Caspase 1

The GSVs from CABG patients and varicose veins were fixed in 10% formalin for at least 24 h. Specimens were dehydrated, embedded with paraffin and sectioned (Tissue-Tek and IVS-410, Sakura Company, Japan) for further investigations. Hematoxylin-Eosin (HE), Masson trichrome and elastic fiber staining for morphologic analysis were carried out for both GSVs from CABG patients and varicose veins. We measured the thickness at 4 points around the vessel circumference, including 12-o’clock, 3-o’clock, 6-o’clock and 9-o’clock. Then, the histopathological analysis was carried out under an optical microscope (DM4000 B, Leica, German) with Image J software (Version 1.48). The person who performed the histopathological examination was blinded to whether the specimens were GSVs from CABG patients or varicose veins. The immunohistochemistry staining for ASC, Caspase-1 and NLRP3 (NOD-like receptor family, pyrin domain-containing 3) was carried out with antibodies for human ASC (1:200, Proteintech, IL, United States), Caspase-1 (1:200, Proteintech, IL, United States) and NLRP3 (1:100, Abcam, Cambridge, United Kingdom) using EnVison method. Brown or brown-black particles in the cytoplasm were considered as positive expressions.

Total proteins were extracted from heart tissues or endothelial cells using protein extraction reagents. Protein concentrations were quantified, and equal amounts of proteins (60 μg) were separated by 12% SDS-PAGE gel and electro-transferred to nitrocellulose membranes. After blocking with 5% skimmed milk for 2 h, membranes were incubated with the following primary antibodies at 4 °C overnight: NLRP3 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 19771-1-AP), caspase-1 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 22915-1-AP), GSDMD (Santa Cruz, USA, 1:500, Cat. No.: A2315), IL-1β (ABclonal, Boston, USA, 1:1000, Cat. No.: A1112), IL-18 (ABclonal, Boston, USA, 1:1000, Cat. No.: A1115), Transferrin (BIOSS, Beijing, China, 1:1000, Cat. No: bs-2052R), or GAPDH (Proteintech, Chicago, USA, 1:2000, Cat. No: 60004-1-lg). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibody (1:10,000) for 2 h. Western blot bands were analyzed with the ChemiDicTM XRS+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the band densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

The colon tissues were homogenized with the ice−cold RIPA buffer containing protease inhibitors (Beyotime Biotechnology, Shanghai, China). The colon proteins were collected after centrifugation at 14,000× g for 5 min and determined using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by 4–12% SDS−PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking at room temperature with a commercial blocking buffer (Beyotime Biotechnology, Shanghai, China), membranes were incubated overnight at 4 °C with the following primary antibodies: Caspase 3, B cell lymphoma2 (Bcl2), Bcl2 associated X (Bax), cleaved Caspase 3 P17, cleaved Caspase 1 P20, and cleaved IL−1β, which were purchased from Affinity Biosciences (Cincinnati, OH, USA); Occludin, Claudin 1, ZO−1, ACS, NLPR3, Caspase 1, IL−1β, GSDMD, and β−actin, which were purchased from Proteintech (Wuhan, Hubei, China). Then, membranes were incubated with HRP−conjugated secondary antibody for 2–2.5 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Wilmington, DE, USA) and detected by the ChemiDoc™ imaging system (BIO−RAD, Hercules, CA, USA). The results were quantified using Image J software and normalized to β−actin.