Reagents
MgIG was supplied by Chia Tai Tianqing Pharmaceutical Group Co., Ltd. (Lianyungang, China; drug approval number, H20051942). DOX hydrochloride was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. (Zhejiang, China; drug approval number, H33021980).
Animals
A total of 50 male Kunming mice weighing 20.0±2.0 g (4–5 weeks old) were purchased from the Laboratory Animal Center of Hebei Medical University (Shijiazhuang, China). The mice were housed in rust-free cages at 20–22°C and 45–55% relative humidity on a 12 h light-dark cycle, and given a normal pelleted diet and tap water ad libitum. All mice were handled according to the National Institutes of Health Guide for Care and Use of Laboratory Animals (21). All experiments were approved by the Ethics Committee for Animal Experiments of Hebei Medical University (approval number, HEBMU-2015-01; approval date, January 22, 2015).
Experimental design
Following a 1-week adaptation period, mice were randomly and divided into 5 equal groups (n=10/group): A control group [control, saline (normal saline 0.01 ml/g/day), intraperitoneal (i.p.)]; a model group (DOX, 30 mg/kg, i.p.); and treatment groups that received low-dose MgIG (L-MgIG, 10 mg/kg/day, i.p.), middle-dose MgIG (M-MgIG, 20 mg/kg/day, i.p.) and high-dose MgIG (H-MgIG, 40 mg/kg/day, i.p.). Mice in the treatment groups were administered MgIG for 1 week; then mice in the treatment and model groups were administered with 30 mg/kg DOX once. Following 48 h, all mice were weighed and anesthetized with sodium pentobarbital (50 mg/kg, i.p.). When the mice were anesthetized, blood was sampled from the eyes of the mice. The blood stood in the room temperature 30–60 min, and then the serum was separated (centrifuged at 2,200 × g for 10 min at room temperature) for biochemical analysis and liver and heart samples were quickly excised and snap frozen in liquid nitrogen or fixed in a 4% paraformaldehyde solution. The samples were then excised and examined as described below.
Histopathological analysis
Hepatic and cardiac tissue samples were fixed in 4% paraformaldehyde solution for ≥1 week at room temperature and embedded in paraffin. Paraffin-embedded samples were cut to 4-µm-thick sections, mounted on glass slides and stained with hematoxylin and eosin (H&E) for 50 min at room temperature for histopathological analysis, according to the conventional staining steps. To analyze the staining results, micrographs were scanned at ×400 magnification with a digital light microscope (Leica DM750; Leica Microsystems GmbH, Wetzlar, Germany).
Serum biochemical analysis
Changes in the activity of the cardiac-specific enzymes creatine kinase (CK) (22), CK-MB and lactate dehydrogenase (LDH) in the serum were determined. Furthermore, levels of the two hepatic-specific enzymes aspartate aminotransferase (AST) (23) and alanine aminotransferase (ALT), the oxidative stress markers SOD and GSH-Px, and methane dicarboxylic aldehyde (MDA) were measured.
The activity of CK (cat. no. A032), CK-MB (cat. no. E006) and LDH (cat. no. A020-1) in the serum was determined at 37°C following the guidelines of the relevant assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China). Serum levels of AST and ALT were detected by spectrophotometry-based methods following the guidelines of the AST (cat. no. C010-1) and ALT (cat. no. C009-1) assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd.). SOD (cat. no. A001-1) and GSH-Px (cat. no. A005) activity, and MDA (cat. no. A003-1) serum levels were measured following the manuals of the corresponding assay kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd.). All procedures were conducted in strict accordance with the manufacturers protocols.
Western blot analysis
Total proteins were obtained from frozen liver and heart tissue homogenates following the manufacturers protocol. The samples were homogenized and lysed with RIPA lysis buffer containing 50 mM Tris-HCl, 125 mM sodium chloride, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM EDTA, 1 mM dithiothreitol, 0.1% SDS (w/v), 1% TritonX-100 (v/v) and protease inhibitor cocktail (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After incubation for 20 min on ice, the lysis buffer solution was centrifuged at 4°C at 7,800 × g for 20 min. The protein concentration was measured using a Bradford Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). ~50 µg of total proteins were loaded and separated on 10% SDS-PAGE, and then transferred to a nitrocellulose membrane. Membranes were blocked with blocking buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% (w/v) non-fat milk for 2 h at room temperature and then incubated with the membrane was incubated with the primary antibody overnight at 4°C. The primary antibodies were are follows: Rabbit anti-caspase-3 (dilution 1:1,000; cat. no., sc-7148; Santa Cruz Biotechnology, Inc.), rabbit anti-B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax; dilution 1:300; cat. no., BA0315; Wuhan Boster Biological Technology, Ltd., Wuhan, China), rabbit anti-Bcl-2 (dilution 1:1,000; cat. no., BS1031; Bioworld Technology, Inc., St. Louis Park, MN, USA), rabbit anti-nuclear factor (NF)-κBp65 (dilution 1:1,000; cat. no., BS1253; Bioworld Technology, Inc.) and mouse anti-β-actin (dilution 1:2,000; cat. no., TA-09; ZSGB-BIO; OriGene Technologies, Inc., Rockville, MD, USA) antibodies. Horseradish peroxidase conjugated goat anti-rabbit immunoglobulin (Ig)G (dilution 1:3,000; cat. no., ZB2305; ZSGB-BIO; OriGene Technologies, Inc.) or goat anti-mouse IgG (dilution, 1:3,000; cat. no., ZB2301; ZSGB-BIO; OriGene Technologies, Inc.) were used as the secondary antibodies. The membrane was incubated with the secondary antibodies at room temperature for 1 h. The bound antibodies were visualized using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Little Chalfont, UK) and quantified by densitometry using an image analyzer. The gray values of the hybridized bands were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). β-actin was used as an internal protein control for normalization.
Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay
TUNEL staining was performed using an in situ cell death detection kit (Roche Applied Science, Mannheim, Germany) following the manufacturers protocol. Briefly, liver and the heart sections were deparaffinized, dehydrated using a series of increasing concentrations of alcohol, washed in distilled water followed by PBS and deproteinized using proteinase K (20 µg/ml) for 30 min at 37°C. Subsequently, sections were rinsed and incubated with the TUNEL reagent at 37°C for 1 h. Following rinsing, the sections were visualized using a peroxidase-conjugated anti-fluorescein antibody (in the TUNEL kit) with 0.02% 3,3-diaminobenzidine (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and then counterstained with 0.5% hematoxylin at room temperature for 10–30 sec. Neutral balsam (Shanghai Guchen Biotechnology Co., Ltd., Shanghai, China) was used to bond the slides and cover glass together. To analyze the staining results, micrographs were scanned at ×400 magnification with a digital light microscope system (Leica DM750). An image analysis system (Image-Pro Plus software; version 6.0) was used to analyze 20 randomly selected fields per slide at magnification ×400 to determine the area of positive staining.
Data analysis
All data are provided as the mean ± standard error of the mean. Differences among the groups were assessed by one-way analysis of variance followed by Kruskal-Wallis and Tukeys tests. The statistical analysis software, Origin (version 7.5; OriginLab, Northampton, MA, USA) and SPSS (version 22.0; IBM Corp., Armonk, NY, USA) were used. P<0.05 was considered to indicate a statistically significant difference.