A total of 45 male C57BL/KsJ db/db mice (age, 8 weeks; weight, 32-36 g) and 15 age-matched C57BL/6J control non-diabetic db/m⁺ mice (age, 6 weeks; weight, 16-18 g) were purchased from Changzhou Cavans Experimental Animal Co., Ltd., (SCXK2001-0003). All mice were housed in a well-ventilated environment, with a 12-h light-dark cycle, at 23±2°C and 70±10% humidity, with free access to water and food. All animal experiments were performed strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The research protocol was approved by the Traditional Chinese Medicine Guizhou University Animal Care and Ethics Committee.

All mice were randomly divided into four groups as follows: i) Age-matched healthy control (n=15); ii) model control; iii) db/db model + low-dose carvacrol (5 mg/kg); and iv) db/db model + high-dose carvacrol (10 mg/kg) groups. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). Subsequently, the db/db mice were treated with carvacrol (282197-50G, Sigma-Aldrich; Merck KGaA) daily for 6 weeks by gavage. At the same time, the normal control and model control groups were administered 0.9% saline at equal volumes. After 6 weeks, all the mice were euthanized by intraperitoneal injection of pentobarbital sodium (200 mg/kg) according to the recommendations of the animal ethics guidelines. Blood was quickly collected, centrifuged (at 3,000 × g for 10 min at 4°C) to obtain the blood serum samples, and stored at -20°C. The pancreatic tissues and skeletal muscles were removed and immediately immersed in 4% paraformaldehyde for 12 h at 4°C.

After 8 h of fasting, the mice were orally administered glucose solution (1.2 g/kg body weight). Blood was drawn from the tail vein, and the glucose levels were measured using a glucose monitor (Ascensia ELITE; Bayer).

The fasting serum levels of total cholesterol, triglyceride (TG), high-density lipoprotein (HDL) and non-HDL were detected using enzymatic methods (Stanbio Laboratory). Furthermore, the serum insulin concentration was analyzed by enzyme immunoassay (Mercodia).

The thoracoabdominal aorta was fixed at room temperature for 48 h in a buffer solution of 10% formalin, and then embedded in paraffin and sectioned at 20 µm. The sections were subjected to Masson's trichrome and hematoxylin and eosin (H&E) staining and observed under an optical microscope at a magnification, ×200 (BX51, Olympus Corporation). For immunohistochemical analyses, paraffin-embedded sections were incubated with anti-inhibitor of NF-κB kinase (anti-IKK; 1:100, ab32041, Abcam), anti-NF-κB inhibitor-α (anti-IKB-α; 1:100, ab32518, Abcam), anti-NALP3 (1:150, 19771-1-AP; ProteinTech Group, Inc.), anti-NF-κB (1:150, 14220-1-AP; ProteinTech Group, Inc.), anti-phosphorylated insulin receptor (anti-p-InsR; 1:100, ab60946, Abcam), anti-phosphorylated insulin receptor substrate-1 (anti-p-ISR-1; 1:100, ab3690, Abcam) and anti-toll-like receptor (TLR)4 (1:200, ab22048, Abcam) at 4°C overnight. Subsequently, the slides were incubated with secondary antibody (PV-9001; ZSGB-BIO, China) for 2 h at room temperature. The optical density was measured using ImageJ software, v1.8.0 (National Institutes of Health). The semi-quantitative results were evaluated by scoring the percentage of positive cells and staining intensity under the microscope. The percentage of positive cells was scored as follows: <5%, 0 points; 5 -25%, 1 point; 26-50%, 2 points; 51-75%, 3 points; and 76-100%, 4 points. The staining intensity was scored as follows: 0, no staining; 1, light yellow; 2, brown; and 3, tan. The product of the percentage of positive cells and the cell staining intensity was defined as the grade: 0, negative (−); 1-4, weakly positive (+); 5-8, positive (++); and 9-12, strongly positive (+++).

Total RNA was extracted from tissues or cells using a TRIzol reagent kit (15596-026, Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the extracted RNA was reverse-transcribed into cDNA on ice using the TaqMan cDNA Synthesis kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was carried out with the miScript SYBR Green PCR kit (A25742, PowerUp™ SYBR™ Green Master Mix, Applied Biosystems; Thermo Fisher Scientific, Inc.). The conditions for RT-qPCR were as follows: One cycle of 2 min at 50°C; one cycle of 10 min at 95°C; 40 cycles of 15 sec at 95°C and 40 cycles of 1 min at 60°C. The primer sequences of the targeted genes are shown in Table I. The relative expression levels of the genes were normalized to GAPDH expression. The fold changes in expression were calculated using the 2^-ΔΔCq method (PMID: 18546601).

Total protein was extracted from tissues or cells using RIPA lysis buffer (R0010; Solarbio) supplemented with 1% PMSF (P0100; Solarbio), and was determined with BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 120 µg protein was loaded per lane. Subsequently, the protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (IPVH00010; EMD Millipore). The membrane was then blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with the primary antibodies at 4°C overnight, and then incubation with the secondary antibody for 45 min. The primary antibodies included: Anti-IKK (1:1,000, ab32041; Abcam), anti-IKB-α (1:1,000, ab32518, Abcam), anti-NALP3 (1:2,000, 19771-1-AP, ProteinTech Group, Inc.), anti-NF-κB (1:2,000, 14220-1-AP, ProteinTech Group, Inc.), anti-TLR4 (1:2,000, ab22048, Abcam) and anti-β-actin (1:1,000; bs-0061R; BIOSS). β-actin was used as an internal control. Finally, the blots were visualized using an ECL kit (KGP1121; KeyGen Biotech Co., Ltd.).

Human umbilical vein endothelial cells (HUVECs) were cultured for 48 h in Endothelial Cell Growth Medium-2 BulletKit™ (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂, and divided into three groups as follows: i) Control; ii) endothelial cells + HG; and iii) endothelial cells + HG + carvacrol.

Inflammatory cytokines (IL-1β, IL-6, IL-18 and TNF-α), insulin and TG levels in the serum were measured using a 96-well microplate and commercially available ELISA kits (cat. nos. H007, H203 and H266, respectively; all from NanJing JianCheng Bio). The optical density was read at 450 nm using a microplate reader (saf-680t; Thermo Fisher Scientific Inc.). The concentrations of IL-1β, IL-6, IL-18, TNF-α, insulin and TG were quantified based on the standard curves that were constructed by Curve Expert 1.4 software (Beijing Boleide Development of Science and Technology Co., Ltd.). Three wells from each sample were assayed in this experiment.

Vascular endothelial cell apoptosis was evaluated using the Annexin V-FITC Apoptosis Detection kit (BestBio). The cells were incubated with Annexin V-FITC and propidium iodide (PI) in the dark for 15 min at room temperature. The number of apoptotic cells was analyzed using flow cytometry (BD Biosciences).

The cells were seeded in a 96-well plate (3,000 cells/well) and were cultured under different concentrations of carvacrol (20, 50, 100, 200, 500 and 1,000 µM/ml) at room temperature for 24 h. Subsequently, cell viability was evaluated using a CCK-8 Kit (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions.

HUVECs were fixed in 4% paraformaldehyde at 4°C for 15 min and incubated in hydrogen peroxide for 15 min. Subsequently, the cells were blocked with goat serum (Solarbio) at 37°C for 30 min, followed by incubation with anti-IKK (1:100, ab32041; Abcam), anti-IKB-α (1:100, ab32518; Abcam), anti-NALP3 (1:150, 19771-1-AP; ProteinTech Group, Inc.), anti-NFκB (1:150, 14220-1-AP; ProteinTech Group, Inc.), anti-p-INSR (1:100, ab60946; Abcam), anti-p-ISR-1 (1:100, ab3690; Abcam) and anti-TLR4 (1:200, ab22048; Abcam) at 4°C overnight.

Statistical analyses were performed using GraphPad Prism v7.0 (GraphPad Software, Inc.). Data are expressed as means ± standard error of the mean. The differences between two groups were calculated using Student's t-test. Comparisons among multiple groups were performed using one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate statistically significant differences.

Fibrosis was assessed using Masson's trichrome staining on thoracoabdominal aortic sections (Fig. 1A). Compared with the normal control group, severe fibrosis was observed in the thoracoabdominal aorta of the db/db mice in the model group. As described in a previous study, 5 and 10 mg/kg carvacrol did not affect the normal activity and movement of the mice (19). Thus, these concentrations of carvacrol were selected for the present study. Carvacrol treatment (5 and 10 mg/kg) significantly improved vessel fibrosis in db/db mice. The model control group db/db mice exhibited severe pathological changes of the thoracoab-dominal aorta on H&E staining, such as disorderly and loosely arranged HUVECs, hypertrophied, distorted an disordered vascular smooth muscle cells with an increased number of layers, different nucleus sizes, unclear cell membrane and nuclear membrane, uneven cytoplasmic staining, and broken intracellular muscle fibers (Fig. 1B). These histological abnormalities were significantly alleviated in the carvacrol treatment groups (5 and 10 mg/kg) compared with the model group.

It was first observed that carvacrol decreased the levels of fasting blood glucose and serum insulin in the db/db model compared with those in the db/db model control group. Moreover, carvacrol significantly increased the Homeostatic Model Assessment of Insulin Resistance of db/db mice compared with the db/db model control group (Table II). Next, the protein expression of the insulin signaling molecules IRS-1 and InsR was found to be significantly higher in the thoracoabdominal aorta of the db/db model group compared with the control group (Fig. 2A-C). However, carvacrol treatment (5 and 10 µM/ml) significantly reduced the expression levels of these proteins (Fig. 2A-C). Similar results were observed on immunohistochemical examination (Fig. 2D-G). Thus, carvacrol reduces the expression of insulin signaling molecules in the thoracoabdominal aorta of db/db mice.

As shown in Fig. 3A, the serum insulin level in the db/db mice group was significantly higher compared with that in the normal control group, and carvacrol treatment (5 and 10 mg/kg) markedly decreased the serum insulin level compared with the db/db mice of the model control group. It was observed that the serum TG level was significantly higher in db/db mice compared with that in the normal control group (Fig. 3B). However, carvacrol treatment (5 and 10 mg/kg) markedly reduced the TG level compared with that in the db/db mouse control group.

Inflammatory markers, such as IL-1β, IL-6, IL-18 and TNF-α, have been confirmed as the main cause of insulin resistance in diabetic patients. Therefore, the levels of IL-1β, IL-6, IL-18 and TNF-α were measured in the serum of db/db mice. It was observed that the serum levels of IL-1β, IL-6, IL-18 and TNF-α in the db/db mice of the model group were significantly higher compared with those in the normal control group (Fig. 3C-F). However, carvacrol treatment (5 and 10 mg/kg) obviously decreased the serum levels of IL-1β, IL-6, IL-18 and TNF-α compared with those in the db/db mice of the model control group.

It has been demonstrated that the TLR4/NF-κB signaling pathway is involved in the regulation of vascular inflammatory responses (20,21). To explore the potential underlying mechanism, the expression levels of the TLR4/NF-κB pathway molecules, including IKK, IKB-α, NALP3, NF-κB and TLR4, were measured in the thoracoabdominal aorta of db/db mice. The mRNA and protein levels of IKK, NALP3, NF-κB and TLR4 were found to be significantly higher in the db/db mice of the model group compared with those in the normal control group (Fig. 4A-K). However, carvacrol treatment (5 and 10 mg/kg) markedly decreased the levels of IKK, NALP3, NFκB and TLR4 compared with the db/db mice of the model control group (Fig. 4A-K). The results of immunohistochemical analysis were consistent with the results mentioned above (Fig. 5A-L). Moreover, it was observed that the IKB-α and p-IKB-α levels were higher in the thoracoabdominal aorta of db/db mice compared with those in the normal control group, whereas carvacrol treatment (5 and 10 kg/kg) decreased the levels of IKB-α and p-IKB-α.

Endothelial cell apoptosis and viability were then investigated. Flow cytometry was employed to evaluate HUVEC apoptosis. It was observed that carvacrol promoted vascular endothelial cell apoptosis in a dose-dependent manner (Fig. 6A). Cell viability was assessed with the CCK-8 assay. The results demonstrated that carvacrol decreased endothelial cell viability in a dose-dependent manner (Fig. 6B).

The protein levels of IRS-1 and InsR were found to be significantly higher in HG-induced HUVECs compared with those in the HUVECs control group (Fig. 7A-C), and carvacrol treatment (5 and 10 mg/kg) significantly reduced the levels of p-IRS-1 and p-InsR in HG-induced HUVECs (Fig. 7D-G). The results mentioned above indicate that carvacrol reduces the levels of insulin signaling molecules in HG-induced HUVECs.

It was inferred that carvacrol reduces the activation of the TLR4/NF-κB signaling pathway in HG-induced HUVECs. As expected, the mRNA and protein expression of IKK, IKB-α, NALP3, NF-κB and TLR4 were found to be increased in HG-induced HUVECs compared with those in the control group (Fig. 8A-J). However, carvacrol significantly decreased the expression of IKK, IKB-α, NALP3, NF-κB and TLR4 compared with the control group (Fig. 8A-J). These data indicated that carvacrol may be involved in the activation of the TLR4/NF-κB signaling pathway in HG-induced HUVECs. In addition, immunohistochemical analysis was performed. As expected, the results of immunohistochemistry were consistent with the results mentioned above (Fig. 8K-O). The hypothesis diagram of the present study is shown in Fig. 9.