Caspase 1

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.

The rats were sacrificed according to different experimental cycles, and the bilateral hippocampi were immediately removed and stored in liquid nitrogen until further processing. Fifty milligrams of hippocampal tissue were used for Western blotting analysis as previously described [20 (link)]. Experimental procedures are explained in detail in Additional file 7. β-actin was chosen as an internal control to ensure equivalent amounts of protein. Densitometric quantification of the bands was performed using Image J software (version 1.29x: NIH, Bethesda, MD, USA). The following primary antibodies were used: NLPR3 (cat. no. WL02635, 1:1000, Wanleibio, China), Caspase-1 (cat. no. sc-56036, 1:1000, Santa Cruz, USA), ASC (cat. no. sc-514414, 1:500, Santa Cruz, USA), IL-1β (cat. no. sc-12742, 1:500, Santa Cruz, USA), GFAP (cat. no. BM0055, 1:1000, Boster biological technology, China), S100a10 (cat. no. 11250–1-AP, 1:1000, Proteintech, China), C3d (cat. no. AF2655, 1:1000, RD system, USA), and β-actin (cat. no. BM3873, 1:10,000, Boster biological technology, China). The original unedited blots were presented in Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 4: Figure S4 and Additional file 5: Figure S5.