Curcumin

Curcumin was obtained from Cayman Chemical (Ann Arbor, MI, USA) and used without further purification. A 4 mg·mL⁻¹ stock solution of Curcumin was prepared in dimethylsulfoxide (DMSO). A standard curve generated by serial dilutions of the stock solution was used to determine Curcumin concentrations in unknown samples by absorbance at 430 nm. Recombinant human apoA‐I was expressed in Escherichia coli, isolated, and the N‐terminal His‐tag removed as described previously 22. Protein concentration was determined by the bicinchoninic acid assay (Pierce Chemical Co., ThermoFisher, www.thermofisher.com) using bovine serum albumin as standard. ThT was purchased from AnaSpec (Fremont, CA, USA).

MDA-MB-231 human breast cancer cells (ATCC number HTB-26™) were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, U.S.A.) supplemented with 10% fetal bovine serum (Sigma, St. Louis, U.S.A.), 100 U/ml penicillin (Sigma) and 0.1 mg/ml streptomycin (Sigma). The cells were grown at 37°C in a 5% CO₂ humidified incubator. Compounds were purchased from the following companies: celastrol and curcumin (Cayman, Michigan, U.S.A.), L-sulforaphane and resveratrol (Sigma), and betulinic acid (Tocris, Bristol, U.K.). Human recombinant TNFα was purchased from Sigma.

Gentamicin was obtained from the Pharmaceutical Organization, Inc. (Bangkok, Thailand). Curcumin was purchased from the Cayman Chemical Company (Ann Arbor, MI). Biochemical kits for TBARS and glutathione (GSH) assay were obtained from the Cayman Chemical Company (Ann Arbor, MI). Superoxide dismutase (SOD) assay kit was purchased from BioAssay Systems (Hayward, CA). All other chemicals and reagents were purchased from a commercial source at the analytical pure grade.

Crystalline lycopene preparations, purified from tomato extract (>97%), were supplied by Lycored Ltd. (Beer Sheva, Israel). Tetrahydrofuran (THF), containing 0.025% butylated hydroxytoluene (BHT) as an antioxidant, was purchased from Aldrich (Milwaukee, WI, USA). fetal calf serum (FCS), sodium pyruvate, and Ca²⁺/Mg²⁺-free PBS were purchased from Biological Industries (Beth Haemek, Israel). DMEM medium was purchased from Gibco (Grand Island, NY, USA). α-MEM medium, Dimethyl sulfoxide (DMSO), P-nitrophenyl phosphate and acid phosphatase leukocyte kit (387A) were purchased from Sigma Chemicals. Curcumin was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Carnosic acid was purchased from Alexis Biochemicals (Läufenfingen, Switzerland).

The study was approved by the Research Ethics committee of Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences (No. GDREC2016128A). All animals received care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85‐23, revised 1996). Male C57BL/6 mice were purchased from the animal research center of Guangzhou University of Chinese Medicine and housed in a temperature and humidity‐controlled room on a 12 h light/dark cycle, with ad libitum access to food and water. Mice were raised to 18–20 months old to establish the aging model. At 12‐month‐old, mice were randomly received either oral curcumin non‐fat milk powder pill (50 or 100 mg/kg/day curcumin, Cayman, Canada) or an equivalent weight of non‐fat milk powder pill for 6 months. Mice heterozygous for a targeted p300 allele (p300−/+) were generated by crossing p300 floxed mice with CAG‐cre mice (B6‐CAG‐Cre, a mouse model of systemic expression of CRE enzyme driven by CAG promoter; Jiangsu GemPharmatech Co., Ltd, China). Floxed, but Cre‐negative, littermates were used as experimental controls (wild type, WT). The cardiac structure and function were evaluated by echocardiography under isoflurane anesthesia.

The extraction of turmeric (TE obtained by Symrise AG, Holzminden, Germany) was carried out with hexane, and the crude extract was then adjusted to a specified color value of 27% using propylene glycol. To enable further use, the extract was bound to maltodextrin as a carrier substance (60% of the final extract). For our study, the extract was dissolved in distilled H₂O and used in final concentrations of 50–500 µg/mL. Curcumin (10 µM), THC (25 µM), and curcumenol (20 µM) (all Cayman Chemical Company, Ann Arbor, MI, USA, distributed by BioMol, Hamburg, Germany) were dissolved and diluted into their final concentrations using DMSO (Merck KGaA, Darmstadt, Germany). Lipopolysaccharide (LPS) from E. coli (O127:B8; Sigma-Aldrich GmbH, Taufkirchen, Germany) was diluted in phosphate buffered saline (PBS; Roche Diagnostics, Mannheim, Germany) and used in a final concentration of 10 ng/mL in the cultures.