5 bdbd

The MDBK cells were maintained in Dulbecco’s modified eagle medium (DMEM) containing 5% fetal bovine serum (FBS) (ThermoFisher, Shanghai, China), 100 U/mL penicillin, 100 μg/mL streptomycin. Immortalized bovine renal glomerulus epithelial (MDBK) cells were incubated at 37 ℃ and 5% CO₂. Referencing a previous study [26 (link)], we stimulated the MDBK cell with five treatments to mimic abnormal glycemia, including low glucose (LG), 2.5 mM glucose for 48 h; normal glucose (NG), 5 mM glucose for 48 h; high glucose (HG), 25 mM glucose for 48 h; reducing glucose (RG), 25 mM glucose for 24 h followed by 2.5 mM glucose for 24 h; increasing glucose (IG), 2.5 mM glucose for 24 hg followed by 25 mM glucose for 24 h (Figure 1A). In the experiments involving inhibitors, the MDBK cells (60~70% confluent) were pretreated for 30 min with each inhibitor dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich) and then diluted in a culture medium. N-acetyl-L-cysteine (NAC) was purchased from Solarbio Technology Co., Ltd., Beijing, China, and C17H11BrN2O2 (P2X receptor family 4 (P2X4) receptor antagonist, 5-BDBD) and MCC950 sodium salt (NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome inhibitor, MCC950) were purchased from Sigma Chemical Co., St. Louis, MO, USA.

To understand the effect of P2X4-NLRP3 pathway on PND, we treated the animals with the P2X4R-selective antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-be-nzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) (Sigma-Aldrich, USA) (28 mg/kg, i.p.) or saline 3 consecutive days after the surgery [18 (link)]. All the animals were randomly divided into four groups: (1) the control group (CON); (2) the control + 5 − BDBD group (CON + 5 − BDBD); (3) the surgery group (SUR); and (4) the surgery + 5 − BDBD group (SUR + 5 − BDBD). To avoid the putative confounding effects of behavioral tests on inflammatory markers, some animals in each group were sacrificed 3 days after the surgery for biochemistry on the brain tissue, while the others were subjected to behavioral tests. The experimental process is illustrated in Figure 1.

To understand the effect of the P2X4R/BDNF pathway on NTG-induced hyperalgesia, we treated the animals with the P2X4R-selective antagonist 5-BDBD (Sigma-Aldrich, USA) and the inhibitor of TrkB receptors ANA-12 (Sigma-Aldrich, USA) daily for 11 days; on days 3, 5, 7, 9, and 11, the mice were injected with NTG as described above. 5-BDBD (28 mg/kg, i.p.) and ANA-12 (1 mg/kg, i.p.) were administered immediately prior to the administration of NTG [10 (link), 11 (link)].

BSA, EGTA and reagents for producing Ca²⁺ solutions were from Amresco (Solon, OH, USA). Fetal bovine serum (FBS) was purchased from Bovogen Biologicals (East Keilor, Melbourne, Australia) and heat inactivated at 56 °C for 30 min before use. 2-APB, U-73122 and UDP were from Cayman Chemical (Ann Arbor, MI, USA). Primers for RT-PCR were from Integrated DNA Technologies (Coralville, IA, USA). 5-BDBD, ADP (pre-treated with hexokinase as per [41 (link)]), ATP, BAPTA-AM, BzATP, hexokinase from Saccharomyces cerevisae, ivermectin, MEM non-essential amino acid solution, paraformaldehyde, paroxetine, phosphate buffered saline (PBS), poly-_D-lysine hydrobromide (5 µg∙mL⁻¹ working stock), pluronic F-127, saponin, suramin and UTP were from Sigma-Aldrich (St. Louis, MO, USA). DMEM/F12 medium, ExoSAP-IT, Fura-2 AM, GlutaMAX, penicillin-streptomycin and 0.05% trypsin-EDTA were from ThermoFisher Scientific (Melbourne, Australia). 2MeSADP, AR-C118925, thapsigargin and TNP-ATP were from Tocris Bioscience (Bristol, UK).

Drug administration consisted of 5-BDBD, a P2X4R inhibitor, which was purchased from Sigma-Aldrich and diluted to 1.25 mg/mL in 0.5% methylcellulose. The optimal 5-BDBD dose was evaluated (Figure S4). The 5-BDBD at 3 mg/kg/day or an equal volume of vehicle was administered via oral gavage 30 min after ICH and was subsequently administered once daily for 7 consecutive days. ANA-12 was obtained from MedChem Express (Monmouth Junction, NJ, USA) and was diluted in DMSO. The optimal dose of ANA-12 was based on a previous report [25 (link), 37 (link)]. The mice received 0.5 mg/kg/day of ANA-12 intraperitoneally. The first injection was performed 1 day before ICH and was subsequently administered daily for 7 days.