Specific pathogen-free (SPF) Sprague-Dawley (SD) experimental rats were purchased from Sleek Venture Experimental Animals Co., Ltd. [license number: SCXK (Xiang) 2019-0004]. The experimental unit used the license number SYXK (Gan) 2018-0004. A total of 30 male SPF SD rats (weighing 200-220 g, 6-to 8-week-old) were housed at the Laboratory Animal Center of Gannan Medical University (Ganzhou, China) under standard conditions of temperature (20-25˚C), humidity (50-60%) and lighting (12-h light/dark cycle), with free access to food and water. The rats were allowed to adapt for 3-5 days before the experiment. All animal experiments were conducted in the animal laboratory of the Laboratory Animal Center of Gannan Medical University and were approved by the Ethics Committee of Gannan Medical University (approval no. LLSC-2022110701). All animal procedures were performed following the guidelines of the Council on Animal Care (18). The research complied with the Five Freedoms (freedom from hunger and thirst, freedom from discomfort, freedom from pain, injury and disease, freedom to express normal behavior, freedom from fear and distress) and the 3R principles (Replacement, Reduction and Refinement) for experimental animals was observed. The present study strictly followed the regulations and guidelines set by the ethics committee to ensure that the welfare and rights of the animals were respected and protected.

On the day of the experiments, the animals were anesthetized with intravenous pentobarbital sodium (30 mg/kg). The rats were anesthetized and maintained under anesthesia throughout the experiment. At the end of the experiment, deep anesthesia was induced for sample collection, followed by euthanasia via intraperitoneal injection of 3% pentobarbital sodium (100 mg/kg) to ensure a painless and humane procedure. Experimental procedures and handling were conducted by trained professionals who ensured the scientific validity and reliability of the experiment.

The TDVT model, widely used in the present study, was established as follows: 28 male SPF SD rats weighing 200-220 g were randomly divided into sham operation group, model group, C29 group and Pam3CSK4 group. Except for the sham operation group, all SD rats were placed in the supine position and anesthetized with 3% sodium pentobarbital solution at a dose of 1 mg/kg via intravenous injection. Following anesthesia induction, a self-made traumatic quantifiable impact device (19) was used to deliver an instantaneous energy of 5 J striking each side of the proximal outer thigh of the rats once (1 cm below the greater trochanter) (19). Fractures of the femur were confirmed by bone scraping sensation or abnormal activity and confirmed by X-ray imaging. A window was created on the surface of the plaster in the superficial femoral vein area of the inner thigh on both sides to observe the formation of thrombus. After modeling, the experimental rats were allowed free access to food and water without the use of hemostatic agents or antibiotics. At 0, 24, 48 and 96 h post-modeling, rats in the sham operation and model groups were intraperitoneally injected with an equal volume of physiological saline; the C29 group received 5 mg/kg C29 (cat. no. HY-100461; MedChemExpress) (20) and the Pan3CSK4 group received 1 mg/kg Pam3CSK4 (InvivoGen) (21,22), Based on the results of preliminary experiments, 5 mg/kg of C29 and 1 mg/kg of PAM3CSK4 were selected to achieve a balance between the mortality rate of experimental animals and experimental effectiveness.

The rats were anesthetized via intraperitoneal injection of 3% pentobarbital sodium solution (30 mg/kg) and routine disinfection. At 24 h after the last administration, deep anesthesia was induced for sample collection, followed by euthanasia via intraperitoneal injection of 3% pentobarbital sodium (100 mg/kg). The size, length and weight of deep vein thrombosis and thrombus formation rate were measured.

Human umbilical vein EC (HUVECs; immortalized human umbilical vein EC Line Certificate of STR Analysis:FH1122, STR 20170721-05)were purchased from Shanghai Fuheng Biology Co, Ltd. and cultured in RPMI-1640 (cat. no. 22400089; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (cat. no. 10099141; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. P1400-100; Beijing Solarbio Science & Technology Co., Ltd.) in a 5% CO₂ incubator at 37˚C. At the logarithmic growth stage, the cells were divided into control, oxygen-glucose deprivation (OGD) model (model), Pam3CSK4 and C29 groups. All the cells were subjected to OGD, with the exception of those in the control group. The HUVECs were treated with Pam3CSK4 (1 µmol/l) or C29 (1 µmol/l) for 1 h before OGD (23). Meanwhile, an equal volume of RMPI-1640 was added to the control and OGD groups. To induce OGD, the cells were rinsed and incubated in glucose-free RPMI 1640 (cat. no. 11879020; Gibco; Thermo Fisher Scientific, Inc.) saturated with 95% N₂ and 5% CO₂ for 6 h. The cells were then rinsed and maintained under normal conditions for 24 h.

HUVECs at the logarithmic growth stage were seeded into 96-well plates (3,000 cells/well) and treated with Pam3CSK4 (0.1, 1.0 and 10 µmol/l) or C29 (0.1, 1.0 and 10 µmol/l) for 24, 48 and 72 h. CCK8 proliferation assay was performed using a CCK8 Kit (cat. no. CA1210; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Each well was treated with 10 µl of CCK8 for 4 h and the absorbance was measured at 450 nm using a microplate reader (VLBLATD2 Multifunctional enzyme marker; Thermo Fisher Scientific, Inc.). Each group had six wells and the experiment was repeated thrice.

HUVECs were seeded into six-well plates at a density of 5x10⁴/ml, followed by the addition of Pam3CSK4 (1 µmol/l) or C29 (1 µmol/l) for 24 h. The cells were collected and stained using an Annexin V-FITC/PI kit (cat. no. C1062L; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The HUVECs were exposed to 5 µl of Annexin V-FITC and 10 µl of PI solution for 15 min in the dark. After staining, the cells were run on a Gallios flow cytometer (Beckman Coulter, Inc.) and the data were analyzed using the Kaluza software (perpetual A82959; Beckman Coulter, Inc.).

Total RNA from fresh deep vein tissues from model animals and HUVECs (1x10⁶/well in 6-well plates) was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Additionally, 1 µg RNA was reverse transcribed into cDNA using the HI Script Q RT SuperMix (Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. RT-qPCR was performed using the cDNA templates and SYBR (Takara Bio, Inc.) in a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc.). Specific primers for TLR2, TF and IL-6 were designed and synthesized according to the GenBank (National Center for Biotechnology Information) sequences. RT-PCR was performed using Maxima SYBR Green qPCR Master Mix (cat. no. K0251; Thermo Fisher Scientific, Inc.). RT-PCR was performed using Maxima SYBR Green qPCR Master Mix (cat. no. K0251; Thermo Fisher Scientific, Inc.). The PCR cycling conditions were: denaturation at 95˚C for 30 sec, annealing at 60˚C for 30 sec and extension at 72˚C for 30 sec, for a total of 40 cycles. Gene expression levels were calculated using the 2^-ΔΔCq method (24), normalized to GAPDH. The experiments were replicated three times. The primer sequences are listed in Table I.

Total protein was extracted from HUVECs and vessel tissues using radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA Protein Assay Kit (cat. no. 71285-M; MilliporeSigma) following the manufacturer's protocol. For western blot analysis, 50 µg of lysate was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred onto polyvinylidene difluoride membranes (MilliporeSigma). Equal amounts of proteins were resolved on 10% SDS-PAGE gels and blotted to polyvinylidene difluoride membranes. The membranes were blocked in 5% not-fat dry milk at room temperature for 1 h and incubated overnight with primary antibodies (1:1,000) against TLR2 (cat. no. ab213676; Abcam, Cambridge, UK), NF-kB p65 (cat. no. ab16502; Abcam) and IL-6 (cat. no. ab233706; Abcam). After retrieval of the primary antibody, the membranes were washed thrice with 1XTris-buffered saline with 0.1% Tween 20 for 5 min each. Secondary antibodies conjugated with horseradish peroxidase were diluted 1:1,000 according to the corresponding specifications. Incubation with secondary antibodies was conducted at 4˚C overnight or at room temperature for 4 h. Secondary antibodies (1:3,000; cat. nos. 31460 and 31430; Thermo Fisher Scientific, Inc.) were incubated at room temperature for 1 h. Proteins were visualized using an ECL reagent (cat. no. 34580; Thermo Fisher Scientific, Inc.) Densitometry analysis was performed using Quantity One Software and quantified relative to the loading control, β-actin. Image analysis was performed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

NF-κB translocation in the vein tissues and HUVECs was determined using an NF-κB Activation, Nuclear Translocation Assay Kit (cat. no. SN368; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. HUVECs were seeded onto slides at a density of 1x10⁴ cells per slide. After fixation with 4% paraformaldehyde for 15 min at room temperature (approximately 25˚C). Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. he sections and cells were blocked with 3% bovine serum albumin (BSA; cat. no. A7030; MilliporeSigma) for 1 h. The samples were incubated with primary antibodies, including an NF-κB antibody (1:200, cat. no. ab16502; Abcam) and TLR2 antibody (1:200, cat. no. ab213676; Abcam) for 1 h at room temperature. They were then washed thrice with phosphate-buffered saline (PBS) for 5 min each time. The samples were incubated with a secondary antibody solution (1:500, goat anti-rabbit IgG, cat. no. A-11008; Invitrogen) for 1 h at room temperature. The sections and cells were treated with DAPI solution (1 µg/ml, cat. no. D9542; MilliporeSigma) for 5 min at room temperature. An antifluorescence quencher (cat. no. P0126; Beyotime Institute of Biotechnology) was added. Images were captured under a laser confocal fluorescence microscope (FV1200; Olympus Corporation) at a magnification of 40x and analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

For histological examination, tissues were fixed with 4% paraformaldehyde at room temperature for 24 h, washed with 70% alcohol, dehydrated through a graded ethanol series (70, 80, 95, and 100%, 10 min each), and cleared in xylene (two changes, 10 min each). Tissues were embedded in paraffin and cut into 4 µm sections. Sections were deparaffinized in xylene (two changes, 10 min each), rehydrated through a graded ethanol series (100, 95, 80, and 70%, 5 min each), and rinsed in distilled water. Hematoxylin staining was performed at room temperature for 5 min, rinsed in running tap water for 5 min, followed by eosin staining at room temperature for 2 min. Sections were then dehydrated through a graded ethanol series and cleared in xylene. Images were acquired under a light microscope (DM 2500; Leica Microsystems GmbH).

Results were presented as mean ± SD and calculated using the SPSS 21.0 software (IBM Corp.) and GraphPad Prism 8.0 (Dotmatics). One-way ANOVA test was employed followed by Tukey post hoc test. P<0.05 was considered to indicate a statistically significant difference.

A DVT model was first established using the quantitative impact and gypsum fixation method (plaster cast) and X-ray examination was performed to examine the effect (Fig. 1A). After successful modeling, the number of thrombus formation cases in each group as well as the length and weight of deep vein thrombosis were compared (Table II). Inflammation was evaluated via hematoxylin and eosin staining of the tissue samples (Fig. 1B). The number of thrombus formation cases, as well as the weight and length of the thrombi, were higher in the Pam3CSK4 group compared with the model and C29 groups (Fig. 1C-E). This indicated that TLR2 activation increases the incidence of DVT in TDVT model rats, whereas TLR2 inhibition reduces DVT incidence.

TLR2 expression was higher in the model group and NF-κB phosphorylation was positively correlated with TLR2 expression. To verify the relationship between TLR2 and its downstream molecules in the TDVT model, immunofluorescence staining, PCR and western blotting of TDVT tissues were conducted. Immunofluorescence staining of TLR2 in the vascular tissues of the animal model showed that TLR2 expression was significantly higher in the model group than in the sham group. In addition, TLR2 expression was significantly higher in the model + Pam3CSK4 group than in the model + C29 group (Fig. 2A and C). The present study also found a positive correlation between NF-κB expression and TLR2 expression as well as intergroup differences in expression (Fig. 2B and D). qPCR revealed that compared with those in the model group, the vascular EC of rats in the model + Pam3CSK4 and model + C29 groups had an increased transcription of TLR2 and the differences were significant (P<0.05; Fig. 2E). These results were confirmed by western blotting, which showed that TLR2 expression was positively correlated with P-65 and phosphorylated (p-)P65 expression in the TDVT animal model (Fig. 2F-I).

To validate the relationship between TLR2 and its downstream molecules in the TDVT model, the PCR and western blotting data from the tissues of the TDVT animal model were analyzed. It was found that TLR2 expression was significantly higher in the model group than in the sham group, along with a significant increase in the gene expression of IL-6, COX-2 and P-selectin. In the model + Pam3CSK4 group, the gene expression levels of IL-6, COX-2, P-selectin and TF were significantly higher than those in the model + C29 group (Fig. 3A and D). Western blotting analysis revealed that the protein levels of IL-6 and COX-2 in the TDVT model were higher than those in the sham group. Furthermore, TLR2 activation led to an increase in protein levels, whereas TLR2 inhibition resulted in a significant decrease in protein levels (Fig. 3E-G). Based on these results, it was considered that IL-6, COX-2 and P-selectin are the downstream molecules of TLR2/NF-κB in the TDVT model.

To validate the specific cellular changes responsible for alterations in thrombus formation in the TDVT model, the present study focused on EC injury induced by inflammation as a key factor in TDVT pathogenesis. EC are an important component of the vascular wall that maintain the integrity and normal blood flow of the vessel by controlling platelet activity and clotting factors in the blood. The present study used HUVECs and established an OGD model for analysis. The appropriate drug concentration for experimentation was determined using the CCK8 method. Pam3CSK4 at 1 µmol/l showed inhibitory effects on HUVECs and this effect increased with increasing Pam3CSK4 concentration. At low concentrations, C29 promoted HUVEC proliferation, but it peaked at 1 µmol/l and then declined. Pam3CSK4 and C29 both had the most significant promoting or inhibitory effects at 1 µmol/l and the differences compared with the control group were significant (Fig. 4A). In addition, the effects of TLR2 activation and inhibition on EC apoptosis under OGD conditions were observed. It was found that the apoptosis rate in the vehicle group was significantly higher than that in the control group but was lower than that in the Pam3CSK4 (Fig. 4B-D).

To verify the relationship between TLR2 and its downstream molecules in the HUVEC OGD model, PCR analysis weas performed. It was found that IL-6 expression increased in the vehicle group compared with the sham group and that the gene expression of IL-6, COX-2, P-selectin and TF increased significantly. In the model + Pam3CSK4 group, the gene expression of IL-6, COX-2, P-selectin and TF was significantly higher than that in the model + C29 group (Fig. 5A-D). Western blotting revealed that the protein levels of IL-6 and COX-2 in the TDVT model were higher than those in the sham group. TLR2 activation increased their protein levels, whereas TLR2 inhibition significantly decreased their levels (Fig. 5E-G). These results indicate that IL-6, COX-2 and P-selectin are the downstream molecules of TLR2/NF-κB.

To investigate the relationship between TLR2 and its downstream molecules in the HUVEC OGD model, immunofluorescence staining, PCR and western blotting analysis were performed. Immunofluorescence staining of TLR2 in the vein tissue of the cellular OGD model showed that TLR2 expression significantly increased in the vehicle group compared with that in the sham group. TLR2 expression in the OGD + Pam3CSK4 group was significantly higher than that in the OGD + C29 group (Fig. 5A and C). Meanwhile, NF-κB expression was positively associated with TLR2 expression and showed intergroup differences (Fig. 5B and D). qPCR showed that the transcription level of TLR2 in the EC of the model + Pam3CSK4 group was greater than that in the model group. The transcription levels of TLR2, P65 and p-P65 genes in the EC of the model + C29 group were higher than that in the model group. The differences between the two groups were significant (P<0.05; Fig. 6E). Similar results were obtained in western blotting, which showed a positive correlation between TLR2 expression and the gene expression of P65 and p-P65 in the TDVT animal model (Fig. 6F-I).