Mcc950

0.3% NaCl and 8% NaCl were administered to normal-salt (NS) group and the high-salt (HS) group over a period of 3 months, respectively. The rats were divided into 10 groups: (i) normal salt 2 months (NS2), (ii) high salt 2 months (HS2), (iii) normal salt 3 months (NS3), (iv) high salt 3 months (HS3), (v) NS + Vehicle, (vi) NS + MCC950, (vii) HS + Vehicle, (viii) HS + MCC950, (ix) NS + MCC950’, (x) HS + MCC950’.
After high-salt diet for 4 weeks, bilateral cannulae were implanted into the PVN of (v), (vi), (vii), (viii), (ix), and (x) groups rats for infusion of MCC950 (15 μg/h, Medchem Express), a specific NLRP3 blocker, or vehicle (artificial cerebrospinal fluid, aCSF). The dose applied for MCC950 was assessed from a study in rats with doses of 3, 15, and 65 μg/h [27 (link), 28 (link)]. The highest dose caused mortality while the 15 μg/h produced optimal response but the low dose recorded an incomplete inhibition. After 4 weeks of drug intervention, at the end of 8 weeks rats of (v), (vi), (vii), and (viii) groups were administered an anesthesia of ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture (ip); following this, the brains, hearts, aortas, and peripheral blood were removed and immediately frozen on dry ice. But (ix) and (x) groups were kept, and the rats were fed with high-salt diet for another 1 month without treatment until the end of the experiment.

The EAE model was induced as previously described [19 (link)]. Put simply, female C57BL/6 mice were subcutaneously immunized with 200 μl of 100 μg myelin oligodendrocyte glycoprotein (MOG35–55, Wuhan Haode Peptide, China) in incomplete Freund’s adjuvant (Sigma Aldrich, USA) containing 200 μg mycobacterium tuberculosis (strain H37Ra; Difco, USA) on day 0. Pertussis toxin (PTX; List Biologicals, USA) (200 ng) was administered intraperitoneally (i.p.) on day 0 and 2. EAE scores were evaluated as followed: 0.5, partial tail limpness; 1, tail limpness; 1.5, reversible impaired righting reflex; 2, impaired righting reflex; 2.5, paralysis of one hindlimb; 3, paralysis of both hindlimbs; 3.5, paralysis of both hindlimbs and one forelimb; 4, hindlimb and forelimb paralysis; 5, death. Animals were scored daily by two independent investigators in a blind fashion.
The mice were randomly divided into three groups (n = 12 in each group): (1) the control group (Ctrl), mice received saline; (2) the EAE group (EAE), mice were received immunization; (3) the EAE + MCC950 group (EAE + MCC950), EAE mice were intraperitoneally injected with MCC950 (10 mg/kg, Medchem Express, China) at induction of the disease, days 0, 1, and 2 and every 2 days thereafter.

Adult male C57BL/6 mice (7–8-weeks old, 22–25 g) were obtained from LingChang BioTech Co. Ltd. (Shanghai, China). Mice were subjected to CME as described in our previous study^{50 (link)}. Briefly, after the mice were anesthetized and ventilated with oxygen and 1.5% isoflurane (Baxter International Inc., Deerfield, IL, USA), a median thoracotomy was performed to expose the heart and ascending aorta. A total of 500,000 polyethylene microspheres (diameter 9 µm, Dyno Particles AS, Lillestrem, Norway) were injected into the left ventricle chamber during the occlusion of the ascending aorta for 15 s. The sham surgery mice received 0.9% saline instead of the microspheres in an equal volume. For the RVS treatment, mice were orally received 40 mg/kg/d RVS (AstraZeneca, Macclesfield, UK) three days before and after CME induction. To investigate the role of the NLRP3 inflammasome in CME-induced myocardial injury, another group of mice was injected intraperitoneally with a selective NLRP3 inflammasome inhibitor MCC950 (20 mg/kg/d, MedChemExpress, Princeton, NJ, USA) three days prior and after CME intervention.

We purchased GC-1 spermatogenic cells from ATCC (American Typical Culture Collection, Manassas, VA, United States) and cultured them in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) under normal oxygen conditions. We replaced glucose-containing DMEM medium with glucose-free DMEM medium and maintained the cells under hypoxic conditions for 3 h to simulate OGD/R (oxygen-glucose deprivation/reoxygenation) injury in vitro. Next, the cells were then switched to glucose-containing medium and incubated under normal oxygen conditions for 0, 6, 12, or 24 h. In addition, in the NLPR3 inhibition experiment, 50 μM MCC950 (MedChem Express, Shanghai, China) dissolved in dimethylsulfoxide was given in GC-1 cells 1 h before OGD/R treatment.

Cells were planted into the 12-well plate (1 × 10⁵ cells/well) and stimulated with 1 mM sevoflurane for 12 h. To assess the role of cGAS-STING pathway in sevoflurane-induced inflammation in BV2 cells, we treated cells with the cGAS inhibitor (RU.521, 1 µM, MedChemExpress, USA), the STING inhibitor (H151, 5 µM, MedChemExpress, USA) and the NLRP3 inhibitor (MCC950, 10 µM, MedChemExpress, USA) 30 min before sevoflurane stimulation. To evaluate the role of mitochondrial DNA in sevoflurane-induced inflammation in BV2 cells, we treated cells with the DRP1 inhibitor (Mdivi-1, 100 nM, MedChemExpress), the mPTP inhibitor (CsA, 1 µM, TargetMol, China) or the VDAC inhibitor (VBIT-4, 10 µM, TargetMol) 30 min before sevoflurane stimulation.