Tert butyl hydroperoxide tbhp

EZ-Cytox reagent and KC (≥98%, HPLC) came from Dogenbio (Seoul, Korea) and ChemFaces (Wuhan, China), respectively. APAP, silymarin, tert-Butyl hydroperoxide (tBHP), ImmunoHistoMountTM, LY294002 PI3K/Akt inhibitor, ML385 Nrf2 inhibitor, and O8 OGG1 inhibitor came from Sigma-Aldrich (St. Louis, MO, USA). 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA, C400) came from Invitrogen (Carlsbad, CA, USA). Antibodies for 8-Oxoguanine DNA Glycosylase (OGG1), Nrf2, Bcl-2, Bax, phosphor-p38, phosphor-JNK, and β-actin came from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for caspase-3, cleaved caspase-3, Akt, phosphor-Akt, and phosphor-ERK came from Cell Signaling Technology Inc. (Beverly, MA, USA). JC-1 dye and MEBCYTO apoptosis kits came from Thermo Scientific (Rockford, IL, USA) and MBL International (Nagoya, Japan), respectively. Horse serum, ImmPRESSTM HRP, and 3-amino-9-ethylcarbazole (AEC) peroxidase substrate came from Vector laboratory (Burlingame, CA, USA). All other chemicals used were of reagent grade and came from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.

Cells were lysed in homogenization buffer (100 mM Tris pH 7.6, 300 mM KCl, 0.1% Triton X-100), sonicated, and centrifuged (20,000×g, 10 min, 4 °C). Total GPx activity was measured by the glutathione reductase-coupled test using 96-well plates [24 (link)]. After 10 min preincubation at 37 °C with all the assay components, the reaction was started by the addition of different substrates. Herein, H₂O₂ (Sigma-Aldrich), hydroxy-octadecadienoic acid (HPODE), hydroxy-eicosatetraenoic acid (HPETE) or tert-butyl hydroperoxide (TBHP, Sigma-Aldrich) were used to reach a final concentration of 50 μM. The reaction was measured for 2 min at 340 nm using a microplate absorbance reader (Biotech Instruments, Bad Friedrichshall, Germany). According to Lambert–Beer's law, one unit generally is defined as consumption of 1 μmol NADPH/min and expressed as mU/mg protein.

Male C57/B6 mice aged 8‐12 weeks were supplied by the Animal Center of the Wenzhou medical university. The animal use and care protocols were in line with the National Institutes of Health Guide of the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wenzhou medical University (Number: 2017‐010). Rat cardiomyocyte H9C2 cells were purchased from the American Type Culture Collection. DMEM and foetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Anti‐p‐PI3K (ab182651) was purchased from Abcam (Cambridge, MA). Anti‐PI3K (C73F8), anti‐AKT (C67E7), p‐AKT (Ser473), anti‐BAX (D3R2M), Bcl‐2 (D17C4), anti‐CHOP (D46F1), GRP‐78 (C50B12), ATF‐6 (D4Z8V), GAPDH (D16H11), β‐actin (13E5) antibodies and goat anti‐rabbit secondary antibody were purchased from Cell Signaling Technology, Inc (Danvers, MA). An enhanced chemiluminescence (ECL) kit was purchased from Bio‐Rad (Hercules, CA). Tert‐butyl hydroperoxide (TBHP) and PI3K/AKT inhibitor (LY294002) were purchased from Sigma‐Aldrich.

Curcumin (95% purity) was obtained from Alfa Aesar (Ward Hill, MA, USA); PLGA (Resomer RG 503 H) was supplied by Evonik (Essen, Germany); poly(vinyl alcohol) (PVA, Mowiol 4-88) was purchased from Kuraray (Hattersheim, Germany); polysorbate 80 (Tween 80), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2’,7’-dichlorofluorescin diacetate (DCFDA) and tert-butyl hydroperoxide (TBHP) were obtained from Sigma-Aldrich Chemie (Taufkirchen, Germany). Inhibitors of cellular uptake, viz. Filipin III, chlorpromazine and dynasore were also obtained from Sigma Aldrich. Ultrapure water, generated by a PURELAB flex 4 device (ELGA LabWater, High Wycombe, UK) was used for all experiments. For cell culture studies, ultrapure water was additionally autoclaved and filter-sterilised using 0.2 μm polyethersulphone membrane filters (Sarstedt, Nümbrecht, Germany) prior to use. HPLC-grade ethyl acetate (VWR, Darmstadt, Germany) was used to prepare the organic phases. All other chemicals used were of analytical grade. All buffers used in this study were prepared in the laboratory, unless stated otherwise.

Dosed mitochondria were pelleted and resuspended at a concentration of 1.5 mg of mitochondrial proteins/mL in PBS containing 500 µM of the oxidant tertbutyl hydroperoxide (TBHP) (Sigma-Aldrich). Mitochondria were oxidized for 1 h 30 min at 37 °C under gentle agitation then rinsed twice with ice-cold PBS. Oxidized mitochondria were subsequently quantified by BCA.