Patients and healthy donors
The study population consisted of six patients (three females and three males) diagnosed with thalassemia major. Six age-matched healthy donors were enrolled as controls. This study was carried out with approval from the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, and written consent was obtained from all subjects prior to participation in this study, in accordance with the Declaration of Helsinki.
Sixty mice weighing 20–25 g were obtained from the Laboratory Animal Centre of Wenzhou Medical University. They were raised at the certified animal care facility of the First Affiliated Hospital of Wenzhou Medical University. The mice were randomized into an iron loading group (200 mg/kg iron dextrin intraperitoneal injection, 1 ml/kg corn oil gavage, n = 15), a control group (0.2 ml normal saline intraperitoneal injection, 1 ml/kg corn oil gavage, n = 15), a curcumin group (0.2 ml normal saline intraperitoneal injection, 200 mg/kg curcumin gavage, n = 15), or a curcumin + iron loading group (200 mg/kg iron dextrin intraperitoneal injection, 200 mg/kg curcumin gavage, n = 15). The iron overload groups received an intraperitoneal injection of iron dextran every 3 days for 4 weeks. The curcumin groups were gavaged with 200 mg/kg curcumin every day for 4 weeks.
Peripheral blood cell and bone marrow mononuclear cell (BMMNC) counts
We obtained peripheral blood from mice via the orbital sinus and collected blood samples in ethylenediaminetetraacetic acid (EDTA) tubes. Complete blood counts were analysed by a pocH-100i haematology analyser (Sysmex, Kobe, Japan). The cell counts included red blood cells (RBCs), haemoglobin (Hb), white blood cells (WBCs) and platelets (PLTs). The BMMNCs were flushed from the bones as described previously 18,19 and counted using the haematology analyser.
BMMNCs were isolated from bone marrow aspirates from healthy donors or patients with thalassemia major by Ficoll-Hypaque density gradient centrifugation, and immediately cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 20% fetal bovine serum (FBS; Gibco), which mainly consisted of progenitor and stem cells as determined by flow cytometric analyses of CD34 and CD38 expression. Cells were grown in a humidified atmosphere of 5% CO2 at 37 °C. After 3 days, medium was changed. When cells reached confluence, they were replated at a density of 1 × 104 cells/cm2. 1 × 104 cells were cultured in RPMI 1640 medium containing 1.0% of methylcellulose, 30% of FBS, 1% of bovine serum albumin (BSA; Sigma-Aldrich) and 10− 4M of 2-mercaptoethanol (Sigma-Aldrich) for following experiments.
Cell experimental protocol
The effects of ferric ammonium citrate (FAC) on autophagy in BMMNCs were evaluated. The cells were treated with FAC (F879; Sigma, St. Louis, MO) at different concentrations (0, 100, 200, 400 µM) for 24 hours. Next, we investigated the ability of curcumin (targetmol, T516) to alleviate FAC induced myelotoxicity. BMMNCs were pretreated with 30 µM curcumin for 2 hours prior to FAC treatment. Finally, the role of the SIRT3–SOD2 pathway in mononuclear cell protection after curcumin pretreatment was investigated.
Cell viability was analysed using a Cell Counting Kit-8 according to the manufacturer’s instructions (CK04; Dojindo Molecular Technologies, Kumamoto, Japan). The results are expressed as percentages relative to the control.
Determination of mitochondrion-derived ROS
To assess mROS, BMMNCs were incubated with culture medium containing 10 mM MitoSOX (M36008; Invitrogen, Carlsbad, CA, USA) for 20 minutes at 37 °C. After incubation, fluorescence intensity was measured at an excitation wavelength of 492 nm and an emission wavelength of 595 nm using an Infinite™ M200 Microplate Reader (Tecan, Männedorf, Switzerland).
Measurement of SOD2 enzyme activity
SOD2 enzymatic activity was assayed using a SOD1 and SOD2 Assay Kit with WST-8 (S0103; Beyotime, Haimen, China) in accordance with the manufacturer’s instructions. The A450 was measured using an Infinite™ M200 Microplate Reader (Tecan).
Plasmids and transfection
The plasmid LV5-SIRT3 was designed by Yuxi Biotechnology Corp. (Yuxi, China). Mononuclear cells grown in Dulbecco’s modified Eagle’s medium (DMEM) with 20% FBS for 24 hours were transfected with SIRT3 and control plasmids using Opti-MEM I-reduced serum media and Lipofectamine 2000 according to the manufacturer’s instructions (11668-019; Invitrogen). Twenty-four hours after transfection, the cells were washed and processed for immunoblotting and other assays.
Real-time PCR analysis to detect SIRT3 mRNA
All reagents used for real-time PCR were obtained from Life Technologies (Carlsbad, CA, USA). The SIRT3 probes were 5'-GACATTCGGGCTGACGTGAT-3' and 5'- ACCACATGCAGCAAGAACCTC-3'; the GAPDH probes were 5'-TGACAACAGCCTCAAGAT-3' and 5'-GAGTCCTTCCACGATACC-3'.
SIRT3 enzymatic activity was assayed by following the manufacturer’s instructions of a fluorometric kit (BML-AK557-0001; Enzo Life Sciences, Farmingdale, NY, USA).
Western blotting analysis
BMMNCs were washed twice and transferred to a new tube. Protein concentrations were determined. The protein samples were separated by SDS-PAGE. Following protein transfer onto polyvinylidene difluoride membranes, the membranes were blocked and then incubated overnight at 4 °C with antibodies against LC3, microtubule-associated protein 1 light chain 3( LC3) (1:1,000, L7543; Sigma), SIRT3 (1:100, sc-99143; Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOD2 (1:100, sc-33254; Santa Cruz Biotechnology), and β-actin (1:5,000, A5441; Sigma). The membranes were visualized by enhanced chemiluminescence using Super Signal West Pico blotting detection reagents (34079; Pierce, Rockford, IL, USA) and exposure to Hyper Performance Chemiluminescence film (Amersham, Little Chalfont, UK).
Colony-forming cell assays
To investigate the multipotency of haematopoietic progenitor cells, colony-forming units were assayed using methylcellulose culture medium (Stem Cell Technologies, Vancouver, BC, Canada). Aliquots of 1 × 105 cells were plated in 24-well plates and cultured for 14 days. Colony-forming unit erythroid (CFU-E), burst-forming unit erythroid (BFU-E), colony-forming unit granulocyte–macrophages (CFU-GM), and colony-forming unit Mix (CFU-Mix) were counted. The cells in each group were seeded in triplicate.
Labile intracellular iron pool (LIP) analysis
Cellular labile iron pool (LIP) level was assessed by Calcein-AM fluorescent Dye (Sigma)20. Briefly, aliquots of 3 × 106 cells were inoculated into 6-well plates. After treatment, the cells were washed twice with PBS and incubated with calcein-AM (CA-AM, 0.125 µmol/L) for 10 minutes at 37 °C. After washing twice with PBS, the residue was combined with 0.25 µg/ml trypan blue solution and dispersed, and the fluorescence intensity was measured by fluorospectrophotometry with excitation and emission wavelengths of 495 nm and 530 nm, respectively. Next, the samples were incubated with bipyridine (BIP) (100 µM) for 30 minutes at 37 °C, and the fluorescence intensity was measured again under the same conditions. The difference in cellular fluorescence before versus after incubation with BIP reflects the amount of labile intracellular iron pools.
The data are presented as the mean ± SEM and were analysed by t test or one-way ANOVA. Data were analysed using GraphPad Prism-5 software (GraphPad Software, San Diego, CA, USA). In all analyses, P < 0.05 was considered to indicate statistical significance.