Carnosic acid was purchased from J&K Scientific Ltd. (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were supplied by Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Fluo-3 acetoxymethyl (AM), ROS assay kit, lactate dehydrogenase (LDH) activity assay kit, BCA protein concentration assay kit, MMP assay kit with JC-1, and Caspase-3 Activity assay kit, as well as the cleaved caspase-3 (cat no. AF0081), B-cell lymphoma 2 (Bcl-2; cat no. AF0060), Bcl-2-associated X protein (Bax; cat no. AF0054) and β-actin (cat no. AF003) antibodies, were obtained from Beyotime Institute of Biotechnology (Nantong, China). Calcein-AM was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).

Rat H9c2 cardiomyocytes were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin under humid conditions with 5% CO₂ and 95% air at 37°C. Next, the cells in logarithmic phase were incubated in 96-well plates at a density of 1×10⁵ cells/ml. The cells were divided into the control group, hypoxia/reoxygenation model group (H/R group), and three experimental groups pretreated with 0.1, 1 and 10 µM carnosic acid in DMSO for 4 h prior to hypoxia/reoxygenation. To establish the hypoxia/reoxygenation model, H9c2 cells in the H/R and experimental groups were incubated in an atmosphere with 95% N₂ and 5% CO₂ at 37°C for 4 h, and then exposed to 95% air and 5% CO₂ at 37°C for a further 4 h. The control group was cultured under normoxic conditions.

To detect the protective effects of carnosic acid on H9c2 cardiomyocytes exposed to hypoxia/reoxygenation, an MTT assay was performed. Subsequent to the aforementioned treatments, the cells were incubated with 0.2 ml MTT (0.5 mg/ml) for 4 h at 37°C. Next, 200 µl DMSO was added into each well in order to dissolve the formazan crystals. The optical density (OD) was recorded on a BioTek ELx800 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm. The results are expressed as the relative percentage of the control group.

To further confirm the protective effects of carnosic acid, the LDH activity in the culture medium was measured by an LDH assay kit, according to the manufacturer's protocol. Briefly, following treatment, the culture medium was centrifuged at 400 × g and room temperature for 5 min, and then 20 µl supernatant was collected and mixed with 20 µl 2,4-dinitrophenylhydrazine. After incubation at 37°C for 15 min, 250 µl NaOH (0.4 M) was added into the mixture and incubated for a further 15 min at 37°C. The mixture was maintained at room temperature for 5 min, and subsequently the OD was recorded on a microplate reader at 450 nm. The activity of LDH was derived from the OD values and expressed as U/l.

To monitor the intracellular calcium in H9c2 cardiomyocytes, the fluorescence dye Fluo-3 AM was employed, following the manufacturer's protocol. Briefly, the pretreated H9c2 cardiomyocytes were loaded with 5 µM Fluo-3 AM at 37°C for 30 min in the dark, and then washed with PBS for three times to remove any excessive dye. The fluorescence intensity of Fluo-3 chelated with calcium was recorded on a PerkinElmer EnVision fluorescence microplate reader (PerkinElmer, Llantrisant, UK) at excitation and emission wavelengths of 488 and 525 nm, respectively.

The production of ROS was detected by a fluorescence method using a ROS assay kit. Subsequent to treatment, the medium was replaced and the cells were rinsed with PBS. Next, 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in DMEM was added and incubated at 37°C for 30 min. The cells were then washed again with PBS to remove the excessive dye, and the fluorescence intensity was recorded on a fluorescence microplate reader at 485 nm (excitation wavelength) and 520 nm (emission wavelength).

The MMP was also detected using a MMP assay kit with JC-1. JC-1 accumulates in the mitochondrial matrix of normal cells and forms aggregates, which emit red fluorescence under excitation. When the MMP collapses, JC-1 exists as a monomer and thus no red fluorescence is observed under excitation (18). Following the treatment, the H9c2 cardiomyocytes were washed with PBS and then loaded with JC-1 at 37°C for 20 min. Subsequent to washing with JC-1 buffer solution, the fluorescence intensity was read at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The MMP is expressed as a percentage compared with the fluorescence intensity of the control group.

The mPTP opening was directly evaluated through monitoring the release of mitochondrial calcein. Briefly, cardiomyocytes were incubated with 2 µM calcein-AM and 1 mM CoCl₂ at room temperature for 30 min. The free calcein-AM and CoCl₂ were washed with PBS to be removed, and the cells were incubated with 1 mM CoCl₂ for a further 20 min at 37°C to specifically quench the fluorescence of free calcein in the cytosol. Subsequently, the fluorescence intensity of mitochondrial calcein in the cardiomyocytes was measured on a fluorescence microplate reader at 490 nm for excitation and 515 nm for emission. The loss of calcein fluorescence in cardiomyocytes indicated the opening of mPTP. The results are expressed as the fluorescence intensity percentage of the control group.

Caspase-3 activity was assessed by a colorimetric assay kit following the manufacturer's instructions. H9c2 cardiomyocytes were pretreated as described earlier and washed with PBS, followed by lysis using a Caspase-3 Assay kit (cat no. C1116; Beyotime Institute of Biotechnology) and centrifugation at 16,000 × g for 10 min at 4°C. The supernatant was then collected and incubated with substrate (Ac-DEVD-pNA) at 37°C for 2 h. The OD values were measured on a microplate reader at 405 nm, and the activity of caspase-3 is expressed as the relative percentage of the OD value of the control group.

H9c2 cardiomyocytes were treated as described earlier and then subjected to western blot analysis to detect the expression levels of caspase-3, Bcl-2 and Bax. Briefly, the cells were lysed with lysis buffer solution containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton and 1 mM phenylmethane sulfonyl fluoride on ice for 30 min. Next, the cell lysate was centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was collected as the total protein for the subsequent analysis of cleaved caspase-3, Bcl-2, and Bax. Following quantification by a BCA assay kit, the samples were separated by 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Subsequent to blocking with defatted milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against cleaved caspase-3, Bcl-2, Bax and β-actin (diluted at 1:1,000). The membranes were then incubated with the respective secondary antibody conjugated to horseradish peroxidase (diluted at 1:3,000; cat no. A0192; Beyotime Institute of Biotechnology) at room temperature for 1 h, and visualized by an enzyme-link chemiluminescence substrate (cat no. P0203; Beyotime Institute of Biotechnology) on a Bio-Rad ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used as the internal control.

The results are expressed as the mean ± standard deviation, and were analyzed by GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons were implemented by Student's t-test for single components or one-way analysis of variance followed by Dunnett's test for multiple components. P<0.05 was considered to be an indicator of statistically significant differences. All data are the result of at least six independent experiments.

As shown in Fig. 2, the viability of H9c2 cardiomyocytes was decreased when exposed to hypoxia/reoxygenation (P<0.001). In the presence of different carnosic acid concentrations, the poor survival of H9c2 cardiomyocytes was significantly ameliorated compared with the H/R group, in a dose-dependent manner. In particular, the viability in the group treated with 10 µM carnosic acid reached 81.55±7.31% of the control group value (P<0.001). These results indicated that carnosic acid exhibited protective effects on H9c2 cardiomyocytes injured by hypoxia/reoxygenation.

Under hypoxia/reoxygenation, the activity of LDH in the culture medium of H9c2 cardiomyocytes increased to approximately three times greater than the control group activity, which demonstrated that leakage of LDH from the cytosol occurred in the H/R group and cell survival decreased (P<0.001). When treated with carnosic acid, the release of LDH in H9c2 cardiomyocytes was significantly decreased in a dose-dependent manner (P<0.05; Fig. 3). These results indicated the carnosic acid affected the LDH release in H9c2 cardiomyocytes induced by hypoxia/reoxygenation.

To determine the intracellular calcium level, the fluorescence probe Fluo-3 AM was used. Following treatment of hypoxia/reoxygenation, the fluorescence intensity of the intracellular calcium in the H/R group was significantly elevated (2,352.85±185.48) when compared with the control group (850.00±96.86; P<0.001; Fig. 4). However, compared with the H/R group, carnosic acid treatment dose-dependently attenuated the overload of intracellular calcium as observed by the reduced fluorescence intensity (2,090.10±218.08, 1,609.07±98.73 and 1,224.07±76.41 at 0.1, 1 and 10 µM, respectively; P<0.05; Fig. 4). These results suggested that carnosic acid was able to reduce the overload of intracellular calcium in H9c2 cardiomyocytes induced by hypoxia/reoxygenation.

The production of intracellular ROS was measured with the fluorescence probe DCFH-DA according to the manufacturer's protocol. DCFH-DA passes through the cellular membrane and is hydrolyzed to DCFH by intracellular esterases. DCFH is then unable to cross the membrane and is thus reserved in the cytosol. With the excessive production of ROS, DCFH is quantitatively oxidized into the fluorescent dichlorofluorescein (DCF) (19). The results of the present experiments revealed that the fluorescence intensity in the H/R group (238.12±20.51) was markedly higher in comparison with that in the control group (90.00±9.43). Upon treatment with different concentrations of carnosic acid, the fluorescence intensity of DCF was reduced in a dose-dependent manner (Fig. 5), implying that carnosic acid was able to alleviate the overproduction of intracellular ROS in H9c2 cardiomyocytes induced by hypoxia/reoxygenation.

The MMP in H9c2 cardiomyocytes was evaluated to investigate the effects of carnosic acid on the mitochondrial function of H9c2 cardiomyocytes induced by hypoxia/reoxygenation. In contrast to the control group, the fluorescence intensity in the H/R group (42.78±6.54%) was evidently diminished (P<0.001), which implicated the collapse of MMP in the H/R group after induction of hypoxia/reoxygenation. However, in the presence of different carnosic acid concentrations, the fluorescence intensity was significantly increased to different extents (Fig. 6). In particular, the experimental group with 10 µM carnosic acid treatment exhibited ~80% of the fluorescence intensity of the control group. These results revealed that the collapse of MMP in hypoxia/reoxygenation-treated H9c2 cardiomyocytes was relieved by carnosic acid.

The mPTP opening was assessed based on the fluorescence intensity of mitochondrial free calcein. As shown in Fig. 7, following induction of hypoxia/reoxygenation, the fluorescence intensity of mitochondrial calcein in the H/R group was close to half that of the control group (53.71±6.49%; P<0.001), which revealed the opening of the mPTP. Upon exposure with increasing carnosic acid doses, the opening of the mPTP was ameliorated in a dose-dependent manner, as indicated by the reduced fluorescence intensity of mitochondrial free calcein. Treatment of 10 µM carnosic acid resulted in a fluorescence intensity at 81.74±6.40% of the control group value (P<0.001). These results implied that carnosic acid inhibited the mPTP opening that was induced by hypoxia/reoxygenation.

Caspase-3 is an executioner enzyme in apoptosis and is activated through hydrolysis. The results of the colorimetric assay revealed that the activity of caspase-3 was markedly enhanced in H9c2 cardiomyocytes following treatment of hypoxia/reoxygenation. However, when pretreatment with carnosic acid was performed, the activity of caspase-3 was significantly inhibited (Fig. 8A). Western blot analysis also demonstrated that hypoxia/reoxygenation upregulated the expression of caspase-3, while carnosic acid treatment suppressed this expression in a dose-dependent manner (Fig. 8B). In addition, western blot analysis indicated the expression changes of Bcl-2 and Bax, two proteins associated with apoptosis. Hypoxia/reoxygenation downregulated the expression of Bcl-2 and upregulated Bax expression. On the contrary, different concentrations of carnosic acid resulted in the upregulation of Bcl-2 and downregulation of Bax (Fig. 8B).