′ and reverse, 5′-GACCAAAAGCCTTCATACATCTC-3′.

The normal and injured carotid arteries of the rats were homogenized and lysed in NP-40 buffer (Beyotime Institute of Biotechnology, Haimen, China). Following 5–10 min of boiling, the cells were centrifuged at 10,000 × g at 4°C for 10 min to obtain the supernatant. Protein concentrations were determined using the Bicinchoninic Acid kit for Protein Determination (Sigma-Aldrich; Merck KGaA). Protein samples (50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylimide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline and 0.1% (w/v) with Tween-20, and incubated with the following primary antibodies: NLRP3 (cat. no. ab210491, 1:500; Abcam), ET-1 (cat. no. sc-57116, 1:10,00) caspase-1 (cat. no. sc-398715, 1:500), IL-1β (cat. no. sc-515598, 1:10,00) and β-actin (cat. no. sc-130065, 1:2,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following washing with PBS three times (5 min/each), the membranes were incubated with HRP-conjugated anti-IgG (cat. no. sc-516102, 1:10,000; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Signal detection was performed using an ECL system (GE Healthcare Life Sciences, Chalfont, UK). Quantitative analysis of protein was assessed using Quantity One software version 4.5 (Bio Rad Laboratories, Inc.).

Data are expressed as the mean ± standard error of the mean. All statistical analyses were performed using GraphPad Prism software, version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Groups were compared using one-way analysis of variance, followed by Tukey's multiple comparison post hoc test to compare the mean values of each group. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A and B, body weights steadily increased, and the fasting blood glucose (FBG) level was maintained within the normal range of 3.9–6.1 mmol/l in the non-diabetic control rats during the experimental period. In the rats fed with a HFD 4 weeks prior to STZ injection, the body weights of the rats significantly increased, however, injection of STZ markedly decreased the body weights of the rats (Fig. 1A). At 2 weeks post-STZ injection, the FBG values of the diabetic rats and oleanolic acid-treated diabetic rats increased from 4.6 mmol/l at week 4 to 16.7 mmol/l at week 6, and 4.7 mmol/l at week 4 to 16.9 mmol/l at week 6, respectively. These increases were significantly higher than that of the control group (5.0 mmol/l) at week 6 (Fig. 1B). Oleanolic acid administration reversed the HFD- and STZ-induced changes in body weight and glucose metabolic disorder (Fig. 1A and B).

The degree of neointimal hyperplasia was evaluated by morphologic analysis with H&E staining. The results demonstrated that the injured carotid arteries of the model group developed severe stenosis and neointimal hyperplasia, compared with the non-diabetic normal rats (Fig. 2A). Oleanolic acid treatment significantly reduced the intimal area, compared with that in the model group (Fig. 2B). The intima/media ratio was also lower in the oleanolic acid-treated group, compared with that in the model group (Fig. 2C).

ET-1 is a potent vasoconstrictor, and its increase in endothelial cells leads to endothelial dysfunction and cardiovascular disorders (20). NO is the most important vascular relaxing factor, which is also regulated in the endothelium, and alterations in the endothelial production of NO are known to correlate with endothelial dysfunction (21). Compared with the normal rats, an increase in the serum level of ET-1 was observed in the model group, whereas oleanolic acid administration significantly downregulated the level of ET-1 in the diabetic rats (Fig. 3A). The results also showed that the levels of NO were significantly decreased in the serum of diabetic rats, compared with the non-diabetic control group. Oleanolic acid administration significantly prevented the hyperglycemia-induced decrease NO in diabetic rats (Fig. 3B). The serum concentration of vWF, a well-known marker of endothelial function/injury, was significantly higher in the diabetic rats, compared with that in the control group rats (Fig. 3C). Oleanolic acid treatment significantly reduced the levels of vWF in the diabetic rats. The findings also demonstrated that oleanolic acid significantly reversed the hyperglycemia-induced increase in vascular permeability (Fig. 3D), which is a characteristic of diabetic vasculopathy (22).

Consistent with the results described above, the present study confirmed that the mRNA and protein expression levels of ET-1 were markedly upregulated in the carotid artery of diabetic rats, compared with those of non-diabetic control rats. Oleanolic acid administration significantly downregulated the mRNA and protein expression of ET-1 in the diabetic rats (Fig. 4A and B).

As shown in Fig. 5A, hyperglycemia resulted in upregulation of the serum level of TNF-α in the rat model of carotid artery injury, compared with that in the control group. Similarly, the serum levels of IL-1β (Fig. 5B), IL-6 (Fig. 5C) and IL-18 (Fig. 5D) were significantly increased in the diabetic rats, compared with those in the control group rats. However, oleanolic acid administration led to significant decreases in the levels of TNF-α, IL-1β, IL-6 and IL-18 (Fig. 5A-D). These results suggested a major role for oleanolic acid in reducing the circulating levels of pro-inflammatory cytokines. To further investigate the anti-inflammatory effect of oleanolic acid and its molecular mechanisms in the progression of hyperglycemia-induced carotid artery injury, the expression levels of NLRP3 inflammasome components were examined in the carotid arteries of the rats. RT-qPCR and western blot analyses of the expression levels of NLRP3 inflammasome components in the carotid arteries revealed that the mRNA levels of NLRP3 (Fig. 6A), caspase-1 (Fig. 6B) and IL-1β (Fig. 6C), and their protein levels (Fig. 6D and E) were higher in the diabetic rats, compared with those in the control group rats. Oleanolic acid administration led to significant decreases in the levels of these NLRP3 inflammasome components.