Materials and reagents
Superoxide dismutase (SOD, A001-3-2), malondialdehyde (MDA, A003-1-2) and glutathione peroxidase (GSH-Px, A005-1-2) reagents kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA detection kits of tumor necrosis factor-alpha (TNF-α, K1051-100), Interleukin (IL)-6 (K4145-100) and IL-1β (K4796-100) were purchased from BioVision (USA). NF-κB p65 acetyl K310 (ab19870), Sirt1 (ab110304), F4/80 (ab100790) antibodies and TUNEL assay kit (ab66108) were purchased from Abcam (USA). Caspase 3 (9662), caspase 9 (9508), caspase 12 (2202), AMPK (2532) and p-AMPK (2535) antibody were purchased from cell signaling technology (USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies were provided by Proteintech Group, Inc. (USA). BCA protein assay kit was from Zhongshan Institute of Biotechnology (Beijing, China).
Preparation of TST 
The root and rhizome of TTM was purchased from Enshi Chinese Medicine Co. Ltd. (Enshi, Hubei Province, China) and authenticated by Xiujing Zhang (Beijing University of Chinese Medicine Third Affiliated Hospital). The voucher specimens (No. TTM-201802) were deposited at Beijing University of Chinese Medicine.
Take about 10 kg of air-dried plant materials, crushed into powder and through 60 mesh sizes. Soak the powder with 70% ethanol, heat and reflux for 3 hours with three times (1:8 for the first extraction and 1:6 for the second and third extraction, w/v for drug powder/ethanol solvent), then filtered. Next, the drug filtrates were evaporated and concentrated to remove ethanol under vacuum. Suspend the concentrated drug extract with distilled water and then add petroleum ether and n-butanol successively to partition, the n-butanol extract was considered as TST. Finally, collected the n-butanol extract, concentrated, vacuum dried, and finally got TST. The content of TST was 19.1 mg/g (based on crude drugs).
Forty-eight male Sprague Dawley (SD) rats with body weight 200±20 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK 2009-0011, Beijing, China). Six rats were kept in one polyacrylic cages. The rats were free access to food and water, housed in the standard controlled conditions with the temperature in 23 ± 2°C, the humidity in 50 ± 5%, and a 12 h day/night cycle. The rats were quarantined for one week before the experiments. The human care of the rats was according to the National Institutes of Health Guidelines of China and related ethical regulations of Beijing University of Chinese Medicine (Beijing, China).
All the rats were raised for one week before the formal experiment, in order to adapt the environment. The rats were randomly divided into four groups (n=12): Sham group, ischemia reperfusion injury (IR) group, TST-L 100 mg/kg group and TST-H 200 mg/kg group. The rats in TST group were oral administrated with the related dosage of TST for 14 days. The rats in Sham and IR group were oral administrated with normal saline for 14 days. After drug administration, the rats except Sham group were established IR model according to the method of Xu et al. reported . The ischemic time is 30 min, the reperfusion time is 120 min. The rats in Sham group were surgically as IR rats except the suture was in the coronary artery without ligation. At the end of the experiment, all the rats were sacrificed by cervical dislocation.
Hemodynamic indexes detection
After myocardial ischemia and reperfusion, all the rats were detected the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVDP), the maximum rate of left ventricular pressure rise (+dp/dtmax) and the maximum rate of decline (-dp/dtmax) by BL420F physiological instrument.
Anti-oxidative index detection
Heart homogenate (10%, w/v) was prepared by homogenizing the heart tissue in 150 mM Tris-HCl buffered saline (pH 7.2) with a polytron homogenizer. The enzyme activity of SOD and GSH-Px in heart tissue was measured according to the commercial kit. The data are read at the wavelength of 550 nm and expressed as U/L and nmol/L respectively. The lipid peroxidation products MDA in heart tissues was measured at 532 nm according to the protocol of commercial kit. The data of MDA was expressed as μmol/g protein.
Inflammatory factors detection
The levels of TNF-α, IL-1β and IL-6 in heart tissue homogenate were measured by ELISA kit from BioVision following the protocol provided by manufacture. The data were obtained at the wavelength of 450 nm and expressed as pg/mL. The preparation of heart tissue homogenate was followed the section of “Anti-oxidative index detection”.
Heart specimens were fixed in 4% neutral formaldehyde buffer for overnight, and then embedded in paraffin, cut into 5 µm thickness for hematoxylin and eosin (H&E) staining. The H&E sections were examined and photographed under a Olympus BX-50 light microscope at 200× magnification.
For terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, the hearts of rats were fixed in 4% paraform-aldehyde and dehydrated in 20% sucrose, embedded in paraffin and sectioned (5 μm). The sections were treated as indicated in the situ cell death detection kit (Nanjingjiancheng, China). Thereafter, nuclei were co-stained with hematoxylin.
Protein levels of caspase-3 (1:1000), caspase-9 (1:1000), caspase-12 (1:1000), AMPK (1:1000), p-AMPK (1:1000), acetylation NF-κB p65 (1:1000), and sirt1 (1:1000) in heart tissues were detected by Western blot. Heart tissue was washed with pre-cold PBS buffer, then homogenized with the pre-cold tissue homogenizer in homogenate buffer (50 mmol/L, pH 7.5 Tis-HCl, 150 mmol/L NaCl, 1 mmol/L phenyl methyl sulfonyl fluoride, 1 mg/mL aprotinin, 4 mg/mL leupeptin) on ice bath. The homogenate centrifuge at 4°C, 10000 rpm/min, then collected the supernatant, and detect the protein concentration by BCA kit. 40 μg protein samples were loaded at SDS-PAGE electrophoresis gel for protein separation, then transferred the protein from gel to PVDF membrane at 4°C, 100 V for 1.5 h. First antibody incubated overnight at 4°C, second antibody incubated 1h at room temperature, then the membrane was exposure and imaged at Bio-Rad imager (Bio-Rad ChemiDoc MP, USA).
The biological data presented as mean ± SD. The statistical comparisons were made by One-way ANOVA test followed by Dunett’s t-test with GraphPad Prism 6.0 statistical software. P < 0.05 and P < 0.01 showed a statistically significant.