Zinc protoporphyrin 9 znpp

KGF-2 was provided by the School of Pharmacy, Wenzhou Medical University (Wenzhou, China). Antibodies of p-Akt (#4060), Akt (#4685), cleaved caspase-3 (#9664), Bcl-2 (#4223), and Bax (#2772) were provided by Cell Signaling Technology (Danvers, MA, USA). Antibodies of Nrf2 (ab137550) and the PI3K/Akt inhibitor LY294002 (ab120243) were provided by Abcam (Cambridge, MA, USA). Antibodies of β-actin (20536-1-AP), HO-1 (27282-1-AP), NQO-1 (11451-1-AP), CAT (21260-1-AP), SOD2 (24127-1-AP), and horseradish peroxidase-conjugated goat antimouse/rabbit secondary antibodies (SA00001-1 or SA00001-2) came from ProteinTech (Rosemont, USA). Hydrogen peroxide (H₂O₂, #349887), zinc protoporphyrin IX (ZnPP, #282820) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, M2128), and dimethyl sulfoxide (DMSO, D8418) came from Sigma-Aldrich (St. Louis, MO, USA). The reagents of cell culture were obtained from Gibco (Grand Island, NY, USA). The fluorescent dyes 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, S0033) and Annexin V-FITC/PI apoptosis detection kit (C1062L) were provided by Beyotime (Shanghai, China). Nrf2-small interfering RNA and fluorescein-conjugated control siRNA (sc-37030 and sc-36869) were provided by Santa Cruz Biotechnology Inc. (CA, USA).

Cardiac I/R injury experiments in animals were performed as described previously (21 (link), 22 (link)). In brief, the rats were anaesthetized with pentobarbital (70–80 mg/kg ip) every 2 h. Under sterile conditions, the heart was exposed by a left thoracotomy in the fourth intercostal space. I/R was achieved by a 30-min occlusion of the left anterior descending coronary artery (LAD), followed by 4-h reperfusion. The rat received a single intraperitoneal injection of PBS (vehicle) or zinc protoporphyrin-IX (ZnPP, an HO-1 inhibitor) (Sigma-Aldrich, St. Louis, MO) at a dose of 50 µmol/kg, and then underwent I/R. The maximal slope of systolic pressure increment (+dP/dt) and diastolic pressure decrement (−dP/dt) was measured in anaesthetized rat as described. After the haemodynamic measurements, the rats were sacrificed, the blood was collected, and the hearts were excised. CK release was measured by a commercially available kit (Sigma).

For the cell viability study, RAW 264.7 cells were seeded at 200 μL in a 96-well plate at a density of 5 × 10³ cells per well. After a 24 h incubation, the cells were incubated with the control medium (0.05% DMSO) or different concentrations of H₂O₂ for another 24 h or pre-incubated with various concentrations of glutathione for 1 h before a 500 μM H₂O₂ treatment for 24 h. The cells were also treated with 10 μM zinc protoporphyrin IX (ZnPP; Sigma-Aldrich Chemical Co.), a well-established HO-1 inhibitor, for 1 h in the presence or absence of H₂O₂. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Chemical Co.) solution (0.5 mg/mL) was added to each well incubated at 37°C for 3 h. After removing the medium, 200 µL of DMSO were added to each well to dissolve the formazan for 10 min. The effect for cell viability was assessed by measuring the optical density at a wavelength of 560 nm with a microplate reader (Dynex Technologies, Chantilly, VA, USA). The experiment was repeated in triplicate and the cell viability was determined by dividing the absorbance values of the treated cells to that of untreated (control) cells.