Nlrp3

A total protein extraction kit (Keygen Biotech, Nanjing, China), which consists of lysis buffer, phosphatase inhibitor, protease inhibitor and phenylmethylsulfonyl fluoride (PMSF), was used to extract protein from cells and heart tissues. Protein samples were quantified by BCA assays (Thermo Fisher, USA). The samples were separated using 10% or 12.5% SDS‐PAGE and transferred onto PVDF membranes. Then, 5% non‐fat milk was used as a blocking buffer. The membranes were incubated with primary antibodies against tubulin (1:10,000; Bioworld; cat. no. AP0064), caspase‐1 (1:1000; Proteintech; cat. no. 22915–1‐AP), cleaved caspase‐1 (1:500; Cell Signaling Technology; cat. no.89332), IL‐18 (1:1000; Proteintech; cat. no. 10663–1‐AP), GSDMD (1:1000; Cell Signaling Technology; cat. no.93709), NLRP3 (1:1000; ABclonal; cat. no. A5652), IL‐1β (1:1000; Proteintech; cat. no. 16806–1‐AP), PAK1 (1:1000; Proteintech, cat. no. 21401–1‐AP), PAK2 (1:200; Santa Cruz Biotechnology, cat. no. sc‐373740) and caspase‐7 (1:1000; Cell Signaling Technology; cat. no. 9492) at 4℃ overnight. The immunoblots were visualized by an Enhanced Chemiluminescence Kit (Thermo Scientific, USA) and semi quantified with tubulin as the internal control.

Immunofluorescence staining was used to examine NLRP3 inflammasome activation in cardiomyocytes. Briefly, cells were fixed with 4% PFA at room temperature for 30 min. Following permeabilization with 0.5% Triton X‐100 for 20 min, cells were blocked with 10% goat serum for 1 hour at room temperature and further incubated with α‐actinin (1:150; Sigma; cat. no. A7811) and NLRP3 (1:200; ABclonal; cat. no. A5652) at 4°C overnight in the dark. Then, the cells were stained with the proper secondary antibody. DAPI (Sigma, USA) was applied to label the nucleus. The staining results were acquired via fluorescence microscopy (Carl Zeiss, Germany).

DMEM was purchased from Hyclone (Beijing, China). Fetal bovine serum (FBS) was purchased from BI (Israel). Griess reagent was purchased from Beyotime (Shanghai, China). LPS, RIPA buffer, and CF3COOH (TFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibodies against mouse iNOS, COX-2, Tau, p-Tau, IBA-1 TREM2, DAP12, TLR4, MyD88, Caspase-1, and NLRP3 were obtained from Abclonal (Wuhan, China). HPLC-grade methanol and acetonitrile were supplied from Merck KGaA (Darmstadt, Germany). PBS, CM5 sensing chips were obtained from from GE Healthcare (Sweden). HP-20 macroporous resin was purchased from Mitsubishi Chemical Company (Japan).
An ultrasonic cleaner, high-speed freezing centrifuge, and electric heater were obtained from Branson (EYELA, Tokyo, Japan), and Biacore T200 (GE Healthcare, Sweden). The Xevo G2-XS Qtof was acquired from Waters Inc (Milford, USA.). Deionized water was collected from a Milli-Qwater purification system (Merck KGaA, Darmstadt, Germany). The rotavapor tandem cold trap was from EYELA (EYELA, Tokyo, Japan). The Epoch 2 microplate reader was got by BioTek (USA). The Electrophoresis Device and scanning imager were acquired from BIO-RAD.

The sterilized coverslips were placed into the wells of a 24-well plate, and then RIMVECs were added to each plate at the concentration of 1 × 10⁵ cells/well in 600 μL DMEM. When the cells reached 90% confluence onto the coverslip, they were divided into the same groups as described in the in vitro experiment paragraph. At the end of the incubation time, the cells were fixed with 500 μL paraformaldehyde (PFA) for 40 min, followed by permeabilization with 0.2–0.5% Triton X-100 for 20 min. The cells were washed three times with phosphate buffered saline (PBS) before and after each step and then incubated with the following primary antibodies at 4°C overnight: NLRP3 (1:1000, ABclonal), GSDMD (1:1000, ABclonal), caspase-1 (1:1000, ABclonal), and ZO-1 (1:1000, Affinity Biosciences). Next, the secondary antibody (1:5000 dilution in 5% BSA, ABclonal) was added, and the cells were incubated at room temperature for 1 h in the dark. One μg/mL 4’,6-diamidino-2-phenylindole (DAPI) was added to each well, and the cells were incubated at room temperature for 20 min in the dark. After washing, the coverslips were removed from the wells and blotted to remove any excess water. Each coverslip was placed onto a microscope slide, and the slides were sealed with nail polish and observed under a confocal fluorescence microscope (NIKON Ti-S, Tokyo, Japan).

RIMVECs were seeded, divided into groups, and treated as described in the in vitro experiment paragraph. Total proteins were extracted using the radio immunoprecipitation assay (RIPA) cell lysis buffer for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology, China). A standard western blot protocol was performed, and the following primary antibodies were used overnight at 4°C: TLR4 (1:800) (ABclonal, Wuhan, China), NLRP3 (1:1000) (ABclonal), GSDMD (1:1000) (ABclonal), caspase-1 (1:1000) (ABclonal), ASC (1:1000) (ABclonal), NF-κB p65 (1:1000) (Abmart, Shanghai, China), p-NF-κB p65 (1:400) (Abmart), p-I-κB (1:1000) (Abmart), I-κB (1:1000) (Abmart), IL-18 (1:1000) (Affinity Biosciences, Colorado, USA), IL-1β (1:1000) (Affinity Biosciences), claudin-1 (1:1000) (Affinity Biosciences), claudin-2 (1:1000) (Affinity Biosciences), ZO-1 (1:1000) (Affinity Biosciences), and β-tubulin (1:1000) (Affinity Biosciences) used as loading control, followed by the incubation with HRP (Horseradish Peroxidase)-conjugated secondary antibodies (1:5000) (ABclonal) for 1 h at room temperature. The blots were visualized using a Tanon 5200 chemiluminescence imaging system (Shanghai, China) and quantified using the ImageJ software (National Institutes of Mental Health, Bethesda, MD, USA).

Cells were lysed using the PRO-PREPTM Protein Extraction solution (iNtRon Biotech, Seongnam-Si, Republic of Korea). Total proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skimmed milk for 1 h. Specific proteins were detected with primary antibodies: NLRP3 (A12694), GSDMD (A20197), IL-1β (A1112), caspase-3 (A19654), caspase-9 (A11451), BNIP3 (A5683), Cyt-c (A1561), Bax (A19684), Bcl-2 (A19693), β-actin (AC026), p-mTOR (S2448) (AP0115), mTOR (A2445), p-PI3K (AP0845), PI3K (A4992), p-Akt (AP0637), Akt (A17909), P62 (A19700), and LC3B (A19665), which were all bought from ABclonal Technology Co. Ltd., Wuhan, China. The blots were then treated with horseradish peroxidase-conjugated secondary antibodies. Bands were visualized using the ECL reagent and FluorChem M system (ProteinSimple, SFO, San Jose, CA, USA)