Il 18

Jejunum tissues were homogenized at 4°C in RIPA lysis buffer which contained protease inhibitors (PMSF) (Beyotime, Shanghai, China), and concentrations were determined using a BCA assay (35 (link)). Samples were further diluted, and 5× SDS-PAGE loading buffer was added and boiled for 5 min. Equal amounts of protein (10 μg) were loaded onto 12% SDS-polyacrylamide denaturing gels before being transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with tris-buffered saline Tween (TBST) containing 5% non-fat milk powder for 1 h at room temperature, the membrane was incubated overnight with diluted primary antibodies against ZO-1 (1:1,000; ABclonal, Wuhan, China), occludin (1:1,000; Selleck Chemicals, USA), claudin-1 (1:1,000; ABclonal, China), TLR4 (1:500; Proteintech, Wuhan, China), MyD88 (1:500; Wanleibio, Shenyang, China), NF-κB (1:500; Wanleibio, China), NLRP3 (1:1,000; Wanleibio, China), ASC (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), caspase-1 (1:500; Wanleibio, China), IL-1β (1:500; Wanleibio, China), IL-18 (1:1,000; Wanleibio, China), and GAPDH (1:5,000; Proteintech, China). Electrochemiluminescence liquid (ECL) (Tanon, Shanghai, China) was used to detect the signal. ImageJ software was used to assess protein levels. The target protein levels were normalized to GAPDH, and the radioactivity was compared with the control group.

Total proteins were extracted from snap-frozen liver tissues or cells. We use a protein extraction kit (Keygenbio, KGP250) and centrifuge tube (Guangzhou Jet Bio-Filtration Co., Ltd.) for proteins extracting. The protein lysates (30 μg) were separated by electrophoresis in an 8–15% SDS–PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were incubated with appropriate antibodies at 4°C overnight and then incubated with a goat anti-rabbit or mouse conjugated secondary antibody (Sino Biological Inc., 1:3000). All blots were developed using an ECL Plus chemiluminescence system. The following antibodies were used: anti-Trx-1 (CST, #2429, 1:800), anti-NLRP3 (CST, #15101, 1:800), Caspase-1 p20 (Affinity, AF5418, 1:500), IL-1β (Wanleibio, WL0227, 1:500), IL-18 (Wanleibio, WL01127, 1:1000), ASC (Wanleibio, WL02462, 1:500), BAX (CST, #14796S, 1:500), BCL-2 (CST, #3498S, 1:500) and Cleaved Caspase-3 (CST, #9664, 1:500). ImageJ software was used for densitometry analysis and GAPDH was used as an internal control.

The 3μm thick paraffin-embedded kidney sections were dewaxed, and then dehydrated using graded concentrations of alcohol. To inhibit endogenous peroxidase, the sections were incubated with 3% H₂O₂. The sections were microwaved in citric acid for 15 min, and then treated with goat serum for 15 min at room temperature. Afterwards, the sections were incubated in blocking solution with primary antibody at 4°C overnight. After washing with PBS 3 times, the secondary antibody was added and immunostaining was performed using a DAB horseradish peroxidase color development kit (Beyotime, China), and then sections were counterstained with hematoxylin and made transparent with xylene. Finally, sections were observed with the PD37 type microscope (Olympus,Japan). Under 400 × magnification, pictures were taken in 5 random fields. Primary antibodies were used at the following dilutions: IL-1β diluted 1:100 (WL02257, Wanlei, Shenyang, China); IL-18 diluted 1:100 (WL01127, Wanlei, Shenyang, China).