Chemicals and materials
TSLFACP was isolated from Fermented Hemoglobin by professor Bo Yang from Zhejiang Chinese Medical University (Zhejiang, China). The fermentation and extraction process were as follows: the raw material was fermented for seven days at pH 5.5 and 150 r/min. The microbial fermentation broth was filtered to remove mycelia and insoluble components. The filtrate was concentrated to one fifth of the original volume under reduced pressure, and then added equal volume of ethyl acetate for extraction for three times. Then, the TSLFACP was obtained from the obtained aqueous phase which was prepared as described in the previous study .
Doxorubicin (DOX) was purchased from Hanhui Pharmaceuticals Co., Ltd. (Zhejiang, China). The assay kit for cardiactrofoninI (cTnI) was purchased from Meimian company (Jiangsu, China). The Cell Counting Kit-8 (CCK-8, cat. no. C0038), the hematoxylin and eosin (H&E) kit (cat. no. C0105S), DAPI staining solution (cat. no. C1002) and TUNEL apoptosis detection kit (cat. no. C1086) were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies against SQSTM1/P62 (cat. no. #5114), LC3A (cat. no. #4599S), PARP (cat. no. #9532), Beclin-1 (cat. no. #3738S), caspase 3 (cat. no. #9662), GAPDH (cat. no. #2118) and β-actin (cat. no. #4970) were obtained from Cell Signaling Technology (Boston, USA). The secondary antibody of goat anti-rabbit IgM-HRP (cat. no. #7074) and enhanced chemiluminescence (ECL) reagent were obtained from BIO-RAD (Hercules, CA, USA).
Mice and cell lines
All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11). Male BALB/c mice weighing 25-28 g (Laboratory animal license number: SYXK (Zhejiang) 2019-0024; Laboratory animal quality certificate: SCXK (Shanghai) 2017-0005) were purchased from Slac laboratory animal Co., Ltd. (Shanghai, China). The mice were acclimatized at least one week prior to the experiment and housed under controlled environmental conditions of temperature (22 ± 2 ℃) in a 12 h light and dark cycle, and maintained on standard food pellets and tap water ad libitum.
Rat cardiomyocytes H9C2 cells were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). Human embryonic myocardial cell line CCC-HEH-2 were purchased from National Experimental Cell Resources Sharing Service Platform (Beijing Headquarters) (Beijing, China). All the cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin at 37℃ under 5% CO2.
Cell viability assay
Cell viability was detected by CCK-8 assay according to the manufacturer’s instructions as previously reported. Briefly, cells were cultured in a 96-well plate at the density of 3000 cells/well. After TSLFACP (25, 50, 100, 200 μg/mL) or DOX (2.5 μM) treatment, 10 μL CCK-8 reagent was added in each well for additional incubation at 37 ℃ for 2 h, and the optical density (OD) value was detected at 450 nm. Moreover, cell morphologies were imaged using an inverted microscope (100 ×).
Western blotting analysis
Briefly, H9C2 cells were seeded (5 × 105 per dish) in 6 cm2 dishes and pretreated with TSLFACP (200 μg/mL) for 12 h and then treated with DOX (2.5 μM) for another 12 h. Western blotting was performed using antibodies against SQSTM1/P62, LC3A, PARP, Beclin-1, caspase 3 and β-actin. SHST capture was used as an image acquisition tool, and AI 2020 was used as an image processing software package. Image J was used as protein quantitative analysis software.
DOX-induced myocardial injury in vivo
DOX-induced myocardial injury in vivo was performed as described previously [13, 14]. Male BALB/c mice were randomly divided into three groups (n = 6): control group, DOX group and DOX + TSLFACP group. The mice of DOX group and DOX + TSLFACP group were subjected to a single intraperitoneal injection of 100 μL DOX (15 mg/kg), whereas those in the control group was injected with an equivalent volume of saline. The mice in the DOX + TSLFACP group were pretreated with 200 μL TSLFACP (200 mg/kg) for 6 consecutive days by intragastric administration prior to the injection of DOX, and administered for 6 consecutive days after the action of DOX. The daily weight of mice was recorded. Before the termination of the experiment, all mice were anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg / kg), blood samples were obtained by tail-tip phlebotomy and then the hearts were taken for follow-up experiments by opening the thoracic cavity. The mice eventually died due to excessive blood loss in the whole process, and the mice did not feel severe pain. Serum samples were obtained by the centrifugation (3000 rpm, 4 ℃) for 10 min. Part of the heart tissue was fixed in formalin, and the rest were kept at -80 ℃.
Measurement of cTnI level
For in vivo assay to determine the cardiac function, the serum samples of the mice were obtained. The level of cTnI in serum was detected by a commercial ELISA kit.
The heart tissue of mice was fixed in formalin, embedded in paraffin, and cut into slices with a thickness of 4 μm. The sections were then stained with H&E staining reagent. After that, the pathological changes were observed under a light microscope (100 ×).
The heart tissue was fixed in formalin and embedded in paraffin, followed by being cut into 4-μm-thick slices. The slices were dewaxed and hydrated, and the endogenous peroxidase was blocked. Following the antigen repair, the section was sealed with 5% BSA and incubated with primary antibody at 4 ℃ overnight. HRP-labeled polymer was added on the second day, and the section was dyed with DAB, re-stained with hematoxylin, and sealed finally. After that, the pathological changes were observed under a microscope (100 ×).
Cardiac slices were analyzed by TUNEL cell apoptosis assay kit according to the manufacturer’s instructions. Finally, the pathological changes were observed under a microscope (200 ×).
All data were processed and analyzed by GraphPad 5.0. The measurement data were expressed by mean ± standard deviation \(\left(\overline x\;\pm\;s\right)\). T test was used for comparison between two groups, and ANOVA was used for comparison among multiple groups. The difference was statistically significant with the threshold of P < 0.05.