Vcam 1

After 12 h of TMP pretreatment and subsequently12 h with LPS treatment, cells were lysed using cell lysis buffer, and protein concentrations were measured with a BCA assay kit (Beyotime, China). The aorta tissues were collected and homogenized with RIPA buffer. Equal amounts of denatured protein were subjected to 10% SDS-PAGE and blotted onto PVDF membranes (Millipore, United States). Antibodies against β-Actin (Santa Cruz Biotech, United States), MD-2 Monoclonal Antibody (Invitrogen, United States), and Toll-like Receptor four and Phospho-NF-κB p65 (Beyotime, China) were used as primary antibodies. HPSE1 (Signalway Antibody, United States), VCAM-1 (Servicebio, China), Syndecan-1 (Abcam, United States), HS (Amsbio, United Kingdom) and horseradish peroxidase-conjugated goat Anti-rabbit IgG and horseradish peroxidase-labeled Goat Anti-Mouse IgG (Beyotime, China) were used as the secondary antibody. All the primary antibodies were diluted to 1:1000 and the secondary antibodies were diluted to 1:8000. Detection was performed using an ECL kit (Biosharp, China), according to manufacturer’s instructions. The relative amount of protein was determined by densitometry using ImageJ software.

Samples from the left ventriculum were fixed in 4% paraformaldehyde at 25°C, embedded in paraffin, and cut into 3 μm thick sections. After deparaffinization in xylene, the tissues were subjected to antigen retrieval using 10 mM citrate-phosphate buffer (pH 6.0) and incubated in 3% H₂O₂ for 25 min. Then, the sections were blocked with 1% bovine serum albumin in PBS for 10 min. Following incubation with a prediluted biotinylated pan-specific universal secondary antibody (Servicebio, China) for 50 min at room temperature, sections were incubated overnight with mouse monoclonal ICAM-1 (1 : 800, Servicebio), VCAM-1 (1 : 400, Servicebio), MMP-2 (1 : 2000, Servicebio), and MMP-9 (1 : 800, Servicebio) antibody at 4°C. The tissues were washed with PBS, incubated in the streptavidin/peroxidase complex for 5 min and then washed with PBS again, before being washed with 3, 3′-diaminobenzidine (Servicebio, China) for 7 min. Sections were stained with hematoxylin for 3 min, then washed, and mounted. Images were captured using a fluorescent microscope (XSP-C204, CIC, Germany).