All animal experiments were approved by Shaoxing People's Hospital Ethics Committee and performed according to the guidelines from the National Institutes of Health. All efforts were made to minimize suffering.
DOX-induced cardiac injury in rats
24 male Wistar rats (8-weeks-old, 250 g ± 10 g), were purchased from the Experimental Animal Center of Basic Medicine, Zhejiang Chinese Medical University, China. Before being assigned to the experimental groups, rats were allowed to adapt to the environment in a light-controlled room (12 h light/dark cycle) where they had free access to standard chow and water for 7 days. All rats were randomly divided into 4 groups: control (CON), DOX, DOX + Ononin30 (Cat. HY-N0270, MedChemExpress, USA), and DOX + Ononin60 according to previous research. Animals in the DOX, DOX + Ononin30 and DOX + Ononin60 groups were administered 2.5 mg/kg DOX (Cat.HY-15142A, MedChemExpress, USA) via the tail vein once a week for 6 weeks (total dose: 15 mg/kg) to induce cardiac injury. Animals in the DOX + Ononin30 and DOX + Ononin60 groups were intragastrically injected with Ononin (30 mg/kg/day or 60 mg/kg/day, respectively) before receiving the above DOX regimen. The CON group received an equivalent saline solution by gavage for 6 weeks (Fig. 1A).The dosage of DOX was chosen based on previous studies and our preliminary work. The chemical structures of DOX and Ononin were shown in Fig. 1B and C.
At the end of the study, body weight was measured following anesthesia. After euthanasia, blood was collected. All hearts were weighed and cut into several parts for further investigation.
At the end of the 6-week DOX treatment, two-dimensional M-mode echocardiography was performed using the Philips iE33 system (Philips Medical, Best, Netherlands) equipped with an s5-1 probe (12–14 MHz) to evaluate rat cardiac function. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured and calculated with Philips QLab 9 post-processing software.
Measurements of LDH
Cardiac marker enzymes including LDH was measured in serum using commercially available kits (Cat. A020-1-2, Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s protocol.
After the rats were sacrificed, the myocardial tissues were immediately separated and fixed in 10 % neutral buffered formalin for 24 h. To evaluate cardiac morphology and fibrosis, the tissue sections were respectively stained with hematoxylin and eosin (H&E), Masson’s trichrome stain, and wheat germ agglutinin (WGA, Cat.L4895, Sigma Aldrich, USA) according to standard protocols. Subsequently, the sections were imaged using a Leica DM3000 microscope (Leica, Wetzlar, Germany) and Nikon Eclipse Ti-U fluorescence microscope (Minato-ku, Tokyo, Japan).
Immunohistochemistry (IHC) staining
Immunohistochemistry was used to visualize the expression of GRP78. Histologic sections were dehydrated with a series of alcohol and xylene gradients. Then, 3% H2O2was used to quench endogenous peroxidases followed by treatment with citrate buffer for 15 min at 95 ℃. The tissue sections were incubated with anti-GRP78 or anti-SIRT3 at 4 ℃ overnight. The next day, specimens were stained with the secondary antibody for 30 min and 3,3′-diaminobenzidine (DAB) for 5 min at 37 ℃. Finally, images were taken using a Leica DM3000 microscope.
TdT-mediated dUTP nick end-labeling (TUNEL) staining
Cardiomyocyte apoptosis was examined with TUNEL staining, using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. Results were imaged under a fluorescence microscope (Minato-ku, Tokyo, Japan).
Cell culture and treatment
The H9C2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and applied in our experiments. All of these cells were cultured in DMEM (Sigma, USA) supplemented with 10 % FBS (Gibco, USA) and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, Gibco), and maintained at 37 ℃ in a humidified incubator with 5% CO2. H9C2 cells were treated with Ononin (0.5µM) for 24h and then exposed in DOX (1µM) for 24h. In a separate experiment, 3-TYP (SIRT3 inhibitor, 1µM, Cat.HY-108331, MCE) was used 12h before adding DOX. The dosage of 3-TYP was based on previous studies.
Cell viability assay
Cell viability was examined using the CCK-8 assay kit (Cat. HY-K0301, MedChemExpress, USA) according to the manufacturer’s instructions. The results were read with microplate reader at 450 nm.
Heart tissues or H9C2 cells were lysed with RIPA lysis buffer (Beyotime, China) containing protease inhibitors and phosphatase inhibitor (Mce, China). The extracted proteins were separated by 10 % polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, MA). The membranes were blocked for 60 min with 5% skim milk at room temperature and incubated overnight with primary antibodies against GAPDH, Bcl-2, Bax, GRP78, CHOP, Sirt3 (all obtained from Abcam) at 4 ℃. Next day, the membranes were incubated with the corresponding secondary antibody for 1 h. Subsequently, the membranes were visualized using a ECL chemiluminescence detection kit and protein bands were analyzed by Quantity One.
All data were presented as the mean ± standard error of the mean (SEM). Statistical significance was assessed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test for multiple comparisons, using GraphPad Software. P < 0.05 was considered statistically significant.