The study was submitted to and approved by the Key Research and Development Programme of Jiangxi Province ethics committee.
Twelve male nude mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., licence number: SCXK (Xiang) 2019-0004).
H9C2 cardiomyocytes were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, batch number: BNCC295057.
Main reagents and instruments
Complete Dulbecco's Modified Eagle Medium (DMEM) medium (KGM12800S-500, KGI Bio), trypsin-Ethylenediaminetetraacetic acid (EDTA) digestion solution (T1300, Solarbio), Phosphate-buffered saline (PBS) (KGB5001, KGI Bio), hematoxylin staining solution (ZLI-9610, Zhongshan Jinqiao), eosin Staining solution (G1100, Solarbio), ultra-clean advanced mounting glue (YZB, BASO), Scott blue solution (G1865, Solarbio), TUNEL detection kit (C1088, Biyuntian), rabbit anti-Bax, Beclin-1, LCⅠ/Ⅱ Polyclonal antibody, mouse anti-Bcl-2 antibody (A0207, ab62557, bs-8878R, ab692, American Abcam), horseradish enzyme labelled goat anti-rabbit IgG (H+L) (ZB-2301, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Technology Co., Ltd.), DAB colour reagent kit (CW0125, CWBIO), neutral resin (CW0136, CWBIO), Annex V-FITC/PI Apoptosis Kit (AP101-100-kit, MULTI SCIENCES), Amylum Hydrochloride Su (Shenzhen Wanle Pharmaceutical Co., Ltd., batch number: 1812E1), SP (MCE, lot number: 25994), Aprepitant (MCE, lot number: 31982). CO2 incubator (BPN-80CW, Shanghai Yiheng Scientific Instrument Co., Ltd.), multifunctional microplate reader (WD-2102B, Beijing Liuyi Biotechnology Co., Ltd.), fluorescent PCR machine (CFX Connect™ real-time, Bole Life Medical Products ( Shanghai) Co., Ltd.), microtome (BQ-318D, Bernard), fluorescence microscope (CKX53, OLYMPUS), inverted fluorescence microscope (MF53, Guangzhou Mingmei Optoelectronics Co., Ltd.), electrothermal blast drying oven (DHG-9070A, constant Scientific Instruments Co., Ltd.), NovoCyte™ flow cytometer (NovoCyte 2060R, ACEM (Hangzhou) Co., Ltd.).
H9C2 cardiomyocytes were randomly divided into a control group, a myocardial model group (2 μm doxorubicin induction), an SP group (2 μm doxorubicin + 1 μg/ml exogenous SP), and an SP antagonist group (2 μm doxorubicin induction) doxorubicin + 5 μM SP antagonist).
Flow cytometry to detect apoptosis
1 × 106-3 × 106 cells were collected, then added with 1 ml PBS, centrifuged at 1500 rpm for 3 minutes, and washed twice. A 5 × Binding Buffer was diluted to 1 × with double distilled water and 300 ul of pre-chilled 1 × Binding Buffer was used to resuspend the cells. 3 ul Annexin V-FITC and 5 ul PI-PE were added to each tube. After mixing slightly, each tube was incubated for 10 minutes at room temperature in the dark, then 200 ul of pre-cooled 1 × Binding Buffer was added to each tube. After mixing, the mixtures were tested using a flow meter.
Animal model replication and drug treatment
Twelve rats were randomly divided into a control group, a myocardial model group, an SP group, and an SP antagonist group. Except for the control group, the rats in the other groups were modelled: the rats were injected with the prepared adriamycin solution at a dose of 3 mg/kg (the adriamycin concentration was 1 mg/ml, so the injection volume was 3 ml/kg). The injection was carried out once every 3 days, 5 times in a row. In the same period, the control group was intraperitoneally injected with the same amount (3ml/kg) of normal saline once every 3 days for 5 consecutive times. Every 4 days, the SP group was injected with exogenous SP through the tail vein at a dose of 6.7 μg/kg (exogenous SP concentration was 1 μg/ml, so the injection volume was 6.7 ml/kg), the SP antagonist group injected with the SP antagonist at a dose of 6.7 μg/kg (SP antagonist concentration was 1 μg/ml, so the injection volume was 6.7 ml/kg), and both the control and myocardial model group were injected with the same amount (6.7 ml /kg) normal saline.
The tissues were taken out and rinsed with running water for several hours, dehydrated by 70%, 80%, and 90% ethanol solutions, mixed with pure alcohol and xylene for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes (until transparent). Then, the tissues were placed in a mixture of xylene and paraffin in half for 15 minutes, followed by placement in paraffin I and paraffin II for 50-60 minutes each before embedded in paraffin and sectioned. The paraffin slices were baked, dewaxed, and hydrated then put in a hematoxylin aqueous solution, with distilled water added, and stained for 3 minutes. Subsequently, the slices were placed into a hydrochloric acid ethanol differentiation solution for 15 seconds and washed with water. The solution was allowed to turn back to blue for 15 seconds then rinsed with running water and stained with eosin for 3 minutes. Finally, the samples were rinsed with running water, dehydrated, and covered with a transparent film for microscopic examination.
The tissue sections were placed in xylene for 10 minutes. After that, xylene was removed and the samples were allowed to deparaffinise for 10 minutes, before being put in gradient ethanol for hydration. The slices were then placed into a wet box, with 50 μg/ml Proteinase K working solution added dropwise to each sample, and allowed to react at 37 °C for 30 minutes for antigen and antibody retrieval. Following that, the slices were washed thoroughly with PBS 3 times for 5 minutes each time. An absorbent paper was used to absorb the PBS around the tissue, with a sufficient amount of TUNEL detection solution added to each slide, and the samples were incubated for 2 hours at 45 °C in the dark. Then, the samples were washed with PBS 3 times for 5 minutes each time and the liquid was blotted on a glass slide with absorbent paper. Finally, the slides were mounted with anti-fluorescence quenching and observed under a fluorescence microscope.
Western Blot detection
The tissue was taken and ground into a powder with liquid nitrogen, then the lysis solution was added and the tissue was ground again. Following this, the tissue homogenate was sucked into an Eppendorf (EP) tube, lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes and the supernatant was carefully aspirated to obtain the total protein. The protein concentration was determined according to the BCA kit, then the protein was denatured, loaded, and subjected to sodium dodecylbenzene sulfonate gel electrophoresis (SDS-PAGE) for 2 hours. It was then transferred to the membrane with a constant current of 300 mA for 80 minutes. The primary antibody solution was incubated overnight at 4 °C before incubation with the secondary antibody solution for 2 hours at room temperature. The Chemiluminescence (ECL) luminescent liquid was dropped onto the film and exposed in the gel imaging system. The "Quantityone" software was used to analyse the grey value of each antibody band.
Transmission electron microscopy to detect autophagy levels
After the embedded paraffin sections were deparaffinised and dehydrated, the sections were cut into 80 nm thickness using an ultra-thin microtome, and then stained with 3% uranyl acetate-lead citrate, washed with distilled water, and the autophagosomes were observed under a transmission electron microscope.