THP-1 monocytes were cultured in the medium at 5% CO2 and 37℃. The medium was changed every 48h. Before experiments, the cells were transferred to 6-well/96 well plates with 2% FBS RPMI1640. Then we used PMA (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 100ng/ml for 48 hours to induce macrophages: The cells were treated with different concentrations of ox-LDL(Yiyuan Biotechnologies, Guangzhou, China) for 24 hours. Rapamycin(Sigma-Aldrich, USA) in the ox-LDL+rapamycin group was added 2 hours before ox-LDL treatment.
We used SPF ApoE-/- mice(Weitonglihua Laboratory Animal Technology Company, Beijing, China). All mice were divided into 2 groups: The control group was fed with a normal diet, while the AS group was fed with a high-cholesterol diet and received carotid artery perivascular neck ring implantation to build AS models. Mice in AS group underwent carotid artery perivascular neck ring implantation in the third week and were fed a high-cholesterol diet for another 7 weeks after the operation to induce unstable plaque formation quickly. Operation method: First, we fixed the mice then anesthetized the animals by intraperitoneal injection of sodium pentobarbital. Secondly, the common carotid artery is exposed and then separated from the surrounding connective tissue. Next, we spread it on the left and right sides of the common carotid artery with a collar with an inner diameter of 0.3 mm then put it on the common carotid artery and fixed its edges by placing 3 dissolvable sutures. Finally, the surgical incision was closed and the animal was returned to the cage for recovery. All experimental procedures follow the spirit of the Declaration of Helsinki on Animal Management and comply with the Guidelines for the Care and Use of Laboratory Animals.
The mice's carotid artery was surgically removed, rinsed, and placed in 4% formaldehyde solution. Part of the specimens was placed in a refrigerator at 4°C for 24 hours to make paraffin sections and part of the specimens was placed in a refrigerator at -80°C for Western blotting experiments.
Paraffin sections were deparaffinized to water. Carotid artery samples were cut into 5mm for hematoxylin and eosin (HE) staining and IHC analysis. For IHC, paraffin sections were dewaxed to repair the antigen. The slices were blocked with 5% goat serum and then cultured together with primary antibodies of p62 (ab91526, 1: 250, abcam) for 1 hour at 37 °C. The sections were washed with PBS 3 times and incubated with secondary antibody for 1 hour at 37 °C. Sections were stained with VECTASTAIN Elite ABC Kit (Vector Laboratories, USA). The cell nucleus was dyed with Mayer's hematoxylin solution. The staining slides were assessed with a light microscope at 400×magnification.
Oil red O staining
The cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 5-10 min, then washed with PBS three times to remove residual paraformaldehyde solution. The prepared oil red O solution(Sigma-Aldrich, USA) was added (2ml to each well of a 6-well plate or 0.5ml to each well of a 24-well plate) and incubated at 37°C for 15 min. The cells were washed three times with PBS, differentiated with 60% isopropanol for 30s, and washed three times with PBS for microscope observation and photography. Discarded PBS from the plate and the dye were extracted with 100% isopropyl alcohol for 10 min. The OD value of the mixture was quantitatively analyzed at 520 nm.
Transfection of small interfering RNA (siRNA)
We used X-tremeGENE siRNA Transfection Reagent(Roche, Basel, Switzerland) transfection p62 siRNA(GenePharma, China) to inhibit the expression of p62 according to the manufacturer's instructions. After 24 hours we detected whether gene silencing was successful and then conducted other experiments. siRNA-p62-1 is in the following order: GGAGGATCCGAGTGTGAAT. siRNA-p62-2 is in the following order: GGCACAGGGAAAAGA.
qRT-PCR to detect gene expression
We used a high-purity total RNA rapid extraction kit/miRNA column extraction kit to extract RNA/miRNA from each group of cells and reversed transcription of RNA using ReverTra Ace qRT-PCR Master Mix with gDNA Remover kit (TOYOBO, Japan). Real-time quantitative reverse transcription PCR (qRT-PCR) used the SYBR green method and Line Gene 9600 Plus RT-PCR detection system (Hangzhou Bori Company). The reaction mixture (20μL final volume) included 10μ THUNDERBIRD SYBR qPCR Mix (TOYOBO, Japan), 0.4μL ROX reference dye, 0.6μL forward/reverse primers (250nM final concentration), 6.4μL ddH2O and 2μL 1/10 diluted cDNA. Primers were purchased from (Genepharma, China) and the sequences were listed in Table S1. The thermal cycle program consists of holding at 95°C for 1 minute, then performing 15s at 95°C and 40 cycles of 60 s at 60°C. After completing these cycles, melt curve data was collected. The relative gene expression was calculated by the 2-ΔΔCt method, where Ct represents the cycle threshold.
Enzyme-linked immunosorbent assay (ELISA)
The cell culture solution was sucked out and placed in a centrifuge at 1000g for 20 min. The supernatant was sucked and transferred to EP tubes. Used commercially available ELISA kits (Wuhan Elite Biotechnology Company, Ltd, China), the cell supernatant TNF-α, IL-1β, and IL-10 protein were detected according to the manufacturer's instructions.
First, collected macrophages from different groups and made a single cell suspension. Then cells were incubated with cell surface fluorescent antibodies CD86 (ab239075,1:200, abcam) (add IgG antibody to the isotype control group) in the dark at 4°C for 30 min. After washing, the cells were permeabilized and incubated with intracellular fluorescent antibody CD206 (ab125028, 1:200, abcam) for 45 min. For each sample, at least 1 x 105 cells were analyzed using a BD FACS Calibur cytometer (Becton Dickinson, New Jersey, USA).
The total protein was extracted from the lysate buffer (Beyotime, China) by radioimmunoprecipitation containing protease inhibitor PMSF, and the protein concentration was determined using the BCA protein concentration kit (Solarbio, China). The denatured protein was separated by sds - polyacrylamide gel electrophoresis, transferred to PVDF membrane, and blocked with 5% bovine serum albumin (BSA) for 1 hour. Objective The strips were incubated overnight with an antibody 4 and then incubated for 1 hour with AP or HRP. Immune complexes were detected in exposed equipment using enhanced chemical luminescent agents and target protein content was quantified using ImageJ software.The dilution ratios of the various antibodies are: p62/SQSTM (ab91526, 1:1000, abcam), p65 (ab32536, 1:1000, abcam), IκBα (ab32518, 1:2000, abcam), iNOS (ab178945, 1:1000, abcam), Arg1(ab124917,1:1000, abcam), CD206/MRC1 (ab252921, 1:1000, abcam), goat anti-mouse secondary antibody (ab96879, 1:20000, abcam) and goat anti-rabbit secondary antibody (ab96899, 1:20000, abcam).
All data were analyzed with GraphPad Prism 6.0 software. The measurement data are expressed as mean ± standard deviation. Normal distribution data were used paired t-test (comparison between two groups) or analysis of variance (comparison between multiple groups). P<0.05 indicates that the difference is statistically significant.