Hy 10219

Some mouse embryos subjected to in utero electroporation were allowed to be delivered. Among them, some at P30 were closely observed for seizure behaviors and then subjected to EEG recording, and some at 1.5 months old were first treated with rapamycin (HY-10219, MedChemExpress, Shanghai, China) at 10 mg/kg for 11 days and then subjected to the seizure study and EEG recording. EEG data were analyzed using Easy EEG II (version 2.0.1).

The mouse vascular smooth muscle cell line (MOVAS-1) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM (Hyclone, Logan, UT, USA) containing 10% FBS (Gibco, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin at 37 °C in a humidified incubator with 5% CO₂. Cells between passages 6 and 8 were used for all experiments. VSMC calcification was induced according to previous protocols [20 (link), 21 (link)]. Briefly, VSMCs were cultured with growing medium in the presence of β-glycerophosphate (β-GP) (10 mM; G9422; Sigma, St. Louis, MO, USA) for 7 days. To investigate the role of Irisin in VSMC calcification, Irisin (067-29; Phoenix Pharmaceuticals Inc, Burlingame, CA, USA) at different concentrations (50 or 100 ng/ml dissolved in PBS) was used to treat VSMCs in the presence of β-GP medium for 7 days with medium changes every 2–3 days. For pharmacological treatment, the CASP1 inhibitor VX-765 (10 μM; HY-13205), NLRP3 inhibitor MCC950 (100 μM; HY-12815A), ROS scavenger N-acetyl-L-cysteine (NAC, 20 μM; HY-B0215), autophagy inducer rapamycin (200 nM; HY-10219), autophagy inhibitor 3-methyladenine (3-MA, 5 mM; HY-19312), and chloroquine (CQ, 25 μM; HY-17589A) were purchased from MedChem Express (NJ, USA) and used to treat VSMCs according to the experimental design.

In vitro, each group repeated independently for 3 times (n = 3). And all experimental groups received 6 h of oxygen-glucose deprivation and 18 h of reoxygenation (29 (link)). CGS-21680 (A2aR specific agonist, 30μM, Tocris Bioscience, 1036) and dbcAMP (selective PKA activator, 5μM, MedChemExpress, HY-B0764) were added 1 h before reoxygenation. H89 (the PKA selective inhibitor, 10 μM, MedChemExpress, HY-15979) was used 5 min before CGS21680. To verify the effect of autophagy on cell survival, autophagy agonist Rapamycin (100 nM, MedChemExpress, HY-10219) and antagonist 3-Methyladenine (3-MA, 10 mM, MedChemExpress, HY-19312) were used 1 h before reoxygenation (38 (link), 39 (link)).
In vivo study, 36 adult male rats were randomized into six groups (n = 6): Sham group, IR group (30 min LAD occlusion and 120 min reperfusion, 1% DMSO in 1 ml saline, iv), IR+CGS21680 group (30 μg/kg 5min before reperfusion and 30 μg/(kg·min) for 1 h, i.v.), and IR+ZM241385 group (A2aR antagonist, 0.2 mg/kg 5 min before reperfusion, i.v.), IR+dbcAMP group (5 mg/kg, 5 min before reperfusion, i.v.) (40 (link)), and IR+CGS21680+H89 group (20 mg/kg, 5 min before CGS21680, i.v.) (41 (link)).

Purified Ccr2‐KO splenic B cells (2 × 10⁶) were incubated with rapamycin (20 nM, HY‐10219, MedChem Express), XMU‐MP‐1 (3 μM, T4212, TargetMol), fludaradine (5 μg, T1038, TargetMol) or R406 (5 μM, T6174, TargetMol) at 37°C for 2 h (R406 for 4 h) before being activated with sAg. Then western blotting was performed on WT B, Ccr2‐KO B and Ccr2‐KO plus inhibitor B cells.