Urantide (peptide purity >95%) was synthesized by ChinaPeptides Co., Ltd., and has the following amino acid sequence: Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val.

All animals were maintained in accordance with the guidelines of the Certification and Accreditation of the People's Republic of China general requirements for quality and competence of laboratory animal institutions (GB/T 27416-2014) regarding animal care. A total of 45 male Wistar rats (age, 4 weeks; weight, 180–200 g) were purchased from Vital River Laboratory Animal Technology, Beijing, China [License no. SCXK (Jing)-2016-0011] and housed at a constant temperature of 22±2°C with a humidity of 40–60% and under a 12-h light/dark cycle, with free access to food and water. The rats were randomly divided into three groups, with 15 rats per group: The control group was fed basal feed daily, while the model group (AS) and urantide group were injected with 150 U/kg vitamin D₃ (Animal Pharmaceutical Huasheng Technology, Harbin, China) for 3 continuous days to damage the arterial intima (12) and were fed a high-fat diet (Beijing Keao Xieli Feed Co., Ltd.), which was composed of 80.8% basal feed, 10% hog fat, 3.5% cholesterol, 0.5% sodium cholate, 5% refined sugar and 0.2% propylthiouracil. The AS rat model was established using previously described methods (13). Then, 4 weeks later, 5 rats from each group were randomly sacrificed using an intraperitoneal injection of 150 mg/kg pentobarbital sodium [Fortune (Tianjin) Chemical Reagent Co., Ltd.] to detect whether the AS rat model was successfully replicated. Following successful modeling, the urantide group was injected with 30 µg/kg urantide, while the control and model groups were injected with 30 µg/kg normal saline daily for 2 continuous weeks. In the present study, the dosage of urantide was based on two preliminary studies by Zhao et al (10,11). After 6 weeks of treatment, all rats were fasted for 1 day, and the body weight of each rat was measured. Then, the rats were sacrificed using an intraperitoneal injection of 150 mg/kg pentobarbital sodium, and 5–8 ml blood per rat was harvested from the aorta pectoralis. The liver weight of each rat was measured to calculate the liver index with the following formula: Liver index = liver weight/body weight × 100%. The tissues were collected and immediately frozen at −196°C.

Sections of the aorta pectoralis and liver tissues were harvested, fixed with 10% formalin at 4°C overnight, dehydrated in 70–100% ethanol and finally embedded in paraffin. Then, paraffin sections were sliced into 5-µm thick sections and used for H&E staining, immunohistochemistry and immunofluorescence. H&E (Nanchang Yulu Experimental Equipment Co., Ltd.) was performed according to the manufacturer's instructions. Pathological changes in the aorta pectoralis and liver tissues were observed in ten microscopic fields at ×400 magnification using a light microscope (Olympus Corporation).

The presence of positive granules in the atherosclerotic plaques and liver cells were visualized using immunohistochemical staining for UII and GPR14, and immunofluorescence staining for phosphorylated (p)-Erk1/2 and p-JNK. Immunohistochemistry was performed using the SP kit (cat. no. SP-9002; OriGene Technologies, Inc.) according to the manufacturer's instructions. Briefly, sections were blocked with 3% H₂O₂ (cat. no. SP-9002; OriGene Technologies, Inc.) at 37°C for 30 min and then incubated with 1:100 dilutions of anti-UII antibody (cat. no. sc-52299; Santa Cruz Biotechnology, Inc.) and anti-GPR14 antibody (cat. no. sc-514460; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Sections were then incubated with biotin-labeled goat anti-mouse IgG antibody (cat. no. SP-9002; OriGene Technologies, Inc.) at 1:1,000 at 37°C for 30 min, incubated with 100 µl horseradish enzyme labeled streptomycin (cat. no. SP-9002; OriGene Technologies, Inc.) at 37°C for 15 min, incubated with 100 µl DAB (cat. no. ZLI-9017; OriGene Technologies, Inc.) at 37°C for 5 min, and mounted with neutral balsam. For immunofluorescence staining, the isolated paraffinized liver sections were dewaxed with xylene, and subjected to antigen retrieval with the EDTA antigen repair solution (cat. no. ZLI-9071; OriGene Technologies, Inc.) at 100°C for 2 min. The sections were then rinsed with TBS-0.1% Tween-20 (TBST) at 37°C for 5 min, blocked with 10% goat serum (Beijing Solarbio Science & Technology Co., Ltd.) in PBS-0.1% Tween-20 at 37°C for 1 h and incubated with 1:100 dilutions of anti-p-Erk1/2 (cat. no. 4370; Cell Signaling Technology, Inc.) and anti-p-JNK antibodies (cat. no. 9255; Cell Signaling Technology, Inc.) at 4°C overnight. Sections were then incubated with FITC-labeled anti-species-specific antibodies (cat. nos. A0562 and A0568; Beyotime Institute of Biotechnology) in the dark at 37°C for 1 h, and stained with a DAPI solution in the dark at 37°C for 45 min. Sections were then mounted with antifade mounting medium (Beyotime Institute of Biotechnology). Then, immunohistochemical and immunofluorescence staining were observed in ten microscopic fields at ×400 magnification using a fluorescence microscope (Olympus Corporation). Digital quantification (Image-Pro Plus 6.0; Media Cybernetics, Inc.) was performed in a blinded manner.

The plasma was centrifuged at 1,500 × g for 10 min at 4°C, and the serum was stored at −20°C. An automatic biochemical analyzer was used to detect rat serum levels of calcium (Ca²⁺), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), which were determined to assess the extent of AS in rats. The automatic biochemical analyzer was also used to measure alanine transaminase (ALT), aspartate aminotransferase (AST), γ glutamyl transferase (GGT), lactate dehydrogenase (LDH-L), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL), which were determined to assess liver function in rats. The measurements were made on the automatic biochemical analyzer (BS-480; Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) at the 266 PLA Hospital (Hebei, China). Liver tissues (1 g) were homogenized and extracted with 10 ml ice-cold dehydrated alcohol and then centrifuged at 1,500 × g for 10 min at 4°C. The hepatic levels of TC and TG were measured using Ultraviolet-Visible Spectrophotometry kits (cat. nos. BC1980 and BC0620; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol.

Total RNA was isolated from the rat livers using TRIzol^® reagent (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. The mRNA concentrations were measured using a Nano Drop spectrophotometer (Thermo Fisher Scientific, Inc.). The appropriate quantity of cDNA templates (~0.5 mg) was generated with a Fast Quant RT kit (Tiangen Biotech Co., Ltd.) at 42°C for 15 min and 95°C for 3 min, according to the manufacturer's protocol. Reverse transcription-quantitative (RT-q)PCR was performed using a SuperReal PreMix Plus kit (Tiangen Biotech Co., Ltd.) at 95°C for 15 min; followed by 40 cycles of 95°C for 10 sec and 60°C for 32 sec, according to the manufacturer's protocol (14). All samples from 6 rats per group were measured in triplicate, and the mean value was used for the comparative analysis. Quantitative measurements were calculated using the 2^−∆∆Cq method (15). β-actin served as the housekeeping gene for the comparison of the gene expression data. The primer sequences used for RT-qPCR analyses were synthesized by Takara Biotechnology Co., Ltd. and are listed in Table I.

The total protein was isolated from livers of 3 rats in each group using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), and the protein concentrations were evaluated using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). The samples were boiled at 100°C for 7 min, and then 45 µg protein/lane was separated using SDS-PAGE (10% gel) and transferred to a PVDF membrane. The PVDF membrane was incubated with 5% fat-free milk powder dissolved in a TBS solution containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then exposed to the following primary antibodies: Anti-UII (1:800), anti-GPR14 (1:800), anti-p-Erk1/2 (1:1,000), anti-Erk1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-p-p38 (1:1,000; cat. no. 4511; Cell Signaling Technology, Inc.), anti-p38 (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc.), anti-JNK (1:1,000; cat. no. 9252; Cell Signaling Technology, Inc.), anti-p-JNK (1:1,000) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) at 4°C overnight. The PVDF membrane was rinsed with TBST and incubated with horseradish peroxidase-labeled goat anti-rabbit or anti-mouse secondary antibodies (1:5,000; cat. nos. 074-1506 and 074-1806; KPL, Inc.) for 1 h at room temperature. The immunoreactive bands were visualized using the ECL western blotting substrate (Beijing Solarbio Science & Technology Co., Ltd.). A grayscale analysis of the target protein bands was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The experimental data were statistically analyzed using SPSS 20.0 software (IBM Corp.) and are presented as the mean ± SEM of ≥3 independent experiments. One-way ANOVA was used to compare the parameters among all groups followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical charts were produced using GraphPad Prism version 7.0 software (GraphPad Software, Inc.).

The present study examined the pathological changes in the thoracic aorta and liver of rats in each group to determine whether the AS rat model was successfully established, and to assess the effects of vitamin D₃ and a high-fat diet on the livers of AS rats. As shown in Fig. 1, in the control group, the thoracic aortic membrane had a clear boundary between the vascular tunica externa, media and intima. The vascular endothelial cells of the thoracic aorta were arranged in an orderly manner, and the elastic fiber layer structure and vascular smooth muscle cells were intact in the tunica media. In the AS model group, the thoracic aorta exhibited marked calcification and inflammatory cell infiltration, vascular endothelial cells were significantly damaged, elastic fibers were broken and disintegrated, and vascular smooth muscle cells were atrophied in the tunica media. All AS rats presented typical AS pathological characteristics. Following treatment with urantide, the vascular endothelial cells were notably recovered, and calcification and inflammatory cell infiltration were significantly decreased. Moreover, the elastic fibers and vascular smooth muscle cells in the tunica media were restored to normal.

Correspondingly, in the control group, the morphology of hepatocytes and the structure of hepatic plates were normal, and hepatic sinusoids displayed a uniform and regular arrangement. In the AS model group, the hepatocytes were obviously swollen and deformed, the arrangement of hepatic sinusoids was disordered, and the hepatic plates did not have a clear boundary. Inflammatory cell infiltration and vacuole-like lipid droplets were observed in the cytoplasm, and some nuclei were shifted to one side of the cells, which indicated typical hepatic steatosis. In the urantide group, the morphology of hepatocytes was restored to normal, and the hepatic sinusoids were arranged uniformly and regularly (Fig. 1). Based on these results, it was suggested that vitamin D₃ combined with a high-fat diet successfully induced AS and hepatic steatosis in rats, and urantide ameliorated the AS-related pathological changes and hepatic steatosis.

Vitamin D₃ combined with a high-fat diet induces AS and hepatic steatosis, resulting in lipid deposition in the blood and liver (12). The current study measured the serum Ca²⁺ and blood lipid levels to investigate the antihyperlipidemic effects of urantide. Compared with the levels in the control group, the serum levels of Ca²⁺, TC, TG and LDL in the AS model group were significantly increased, and the HDL levels were decreased. Furthermore, the TC and LDL levels in the urantide group were significantly decreased, and no changes in the levels of Ca²⁺, TG and HDL were observed, compared with the model group. Thus, urantide improved TC and LDL levels in rats with AS (Fig. 2A). The hepatic lipid levels and the liver index were also measured to examine the effects of urantide on hepatic steatosis. Compared with the levels in the control group, the hepatic levels of TC and TG in the AS model group were significantly increased, while the levels in the urantide group were significantly decreased (Fig. 2B).

The body weight was significantly decreased, and the liver index was significantly increased in the AS model group compared with the control group. Following treatment with urantide, the body weight and liver index in the urantide groups were both significantly recovered compared with the model group (Fig. 2C and D). Therefore, urantide effectively alleviated hepatic steatosis in rats with AS

Liver function was assessed by measuring biochemical indexes to investigate the degree of liver injury in rats with AS, according to a method described by Lu et al (16). Compared with the levels in the control group, the serum levels of ALT, AST, GGT, LDH-L, ALP, TBIL and IBIL in the AS model group were significantly increased (Tables II and III). Following treatment with urantide, the ALT, AST, GGT, LDH-L and IBIL levels in the urantide group were significantly decreased compared with the levels in the AS model group. Furthermore, the levels of DBIL showed no significant changes in the rats from any group (Tables II and III). In conclusion, rats with AS exhibited liver dysfunction, and urantide improved liver function, particularly hepatic synthesis, storage and metabolic functions, in rats with AS.

Expression levels of UII- and GPR14-positive particles are significantly upregulated in the thoracic aortas and livers of rats with AS. Immunohistochemical staining identified significantly higher expression levels of UII- and GPR14-positive particles in the thoracic aortas of rats in the AS model group compared with in the control group, and those indexes were significantly lower in the urantide group compared with the model group (Fig. 3A and B). Consistent with these findings, the expression levels of UII- and GPR14-positive particles in the livers of rats in the model group were also significantly increased compared with the control group, and urantide significantly decreased those indexes in the livers of rats with AS. Based on these results, the UII/GPR14 system may serve an important role in hepatic steatosis in rats with AS, and urantide-mediated inhibition of the UII/GPR14 system effectively improves hepatic steatosis.

The present study measured the mRNA and protein expression levels of UII and GPR14, as well as intermediates in the MAPK pathway, in the liver tissues of the rats in order to examine the molecular mechanism via which UII/GPR14 affects hepatic steatosis in AS rats. Significantly higher mRNA and protein expression levels of UII and GPR14 were detected in the liver tissues of rats in the model group compared with in the control group, and significantly lower mRNA and protein expression levels of UII and GPR14 were observed in the urantide group compared with in the model group (Fig. 4A and B). Consistent with these findings, compared with the control group, expression levels of the p-Erk1/2 and p-JNK proteins were significantly increased in the liver tissues of rats in the model group, and expression levels of the p-Erk1/2 and p-JNK proteins were significantly decreased in the urantide group compared with the model group (Fig. 4C). Moreover, the expression levels of the Erk1/2, p-p38, p38 and JNK proteins were not markedly changed in the liver tissues of rats in the various groups (Fig. 4C). Thus, hepatic steatosis in rats with AS was closely associated with the Erk1/2 and JNK pathways, and urantide significantly inhibited the activity of the Erk1/2 and JNK pathways. Therefore, urantide may effectively improve hepatic steatosis in rats with AS by regulating the Erk1/2 and JNK pathways.

The localization of UII and GPR14 was further analyzed to determine whether the UII/GPR14 system directly or indirectly affects the Erk1/2 and JNK pathways. These proteins were mainly expressed in perivascular and necrotic hepatocytes, as detected using immunohistochemistry. Consistent with these findings, p-Erk1/2 and p-JNK displayed similar localization patterns, as detected using immunofluorescence staining. Moreover, urantide significantly decreased the expression levels of p-Erk1/2 and p-JNK-positive particles in the livers of rats with AS (Fig. 5A and B). Based on these results, it was suggested that the MAPK/Erk/JNK pathway was substantially activated in the livers of rats with AS. Moreover, the expression levels of p-Erk1/2 and p-JNK are gradually restored in rats with AS treated with urantide.