Txnip

Punicalagin (purity = 98%, CAS: 65995-63-3) was purchased from Herbpurify Co., Ltd. (Chengdu, China). A urea assay kit, a creatinine assay kit (sarcosine oxidase) and a urine microalbumin assay kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). A mitochondrial membrane potential assay kit with JC-1 and a tissue mitochondria isolation kit were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies targeting the following proteins were used in this study: NLRP3 (A12694, Abclonal), Trx (A01219-1, BOSTER), TXNIP (sc-166234, Santa Cruz; A9342, Abclonal), IL-1β (A1112, Abclonal), caspase-1 (BM4291, BOSTER), GSDMD (A10164, Abclonal), β-actin (60008-1-Ig, Proteintech), NOX4 (14347-1-AP, Proteintech).

Recombinant mouse MFG-E8 was obtained from R&D systems (Minneapolis, MN, USA). Hydrogen peroxide (H₂O₂) and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against MFG-E8, Aggrecan, TXNIP, and Caspase-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against Collagen II, MMP13, ADAMTS5, IL-1β, IL-18, NLRP3, GSDMD, PI3K, p-PI3K, AKT, p-AKT and Lamin B were purchased from Abcam (Cambridge, MA, USA). Primary antibodies against β-actin, Nrf2, SOD2, HO-1, and NQO1 were purchased from Proteintech (Rosemont, IL, USA).

Western blotting analysis was performed as reported previously [54 (link)]. In brief, the brain tissues or cells were lysed and centrifuged at 12,000× g for 10 min at 4 °C. Equal amounts of proteins (30 μg) were loaded into 10%–12.5% SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA) by electrophoresis. After blocking with 5% BSA for 1.5 h, samples were incubated overnight at 4 °C with primary antibodies against ZO-1 (1:200, Abcam, Cambridge, MA, USA), occludin (1:200, Abcam), caspase-1 (1:500, Abcam), IL-1β (1:500, Abcam), TXNIP (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), NLRP3 (1:200, Santa Cruz Biotechnology), p38 and phospho-p38 (1:1000, CST, Boston, MA, USA), JNK and phospho-JNK (1:1000, CST),GAPDH (1:8000, Bioworld, Louis Park, MN, USA) or β-actin (1:2000, Bioworld). The blots were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (dilution 1:8000, Bioworld) and developed with enhanced chemiluminescence (ECL, Vazyme Biotech, Nanjing, China). The immune-reactive bands were visualized using the ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA) and the results were quantified by using the Image Lab^TM Software (version 4.1, Bio-Rad, Hercules, CA, USA).

Cell extracts (80 µg/lane) were resolved on 8% SDS-PAGE, transferred to nitrocellulose sheets at 250 mA for 16 h, and probed with the antibodies anti-iNOS (BD Biosciences, Milano, Italy), eNOS/p-Ser¹¹⁷⁷ (Cell Signaling Technology, Danvers, MA, USA), eNOS (BD Biosciences), TXNIP, and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The SuperSignal chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect immunoreactive proteins. All the Western blots were repeated at least three times, and densitometry was performed by the ImageJ software (Java 1.8.0_241).

Three to four coronal sections from each mouse were washed with PBS and permeabilized with 0.4% Triton-X PBS for 20 minutes. The sections were then blocked with 10% normal donkey serum for 1 hour at room temperature in a buffer containing 0.1% Triton. Sections were incubated for 2 nights with the primary antibody at 4°C in the same buffer using the following antibodies: NeuN (1 : 200, Millipore MAB377), ASC (1 : 50, Santa Cruz, sc-22,514-R), NLRP3 (1 : 50, Santa Cruz, sc-66,846), cleaved caspase-1 p20 (1 : 50, Santa Cruz, sc-22,165), cleaved IL-1β (1 : 50, Santa Cruz, sc-23,459), and TXNIP (1 : 50, Santa Cruz, sc-33,099). After the primary antibody incubation, the sections were washed in PBS and incubated with the appropriate secondary antibodies (1 : 500, Alexa Fluor 488/568) for 1 hour at room temperature. Sections were then mounted with water-based DAPI-mounting medium containing antifading agents and observed using confocal microscopy. All images were captured on a confocal laser microscope (Carl Zeiss, Germany) using Zen software at 40x magnification. The intensity above threshold of the fluorescent signal of the bound antibodies was analyzed using NIH ImageJ software. Data were expressed as fold change from sham.