The H9c2 embryonic rat heart-derived cells were purchased from the Chinese Academy of Sciences (Shanghai, China) cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 100 mg/ml penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cells were maintained in serum-free culture and 100 nM deferoxamine to induce ischemia and hypoxic conditions. DMEM, FBS, antibiotics and deferoxamine were purchased from Invitrogen Life Technologies, Carlsbad, CA, USA.

Male Sprague-Dawley rats (age, six weeks; weight, 200–220 g) were purchased from the Chinese Academy of Sciences (Shanghai, China). Rats were housed in a specific pathogen-free laboratory and exposed to a 12-h light/dark cycle at 37°C with free access to food and water. The rats were randomly divided into four groups (10 rats per group). Rats in control group were treated with phosphate-buffered saline, and rats in the experimental groups were administrated intraperitoneally with Tanshinone IIA, or Tanshinone IIA in combination with NSC74859 or AG490. Myocardial ischemia was induced by exposing the heart through a left thoracic incision and placing a 6-0 silk suture around the left coronary artery. Following 30 min of ischemia, the slipknot was released to allow reperfusion. At the end of the experiment, the rats were sacrificed using anesthetic (sodium pentobarbital, 30 mg/kg body weight; Akorn, Lake Forest, IL, USA).

Cytotoxic effects of tanshinone IIA or JAK/STAT inhibitors (Sigma-Aldrich, St. Louis, MO, USA) on H9c2 cells were evaluated using a Cell Counting Kit 8 (CCK-8; Dojindo, Kumamoto, Japan). Cells were seeded onto 96-well microplates at a density of 2×10⁴ cells per well, preconditioned with hypoxia/ischemia (hypoxia, 5% O₂) for 24 or 48 h and exposed to various concentrations of tanshinone IIA (0, 2.5, 10 and 40 μM) or JAK/STAT inhibitors AG490 and NSC74859 (50 and 100 μM, respectively) for 12 h. Cells were then incubated with CCK-8 solution at 37°C for 3 h. Optical density (OD) was measured at 450 nm using a MRX II microplate reader (Dynex Technologies, Inc., Chantilly, VA, USA). Cell viability was calculated as the percentage of viable cells in the drug-treated group versus that of the untreated control.

Following treatment as above, cells (>1×10⁶) were digested with 0.2% trypsin (BD Biosciences, Franklin Lakes, NJ, USA) and collected by centrifugation. Following washing twice with ice-cold phosphate-buffered saline, 10 μl Annexin V-fluorescein isothiocyanate and 5 μl propidium iodide (BD Biosciences) were added to a 85-μl cell suspension and incubated for 15 min in the dark. Stained cells were then analyzed by flow cytometry using a FACS Calibur system with Cell Quest software version 5.1 (BD Biosciences).

Following treatment as above, cells were homogenized in lysis buffer containing 50 mmol/l Tris-HCl (pH 7.3), 150 mmol/l NaCl, 5 mmol/l EDTA, 1 mmol/l dithiothreitol, 1% Triton X-100 and 1% protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged for 15 min at 12,000 × g and the resulting supernatant was transferred to a new tube and stored at −70°C. Protein concentrations were determined using a Bradford protein assay kit (Sigma-Aldrich) and proteins were separated using electrophoresis using 12% SDS-PAGE and transferred to nitrocellulose membranes (Sigma-Aldrich). The membranes were blocked for 1 h in Tris-buffered saline and Tween 20 (TBST; pH 7.6; Sigma-Aldrich) with 5% non-fat dry milk and then incubated overnight at 4°C with anti-mouse monoclonal antibodies against phospho-JAK2, phospho-STAT3, JAK2, STAT3 (dilution, 1:500) or β-actin (dilution, 1:1,000) purchased from Cell Signaling Technologies, Inc. (Beverly, MA, USA) followed by washes with TBST. The membranes were then probed with appropriate secondary antibodies (dilution, 1:5,000) at room temperature for 90 min, followed by washes with TBST. Protein bands were detected using a chemiluminescence kit (Sigma-Aldrich) and were quantified using the Quantity One software package (version 4.6.2; Bio-Rad Laboratories, UK). The result of the control group was defined as 100%.

A portion of the myocardium from the mid-left ventricle of the middle slices was fixed in 4% formalin (Sigma-Aldrich). The apoptotic rate of the myocardial cells was analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using an in situ cell death detection kit (Roche, Basel, Switzerland). A double-staining technique was used; TUNEL staining was used to quantify apoptotic cell nuclei and DAPI staining (Sigma-Aldrich) was used to quantify the total myocardial cell nuclei. TUNEL-positive cells with green nuclear staining and all cells with blue nuclear DAPI staining were counted within five randomly selected fields under high-power magnification (DM-2500; Leica Microsystems, Wetzlar, Germany). The index of apoptosis was expressed as the ratio of positively stained apoptotic myocytes to the total number of myocytes counted ×100%.

Following experimental preconditioning and measurement of hemodynamics, the hearts of mice were quickly removed and dissected into transverse slices from apex to base and processed for histology. Subsequently, the hearts were fixed in a 10% formalin solution (Sigma-Aldrich) for 24 h, embedded in paraffin wax (Sigma-Aldrich). After deparrafinization with xylene, the wax was serially sectioned at 7 μm using routine protocols (17). For the histomorphological analysis, the sections were stained with hematoxylin-eosin (HE; Sigma-Aldrich) and examined using a light microscope (BX40; Olympus, Tokyo, Japan).

Values are presented as the mean ± standard error of the mean. Group comparisons were performed using analysis of variance with SPSS 13.0 software (SPSS, Inc. Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

In order to determine the effect of tanshinone IIA on H9c2 cells, the H9c2 rat myoblast cell line was preconditioned with hypoxia/ischemia (24 and 48 h) and treated with various concentrations (0, 2.5, 10 or 40 μM) of tanshinone IIA for 12 h. A CCK8 assay was then performed (Fig. 1A and B). Hypoxic/ischemic injury significantly reduced the viability of H9c2 cells; however, following treatment with tanshinone IIA, cell viability was markedly increased in a dose-dependent manner compared with that of the untreated group, with the most potent protective effects observed with 10 μM tanshinone IIA. Furthermore, the protective effects of tanshinone IIA were more prominent following 48 h hypoxic/ischemia preconditioning compared to those in the group preconditioned for 24 h.

The JAK-STAT pathway is activated by stress (9,18), including hypoxic/ischemic injury, which was first confirmed in a rodent model study (19). In order to verify whether the protective mechanisms of tanshinone IIA proceeded via the JAK2/STAT3 signaling pathway, the effects of tanshinone IIA on JAK2 and STAT3 activation were examined using western blot analysis. As shown in Fig. 1C, 10 μM tanshinone IIA significantly upregulated the phosphorylation of JAK2 and STAT3 following 24 and 48 h of hypoxia/ischemia treatment, which was consistent with the results of the CCK-8 assay above.

In order to further determine whether the JAK2/STAT3 signaling pathway was responsible for the protective effect of tanshinone IIA, the JAK2 inhibitor AG490 (20) and the STAT3 inhibitor NSC74859 (17) were used to treat H9c2 cells alone (50 and 100 μM, respectively) or in combination with 10 μM tanshinone IIA. As shown in Fig. 2, the viability of H9c2 cells was significantly reduced to 73±1.2% in the 24-h hypoxic ischemia-preconditioned group (Fig. 2A and B) and 44±2.3% in the 48-h hypoxic ischemia-preconditioned (Fig. 2C and D) compared to that of the normoxic and FBS groups (P<0.05). Tanshinone IIA reversed the injury and slightly restored cell viability to 80±1.1% and 49±1.4% in the groups pre-conditioned with 24 and 48 h hypoxic ischemia, respectively, which was significantly increased compared to that of the control group (P<0.05). Conversely, AG490 and NSC74859 reduced cell viability by 8±1.4% (Fig. 2A and C) and 13±2.7% (Fig. 2B and D) in the groups preconditioned with 24 and 48 h hypoxic ischemia, respectively, compared to that of the control group (P<0.05). Of note, co-administration of the JAK inhibitor AG490 with tanshinone IIA restored cell viability by 5.2±1.9% (Fig. 2A and C) in comparison with the tanshinone IIA only treatment group (P<0.05); however, no significant effects were observed following co-administration of tanshinone IIA with the STAT3 inhibitor NSC74859 (Fig. 2B and D). In addition, tanshinone IIA treatment significantly increased the phosphorylation level of JAK2 and STAT3. The level of p-JAK2 was inhibited following treatment with NSC74859 alone or NSC74859 combined with tanshinone IIA (Fig. 2E). As shown in Fig. 2F, AG490 suppressed the activation of JAK2 and STAT3; however, co-administration of tanshinone IIA and AG490 restored the phosphorylation level of JAK2 and STAT3. This therefore demonstrated that contrary to the expected result, the co-application of AG490 and tanshinone IIA did not eliminate the protective effect of tanshinone IIA, but further restored myocardial cell viability following the induction of hypoxic ischemia; furthermore, these results indicated that AG490 may affect the protective function of tanshinone IIA via inhibition of the JAK/STAT pathway.

Flow cytometric analysis was performed in order to investigate the effects of tanshinone IIA and JAK2/STAT3 inhibitors on hypoxic ischemia-induced cardiomyocyte apoptosis following 24 (Fig. 3A) and 48 h (Fig. 3B) of hypoxic ischemia preconditioning. Following treatment with the JAK inhibitor AG490, the percentage of hypoxic ischemia-induced apoptotic cells significantly increased from 2.0% to 11.5% and from 6.0 to 49.7% following 24 and 48 h of hypoxic ischemia preconditioning, respectively (P<0.05). Tanshinone IIA significantly reduced the exacerbation of hypoxic ischemia-induced apoptosis mediated by AG490 from 11.5 to 6.5% and from 49.7 to 18.7% following 24 and 48 h of hypoxic ischemia preconditioning, respectively (P<0.05). Similarly, the STAT3 inhibitor NSC74859 significantly increased the percentage of hypoxic ischemia-induced apoptotic cells from 2.0% to 11.9% and from 6.0 to 81.2% following 24 and 48 h of hypoxic ischemia preconditioning, respectively (P<0.05); however, co-administration of tanshinone IIA with NSC74859 markedly reduced the exacerbation of hypoxic ischemia-induced apoptosis from 11.5 to 5.3% and 81.2 to 64.3% following 24 and 48 h of hypoxic ischemia preconditioning, respectively (P<0.05).

In order to verify the combined effects of tanshinone IIA and JAK/STAT inhibitors in vivo, tanshinone IIA and JAK/STAT inhibitors were administrated to normal mice following hypoxic/ischemia treatment. TUNEL-staining was used to identify apoptotic cells in the myocardium of control and treated mice (Fig. 4A). The number of TUNEL-positive cells observed was reduced in mice treated with tanshinone IIA; this effect was markedly enhanced in mice treated tanshinone IIA and AG490 combined (P<0.05). In addition, co-treatment with NSC74859 slightly decreased the percentage of TUNEL-positive cells compared with that in the tanshinone IIA group.

In order to demonstrate the pathological effects of tanshinone IIA and JAK/STAT inhibitors on ischemia/reperfusion in vivo, mice underwent hypoxic-ischemia preconditioning and treatment with the respective drugs, then histological sections of the myocardium were obtained and stained with HE (Fig. 4B). Staining revealed that ischemia/reperfusion injury was markedly alleviated in the tanshinone IIA group compared with that in the control group; in addition, these beneficial effects were observed to be enhanced by co-treatment with AG490. However, no difference was observed between the ischemia/reperfusion injury in the tanshinone IIA group and the group threated with tanshinone IIA and NSC74859 combined.