A 438079

The human monocytic cell line THP-1 and Raw264.7 murine macrophage-like cell line was obtained from American Type Culture Collection (Rockville, MD). And THP-1 cells was maintained at a density of 10⁶/ml in RPMI 1640 medium, Raw264.7 cells were cultured in DMEM medium, containing 10% FBS, 10 mM HEPES (Sigma, United States) and 1% pen/strep solution at 37°C in a 5% CO₂ incubator. THP-1cells were cultured in six-well plates for 48 h in the presence of 100 nM PMA, which induced differentiation into adherent macrophages (Tsuchiya et al., 1982 (link)). Then, the cells were pretreated with berberine (0–50 μM) or 100 μM A-438079 (a P2X7R inhibitor, Selleck, United States) and 10 μM compound C (AMPK inhibitor), PD98059 (a MAPK/ERK inhibitor), SB203580 (a p38 MAPK inhibitor), and SP600125 (a JNK inhibitor), which are MAP kinase inhibitors (Sigma, United States), for 1 h prior to incubation with oxLDL (50 μg/ml) for 24 h.

We isolated primary cardiomyocytes from Sprague‒Dawley rat heart tissue using previously described procedures [22 (link)]. H9c2 cardiomyocytes were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China). All cells were cultured with DMEM containing glucose (Gibco, USA, 4.5 g/L), and 10 % fetal bovine serum (Gibco, USA) and 1 % penicillin and streptomycin solution were added at 37 °C in a 5 % CO₂ incubator. Cardiomyocytes were pretreated with A-438079 (an inhibitor of P2X7R) from Selleck (#S7705, 10 μM), deferoxamine mesylate from Selleck (#S5742, 50 or 100 μM), or ferrostatin-1 from Selleck (#S7243, 1 or 5 μM) for 2 h before stimulation with Ang II (1 μM).

The P2X7R inhibitor (A438079), PKC inhibitor (LY317615), and ERK-specific inhibitor (PD98095) were purchased from Selleck (Shanghai, China). A RevertAid First Strand cDNA Synthesis Kit was obtained from Thermo Fisher Scientific (Waltham, MA, United States). SYBR Premix Ex Taq^TM (TliRnaseH Plus) was obtained from Takara (Dalian, China). The primary anti-P2X7R antibody (#APR-004) was obtained from Alomone Labs (Israel). Anti-collagen 1 (anti-COL-1, ab34710), anti-matrix metalloproteinase-9 (anti-MMP9, ab38898), and anti-heavy chain cardiac myosin (ab185967) antibodies were obtained from Abcam (Cambridge, United Kingdom). Moreover, antibodies against TGFβ (#3711), GAPDH (#5174), Caspase-3 (#9662), Bcl2 (#2870), phospho-PKCβII (Thr638/641) (#9375), Bax (#5023), and Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204; #4370) were obtained from Cell Signaling Technology (Danvers, MA, United States).