Chemical and Reagents.
Tanshinone I was purchased from Chengdu Pufei De Biotech Co., Ltd, (Chengdu, China), while its purity (over 98%) was measured by HPLC. Verapamil hydrochloride was selected and bought from a Shanghai Macklin Biochemical Technology Co., Ltd, (Shanghai, China). 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH2-DA), t-BHP, Dimethyl sulfoxide (DMSO), MTT and Necrostatin-1 (Nec-1) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Double staining kit (7-AAD & Annexin V-PE) was bought from BD PharmingenTM (Becton-Dickinson, NJ, USA). From Life Technologies/Gibco Laboratories (Grand Island, NY, USA), fetal bovine serum (FBS), as well as dulbecco’s modified eagle medium (DMEM) were bought. JC-1 kit and LDH assay kit were obtained from Beyotime (Shanghai, China). From Thermo Fisher Scientific (Waltham, MA, USA), BCA protein detection kit was obtained. ELISA kits from Neobioscience (Shenzhen, China). MDA, as well as SOD detection kits, were purchased on selection from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies: p-RIP1 (AF7088), p-RIP3 (AF7443), p-MLKL (AF7420), RIP1(AF7877), and RIP3(AF7942) were purchased from Affinity Biosciences (Cincinnati, OH, USA), GAPDH (#5174), p-Akt (#4060), Akt (#4691), HO-1 (#70081), and secondary antibodies from Cell Signaling Technology (Beverly, MA, USA). MLKL (ab196436), Nrf2 (ab31163) and NQO1 (ab80588) were acquired from Abcam (Cambs, UK).
H9c2 cells, purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were placed in DMEM medium (10% FBS and 1% Penicillin/Streptomycin) for cultivation in an incubator with a specified temperature of 37°C and a humidity of 5%.
Cell viability assay
4.0×103 cells per well were cultured in 96-well plates overnight, which were treated with TI (0.125, 0.25, 0.5, 1, and 2 μM), Nec-1 (20, 40, 80, and 100 μM) or t-BHP (50, 100, and 150 μM) for 12 h, 12 h and 10 h subsequently. Our study employed MTT to detect the viability of cells and monitored the absorbance with a microplate reader (BioTek, Winkowski, VT, USA) at a wavelength of 570 nm.
Double staining assay
H9c2 cells were cultured in 6-well plates (1.5 ×105 cells per well). Afterwards, H9c2 cells were pretreated with TI (1 μM) for 2 h and treated with t-BHP (150 μM) for 6 h. After collection, cells were stained with Annexin V-PE/7-AAD and were detected by flow cytometry (Becton-Dickinson, NJ, USA).
Measurement of lactate dehydrogenase (LDH) Release
Inoculated on 96-well plates (4×103 per well) overnight and pretreated with TI (0.25, 0.5, and 1 μM) for 2 h, H9c2 cells were co-cultured with t-BHP (150 μM) for 10 h. Medium was collected to measure LDH level as per the manufacturer’s instruction of LDH. Using a microplate reader (490 nm wavelength), the absorbance was determined.
Intracellular reactive oxygen species (ROS) detection
H9c2 cells were seeded into 12-well plates (7.0×104 cells per well) overnight and treated with t-BHP (150 μM) for a different time, respectively, which subsequently were co-cultured with TI (0.25, 0.5, and 1 μM) for 2 h. Then cells were labeled with DCFH2-DA (1 μM, 0.5 h), which were monitored by flow cytometry at the FITC channel. The images were captured by a fluorescence microscope (Leica, Wetzlar, Germany) .
Mitochondrial membrane potential (MMP)’s detection assay
Based on our anterior study , cells were cultured in 96-well plates (4.0×103 cells in each well) overnight. After that, the cells were exposed to TI (1 μM) for 2 h and cultured with t-BHP (150 μM) up to 4 h. For measuring the MMP, JC-1 (5 µg/mL) was co-cultured for 30 min and relevant images were captured by a fluorescence microscope.
Animal experiments and ethical statement
The study was authorized by the Experimental Animal Management Ethics Committee of Guangxi University of Traditional Chinese Medicine (Approval Document No. SYXK-GUI-2019-0001). As per the “Guidelines for the Care and Use of Laboratory Animals in Guangxi University of Traditional Chinese Medicine”, all animals have received humane care. Healthy SD rats (male, 220–250 g) were purchased from Hunan Saike Jingda Experimental Animal Co. Ltd (Changsha, China) and acclimated for one week. Under normative SPF (specific pathogen-free) circumstances, all animals were housed and had free access to water as well as food with a befitting and controllable humidity (50%) and temperature (25℃).
Conventionally, the animals were divided into the sham-operated group, MI/R group, TI-L group (10 mg/kg), TI-H group (20 mg/kg), and positive drug Verapamil hydrochloride (Ver) group (20 mg/kg) at random and each group consisted of 15 rats. The rats were given pre-administration for one week before modeling. The rats were intraperitoneally injected with TI once a day while the sham operation group and MI/R group were given the uniform amount of normal saline at the corresponding time.
Construction of MI/R SD rat model
The MI/R rats were operated by transient myocardial ischemia for 0.5 h and reperfusion for 2 h therewith. In short, the experimental rats were narcotized by intraperitoneal injection of 10% chloral hydrate (4 mL/kg). These rats were concatenated to a rodent ventilator (respiratory rate of 58-70 breaths/min, respiratory ratio of 5:4, tidal volume of 6-7 mL/time). The third and fourth ribs were cut open, leaving the heart nearly entirely exposed. Then 6-0 silk suture was used to ligature LAD (the left anterior descending coronary artery) at the distal 1/3. The heart grew grey promptly . The ligation was disentangled after occlusion for 30 min, the heart going through reperfusion for 2 h. The sham-operated group underwent the identical operation and was penetrated in absence of ligation. ECG changes were recorded throughout the whole experiment by BL-420N biological signal acquisition and analysis system (Chengdu, China).
Fixed with 4% paraformaldehyde and dehydrated with alcohol gradient, the heart tissue samples were then processed with xylene transparently and paraffin-embedded. Hematoxylin and eosin (H&E) staining were employed after the samples were sectioned. Ultimately, with an optical microscope (UOP, DSZ5000X, China), the study observed the pathological changes of the tissue.
Blood was collected from the abdominal aorta of rats. WBC, Neu, and Lym count from blood were determined by an auto hematology analyzer (Mindray, Shenzhen, China).
Determination of SOD, MDA, TNF-α, IL-6
With a tissue grinder (Tianjin, China), SD rats’ heart tissue was homogenized. The samples were centrifuged. Their supernatant was collected immediately and stored at -80°C. As per the manufacturer’s instructions, the SOD, MDA examined by corresponding kits, TNF-α and IL-6 levels were detected by ELISA kits.
Western blotting analysis.
H9c2 cells were seeded into a dish overnight. TI (0.25, 0.5, and 1 μM) was pretreated for 2 h and co-cultured with t-BHP induction for 4 h. For animal experiments, heart tissue was collected to investigate the related protein expression. The study employed RIPA (1% PMSF and 1% cocktail) to extract total proteins of cells as well as the heart tissue. As per the manufacturer’s instruction, the concentration of the protein was measured by the BCA protein kit. Through 10% or 12% SDS-PAGE gels, the denatured protein was separated, which was transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After 5% skim milk blocking the PVDF membrane up to 2 h, incubate the membrane with 1:1000 primary antibodies at 4°C for over 12 h. The membrane was exposed to ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA) after washing with TBST and incubating with secondary antibody (1:5000) for 2 h at room temperature, of which GAPDH was the specified house-keeping protein.
Dates were presented as means ± SD. All experiments were repeated at least three times. Dates were normally distributed and GraphPad Prism 6.0 software was used to perform one-way-ANOVA or Student’s t-test. When p < 0.05, results were considered to be statistically significant.