Ba0131

Mice were killed and the abdominal aortas were harvested for morphological analysis. To accurately measure the aortic diameter, the periadventitial tissue was carefully removed from aortic wall under a dissecting microscope. The aorta was then embedded in paraffin and cut into 4 µm for hematoxylin & eosin (H&E) staining. The external diameter of abdominal aorta was measured with an electronic caliper, combined with a software (Image J) after H&E staining. Elastin integrity of the aortic wall was evaluated by Verhoeff-van Gieson (VVG) staining and graded as four levels according to the reference [22 (link)]: (1) score 1, the elastic laminae was intact and with no degradation; (2) score 2, there were some breaks in the elastic laminae; (3) score 3, severe elastin fragmentations or loss in aorta; (4) score 4, severe elastin degradation occurred in reputed aortic sites.
For immunohistochemical (IHC) staining, the paraffin-embedded sections of AAA tissues were incubated with primary antibodies at 4 °C overnight. After washing, the secondary antibodies were used for 1 h. Then cell nuclear was stained with DAPI after washing. All images were taken by an inverted microscope and the inflammatory responses were assessed. Primary antibodies of TNF-α (BA0131, Boster, China), IL-1β (ab205924, abcam, UK) and IL-18 (ab71495, abcam, UK), were used for IHC staining.

Nrf2 (ab186825, Abcam), CBR1 (ab89433, Abcam), IL-1β (ab9722, Abcam), IL-6 (21865-1-AP, Proteintech), and TNFα (BA0131, Boster) were used for the IHC detection of protein expression in the trimethylamine (TMA). Nrf2 and CBR1 antibodies were both used at a 1:200 dilution. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. The TMA samples were incubated with biotinylated antibodies and horseradish peroxidase. Staining was developed with diaminobenzidine substrate and the sections were counterstained with hematoxylin. Negative controls were obtained by using PBS instead of Nrf2 or CBR1 antibody. The expressions of Nrf2 or CBR1 were quantified as previously described (Zhang et al., 2016 (link)).

BV-2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Lipopolysaccharide (LPS) was purchased from Sigma−Aldrich (St. Louis, MO, USA). Chlorogenic acid was purchased from Shanghai Yuanye Biotechnology Co. Ltd. (Shanghai, China). Anti-iNOS (1:1000, ab178945) and anti-MMP9 (1:1000, ab228402) were purchased from Abcam. Anti-CD86 (1:1000, BM4121), anti-Arg-1 (1:1000, M01106-4), anti-IL-10 (1:1000, RP1015), anti-CD206 (1:1000, A02285-2), anti-CXCL12 (1:1000, BA1389), anti-CXCR4 (1:1000, A00031-4), anti-PTGS2 (1:1000, A00084), and anti-TNF (1:1000, BA0131) were purchased from Boster Biological Technology Co. Ltd. (Shanghai, China). Cleaved caspase-3 (1:500, GB11532), IBA-1 (1:500, GB12105), and CD206 (1:400, GB113497) were purchased from Wuhan Servicebio Biotechnology Co. Ltd. (Wuhan, China). p-Akt1 (1:1000, 9018S), Akt1 (1:1000, 75692S), p-NF-κB (1:1000, 3033S), NF-κB (1:1000, 8242S), p-ERK1/2 (1:1000, 4370T), ERK1/2 (1:1000, 4696S), p-P38 (1:1000, 4511T), and P38 (1:1000, 8690T) antibodies were purchased from CST.