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Introduction

Ischemic heart disease is a major cause of mortality worldwide, and the World Health Organization predicts that it will be the leading cause of mortality by 2030 (1). For patients that present with acute myocardial infarction, the most effective therapeutic strategy to preserve their myocardial tissue is timely reperfusion; however, the process of reperfusion may prompt further injury, which is a major feature of morbidity and mortality following infarction and has a direct correlation with the occurrence of coronary heart disease (CHD) (2). At present, there is no effective treatment that protects the heart against reperfusion injury. Cyclosporine-A acts by inhibiting the opening of the mitochondrial permeability transition pore (mPTP) and phase II trials are exploring the use of cyclosporine-A immediately prior to percutaneous transluminal coronary intervention (PCI) (3); however, the severe adverse reactions associated with cyclosporine-A limit its clinical use. β-blocker agents have been in use for numerous years. Metoprolol was reported to reduce the infarct area in patients with anterior ST-elevation myocardial infarction undergoing PCI (4); however, it was also previously reported that metoprolol increases mortality in patients with a systolic blood pressure of <120 mmHg (5). Therefore, novel therapies to reduce myocardial ischemia/reperfusion (I/R) injury are required to improve clinical outcomes for patients with CHD.

Although the molecular mechanisms mediating reperfusion injury remain to be fully elucidated, the underlying pathophysiology of myocardial I/R injury may involve reactive oxygen species (ROS) generation, cytosolic and mitochondrial Ca2+ overload, cell apoptosis and inflammatory responses (6). Numerous studies have demonstrated the importance of mitochondrial dysfunction due to opening of the mPTP in I/R injury. Opening of the mPTP results in the non-selective permeability of the inner mitochondrial membrane to small molecules, resulting in the collapse of the mitochondrial membrane potential (ΔΨm) and uncoupling of oxidative phosphorylation. Finally, cell death occurs due to ATP depletion (7). Therefore, inhibition of mPTP opening may be an important cardioprotective strategy. Increasing evidence has indicated that Ca2+ overload in the cytosol and mitochondria, and mitochondrial oxidative stress are the key inducers of mPTP opening, with ROS generation from the electron transport chain appearing within the first few minutes after myocardial reperfusion (8). Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) are components of the reperfusion injury salvage kinase (RISK) pathway, which is thought to be the major signaling pathway involved in cardioprotection following myocardial reperfusion. The pathway is activated by ischemic pre-conditioning (IPC) and post-conditioning, and may be targeted by various pharmacological agents. An association between the activation of the phosphoinositide 3-kinase (PI3K)/Akt and Erk1/2 pathways, and the inhibition of mPTP opening has been previously suggested (9). Activation of the RISK pathway stimulates mPTP, enhances cardiomyocyte survival and reduces I/R injury (10).

Traditional Chinese Medicine (TCM) has received increasing attention regarding applications of multi-target therapies. Radix Ginseng Rubra, Radix Ophiopogonis and Salvia miltiorrhiza Bunge are well-known Chinese herbal medicines frequently used together to enhance their therapeutic efficacy. Shenmai injection (SMI) is composed of water-soluble extracts from Radix Ginseng Rubra and Radix Ophiopogonis. Danshen injection (DSI) is composed of aqueous extracts of S. miltiorrhiza Bunge. Ginsenosides, including protopanaxatriol-type ginsenosides (Re, Rf, Rg1), protopanaxadiol-type ginsenosides (Rb2, Rb1, Rd, Rc) and oleanolic acid-type ginsenosides (Ro), are the major active components of SMI (11). Previous studies by our and other groups have isolated and identified >15 phenolic acids in the water-soluble constituents of S. miltiorrhiza, including salvianic acid, protocatechuic acid, protocatechuic aldehydrate and caffeic acid, as well as salvianolic acid A and B (12,13).

SMI, DSI and their combination, termed Yiqi Yangyin Huoxue (YYH), are clinically used to treat cardiovascular diseases, including CHD, myocardial infarction, congestive heart failure and myocardial I/R injury. Ginsenosides are the primary bioactive components of SMI, and have been confirmed to have various effects, including blocking Ca2+ channels, scavenging oxygen free radicals (14,15), and attenuating I/R injury in cardiovascular and cerebrovascular diseases. Furthermore, SMI has also been demonstrated to inhibit apoptosis and Ca2+ influx in neurocytes subjected to hypoxia-reoxygenation (H/R) (15,16). In vitro and in vivo studies suggest that DSI may be vasoactive, able to scavenge ROS, promote circulation and inhibit platelet aggregation (17). Clinical studies have reported that the combination of SMI and DSI therapy improved myocardial reperfusion injury following IPC in patients with acute myocardial infarction by reducing oxidative stress (18). A previous study by our group has demonstrated the protective effect of pretreatment with YYH, against myocardial I/R injury via the PI3K/Akt and Erk1/2 signaling pathways in isolated rat hearts (Fig. 1) (19). However, the mechanisms underlying the cardioprotective effect of YYH have remained elusive. The present study aimed to investigate the underlying mechanisms by which YYH attenuates H/R- and hydrogen peroxide (H2O2)-induced cardiomyocyte injury, focusing on the inhibition of mPTP opening via the PI3K/Akt and Erk1/2 signaling pathways.

Figure 1. Schematic summarizing the thematic background and questions to be addressed in the present study. The possible implication of PI3K signaling in inhibition of mPTP opening as a mechanism of the protective effects of YYH against cardiomyocytes H/R injury was investigated. SMI, Shenmai injection; DSI, Danshen injection; mPTP, mitochondrial permeability transition pore; PI3K, phosphoinositide 3-kinase; IRI, ischemia/reperfusion injury; PCI, percutaneous transluminal coronary intervention.

Materials and methods

Reagents

SMI and DSI were donated by Chiatai-Qing-Chun-Bao Pharmaceutical Co., Ltd. (Hangzhou, China). Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), fluo-4 acetoxymethyl (Fluo-4/AM) and calcein acetoxymethyl (Calcein/AM) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Collagenase II, bromodeoxyuridine (BrdU), rhodamine123 (Rh123), MTT, H2O2 and PD98059 were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rhodamine-2 acetoxymethyl (Rhod-2/AM) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). LY294002 was purchased from Apollo Scientific Ltd. (Stockport, UK). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium were obtained from Hyclone (GE Healthcare Life Sciences, Logan, UT, USA). Creatine kinase [(CK); cat. no. A0032] and lactate dehydrogenase [(LDH); cat. no. C0016] assay kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Trypsin (1:25) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Isolation and culture of neonatal rat cardiomyocytes

A total of 30 neonatal Sprague Dawley rats (age, 1–3 days; body weight, 10–15 g) were obtained from Beijing HFK Bioscience Co. Ltd. (Beijing, China). Without further housing, these neonatal rats were anaesthetized in a container with metofane-saturated gauze and ventricular cardiomyocytes were isolated (20,21). In brief, after administration of metofane, the newborn rats exhibited no sign of consciousness, indicated by absence of reaction to soft poking and disappearance of skin pinch reaction, which indicated full anesthesia. Furthermore, vital signs and absence of any indications of toxicity were confirmed. The neonatal rats were disinfected with 75% ethanol and the hearts were rapidly harvested. The ventricular myocardium was minced into 1 mm3 pieces using scissors in PBS. Samples were digested in PBS containing 0.0625% (w/v) trypsin and 0.1% (w/v) collagenase II with gentle agitation at 37°C for 5 min. Digestion was repeated for 5–8 cycles in total, and the digestion was stopped by addition of medium containing 10% (v/v) FBS. Subsequently, the cell suspension was filtered through a 200-mesh sieve and centrifuged at 560 × g at 4°C for 10 min. The harvested cell pellet was re-suspended in basic medium containing 10% (v/v) FBS and incubated at 37°C for 90 min to allow for fibroblast adhesion. Non-adherent cells were collected and seeded in a culture flask at 3×105 cells/mm2. Cells were incubated with 95% air and 5% CO2 for 1 h. To enhance the purity of the cardiomyocytes, BrdU (0.1 mM) was added to the culture medium for the first 3 days according to previously described methods (22). Cardiomyocytes were then used for subsequent experiments.

H/R injury in cardiac myocytes

H/R was simulated as previously described (23). In brief, the cardiomyocyte medium for hypoxia was deprived of glucose and serum. Then the cells were deposited into a hypoxia chamber (Stemcell Technologies, Inc., Vancouver, BC, Canada) containing with 95% (v/v) N2 and 5% (v/v) CO2 at 37°C for 20 h of hypoxia. The medium was replaced with high-glucose medium and the cells were transferred to the regular incubator and maintained for 4 h for reoxygenation.

H2O2-induced oxidative stress injury

In the clinic, adult patients receive an i.v. infusion of 30 ml Shenmai injection and 30 ml Danshen injection (18,24), which are diluted with 250 ml saline solution containing 5% glucose. As the maximum concentration, 10 µl/ml [SMI/DSI/culture medium, 5:5:990 (v/v/v)] is equivalently used in the clinic. In the present study, cells were pre-treated with a combination of SMI and DSI (2.5, 5 and 10 µl/ml) for 10 h. Oxidative stress was induced in cultured cardiac myocytes by adding 100 µM H2O2 for 120 min, or 90 min for the mitochondrial Ca2+ experiment. Cells were pre-treated with specific probes and with Hank's solution in the absence or presence of 100 µM H2O2, and fluorescence was monitored.

Cell viability, CK and LDH activity assays

Following reoxygenation, the medium was removed and cells were incubated with a solution of 1.2 mM MTT for 4 h at 37°C. Subsequently, 150 µl dimethylsulfoxide was added to each well following removal of the medium. Mitochondrial dehydrogenase activity, which reflects cell viability, was measured at 490 nm. Cell viability was expressed as a percentage of the control. CK and LDH activity in the medium was measured using the CK or LDH assay kits according to the manufacturer's protocol, respectively.

Assessment of ΔΨm

Fluorescence quenching of Rh123 was used to assess ΔΨm as previously described (25). Cardiomyocytes were cultured with 5 µM Rh123 at 37°C for 30 min and then washed three times with PBS. Fluorescence was measured using a Multimode plate reader (PerkinElmer, Inc., Waltham, MA, USA) at excitation/emission (ex/em) wavelengths of 488/535 nm. Values are expressed as a percentage of the control. Images of the cells were captured under a fluorescence microscope (Olympus IX73; Olympus Corp., Tokyo, Japan).

Measurement of intracellular ROS

Intracellular ROS were detected using the fluorescent dye CM-H2DCFDA. In brief, following the specific treatments, samples were washed with PBS and incubated with 5 µM CM-H2DCFDA for 20 min at 37°C, and then washed again twice with PBS. Fluorescence intensity was measured at ex/em wavelengths of 488/525 nm using a Multimode plate reader (PerkinElmer, Inc.).

Determination of cytosolic and mitochondrial Ca2+

To monitor cytosolic Ca2+, cells were loaded with 4 µM Fluo-4/AM at 37°C for 30 min and then washed three times with dye-free buffer. Following further incubation with 90 µl Hank's solution at 37°C for 20 min, cells were exposed to H2O2 (10 µl H2O2 added to 90 µl Hank's solution) at 37°C for 2 h, and the fluorescence was measured at ex/em wavelengths of 494/516 nm using a Multimode plate reader (PerkinElmer, Inc.).

The Ca2+-sensitive dye Rhod-2AM was used to monitor mitochondrial Ca2+ (26,27). Cells were washed with Hank's solution, followed by incubation with 4 µM dihydro Rhod-2/AM containing 0.05% (v/v) Pluronic F-127 at 37°C for 45 min. Rhod-2 fluorescence was measured at ex/em wavelengths of 552/581 nm as a baseline value. Subsequently, cells were treated with 100 µM H2O2 (diluted with Hank's solution) at 37°C for 90 min and the fluorescence was measured at a series of time-points. Results are expressed as a percentage of the baseline fluorescence intensity.

Monitoring of mPTP opening

mPTP opening was monitored by co-loading cells with Calcein/AM and CoCl2 as previously described (28,29). In brief, cardiomyocytes were incubated with 2 µM Calcein/AM and 1 mM CoCl2 at room temperature for 35 min, and then washed with 1 mM CoCl2 for 25 min. Calcein fluorescence was measured at ex/em wavelengths of 488/515 nm as the baseline value. Subsequently, cells were treated with 100 µM H2O2 (diluted with Hank's solution) at 37°C for 120 min and the fluorescence was measured at a series of time-points. The abrupt loss of fluorescence was regarded as an indicator of mPTP opening. Results are expressed as a percentage of the baseline fluorescence intensity.

Statistical analysis

Statistical analyses were performed using the SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). All values are expressed as the mean ± standard deviation (number of replicate wells, n=6). Differences between two groups were assessed using Student's t-test and one-way analysis of variance followed by the least-significant differences method was employed for comparison between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Cardioprotective effects of YYH in cardiomyocytes subjected to H/R. YYH improves the viability of cardiomyocytes subjected to H/R injury

H/R resulted in an 82.46±3.52% reduction in cardiomyocyte viability compared with that in the control group. YYH pretreatment at increasing concentrations (2.5, 5 and 10 µl/ml) enhanced the cell viability following H/R injury (93.40±3.81, 92.66±3.66 and 113.83±7.57% of the control, respectively; Fig. 2A).

Figure 2. Protective effects of YYH (2.5, 5 and 10 µl/ml) on H/R-induced injury of neonatal rat cardiomyocytes. (A) Effect of YYH on cell viability was determined by an MTT assay. (B) Effect of YYH on the level of extracellular CK was measured using a CK assay kit. (C) Effect of YYH on the level of extracellular LDH was measured using an LDH assay kit. (D) Evaluation of mitochondrial membrane potential of cardiomyocytes using Rh123 fluorescence. (E) Intracellular reactive oxygen species levels were measured using the DCF probe. Values are expressed as the mean ± standard deviation (n=6). ##P<0.01 vs. control; *P<0.05 vs. H/R, **P<0.01 vs. H/R. H/R, hypoxia/reoxygenation; YYH, YiqiYangyinHuoxue, referring to the combination of Shenmai injection and Danshen injection; CK, creatine kinase; LDH, lactate dehydrogenase; Rh123, Rhodamine123; DCF, dichlorodihydrofluorescein.

YYH prevents H/R-induced CK and LDH release in cardiomyocytes

The CK and LDH levels were higher in the H/R group (19.3±1.5 and 278.3±30.0 U/l, respectively) than those in the control group (4.6±2.8 and 25.3±5.5 U/l, respectively; P<0.01). YYH pretreatment (5 and 10 µl/ml) significantly reduced the levels of CK and LDH in a concentration-dependent manner (Fig. 2B and C).

YYH preserves ΔΨm in cardiomyocytes subjected to H/R

H/R increased ΔΨm depolarization to 76.61±3.33% of that in the control group (P<0.01). YYH pretreatment (2.5, 5 and 10 µl/ml) increased mitochondrial depolarization to 89.4±5.4, 91.9±8.1 and 97.2±4.0% of the control, respectively (P<0.01; Fig. 2D).

YYH reduces H/R-induced ROS generation

H/R resulted in increased generation of intracellular ROS compared with the control group (190.82±7.24%; P<0.01). Pretreatment with YYH (2.5, 5 and 10 µl/ml) significantly reduced intracellular ROS generation compared with the H/R treatment group (148.35±2.82, 134.88±1.58 and 130.74±10.01% of the control, respectively) after 4 h of reperfusion following hypoxia (P<0.01; Fig. 2E).

Cardioprotective effect of YYH determined in cardiomyocytes challenged with H2O2

YYH reduces H2O2-induced LDH release in cardiomyocytes

LDH activity was detected at 2 h after treatment with H2O2. Exposure to H2O2 induced an increase in LDH release from 117.5±21.9 to 709.0±37.0 U/l, which was significantly suppressed by YYH preincubation (10 µl/ml) for 10 h (490.0±31.4 U/l; P<0.05; Fig. 3A).

Figure 3. Effects of YYH (2.5, 5 and 10 µl/ml) on H2O2-induced injury of cardiomyocytes isolated from neonatal rats. (A) Extracellular LDH was measured using LDH assay kit. (B) Intracellular reactive oxygen species levels were measured using the chloromethyl H2DCF diacetate probe. (C) Effect of YYH on cytosolic Ca2+ was measured using Fluo-4 acetoxymethyl ester. (D) Time-dependent changes in mitochondrial Ca2+ levels were measured using Rhod-2 (left). Bar graph of Rhod-2 fluorescence intensity at 90 min following exposure to H2O2 (right). (E) Time-dependent changes in mitochondrial permeability transition pore opening indicated by calcein (left). Bar graph of calcein fluorescence intensity at 120 min following exposure to H2O2 (right). Values are expressed as the mean ± standard deviation (n=6). ##P<0.01 vs. Control; *P<0.05 vs. H2O2, **P<0.01 vs. H2O2. YYH, YiqiYangyinHuoxue, referring to the combination of Shenmai injection and Danshen injection; LDH, lactate dehydrogenase; DCF, dichlorodihydrofluorescein; Rhod, rhodamine; F0, baseline value.

YYH attenuates H2O2-induced ROS generation

Aberration in DCF fluorescence reflects the change in ROS levels. H2O2 induced a marked increase in ROS levels compared with those in the control group (P<0.01). Treatment with YYH (2.5, 5 and 10 µl/ml) resulted in a significant decrease in ROS levels compared with those in the H2O2 group (P<0.01; Fig. 3B). ROS levels in middle and high dose of YYH (5 and 10 µl/ml) were closed to those in the control group, indicating a better inhibitory effect on ROS overproduction.

YYH reduces cytosolic and mitochondrial Ca2+ overload in H2O2-challenged cardiomyocytes

The cytosolic Ca2+ level was monitored using Fluo-4AM. The Ca2+ level in the H2O2 group was significantly increased compared with that in the control group (P<0.01), which was reversed by the pretreatment with YYH (P<0.01; Fig. 3C). Among these, the high dose of YYH significantly decreased cytosolic Ca2+ levels, beyond that of the control group (P<0.01).

Mitochondrial Ca2+ overload induced by H2O2 was evaluated using time-lapse fluorescence microscopy, monitoring changes in Rhod-2 fluorescence. There was an obvious increase of mitochondrial fluorescence appeared at 30 min after exposure to H2O2. The high fluorescence was maintained for the remaining time period after 60 min. Pretreatment with YYH did not alter the baseline level of mitochondrial Ca2+. Of note, after 30 min of exposure to H2O2, the associated increases in the mitochondrial Ca2+ levels were reduced in the groups pretreated with YYH (2.5, 5 and 10 µl/ml; Fig. 3D). Rhod-2 fluorescence at 90 min after exposure to H2O2 is presented in the bar graph. Compared with the control group, H2O2 exposure significantly increased fluorescence intensity (P<0.01), indicating increased mitochondrial Ca2+ levels. Pretreatment with YYH (2.5, 5 and 10 µl/ml) significantly inhibited H2O2-induced mitochondrial Ca2+ overload (P<0.01). However, levels of mitochondrial Ca2+ in the pretreatment groups remain higher than those in the control group. With the increasing concentration of YYH, the fluctuating fluorescence intensity indicated that the inhibitory effect of YYH (10 µl/ml) on mitochondrial Ca2+ overloading may be reach the level of saturation.

YYH inhibits mPTP opening induced by H2O2

mPTP opening in intact cells was analyzed by monitoring the fluorescence of mitochondrial-entrapped calcein. A rapid decrease in calcein fluorescence was detected at ~35 min after exposure to H2O2, indicating mPTP opening. YYH pretreatment (2.5, 5 and 10 µl/ml) suppressed the sudden drop of the fluorescence value and the 10 µl/ml dose exerted the greatest effect (Fig. 3E). Differences in calcein fluorescence at 120 min after exposure to H2O2 are presented in the bar graph. Compared with the control group, H2O2 exposure decreased the fluorescence intensity from 65.8±7.8 to 21.0±4.3% of the baseline value (P<0.01), indicating increased mPTP opening. By contrast, YYH pretreatment (2.5, 5 and 10 µl/ml) inhibited mPTP opening, as indicated by the increase in fluorescence from 21.0±4.3 to 44.1±3.9, 46.6±4.7 and 52.9±3.2% of the baseline value, respectively (P<0.01). Therefore, YYH inhibited H2O2-induced mPTP opening.

Inhibition of PI3K/Akt and ERK1/2 pathways attenuates YYH cardioprotection from H/R injury

To further explore whether the cardioprotective effect of YYH is associated with the activation of PI3K/Akt and Erk1/2 signaling, a PI3K-specific inhibitor, LY294002, and an ERK1/2-specific inhibitor, PD98059, were used to investigate cell viability and ΔΨm. Compared with that in the H/R group, pre-treatment with YYH (10 µl/ml) resulted in a marked increase in cell viability (P<0.01; Figs. 4A and 5A). However, these effects were partially attenuated by LY294002 (P<0.01) or PD98059 (P<0.01) compared with combination group LY294002 reduced cell viability compared with that in the H/R group, indicating that the protective effect of the PI3K signaling pathway was inhibited by LY294002.

Figure 4. LY294002 antagonizes the protective effects of YYH (10 µl/ml) on cardiomyocytes that were subjected to H/R. (A) Cell viability was measured via an MTT assay and cells were incubated with a solution of 1.2 mM MTT for 4 h at 37°C. (B) Mitochondrial membrane potential was assessed via Rh123 fluorescence. Quantified Rh123 fluorescence intensity is provided in a bar graph. (C) Fluorescence microscopy images of cells in the different groups subjected to Rh123 staining (scale bar, 100 µm). Values are expressed as the mean ± standard deviation (n=6). ##P<0.01 vs. Control; **P<0.01 vs. H/R; ▲P<0.05, ▲▲P<0.01 vs. YYH group. H/R, hypoxia/reoxygenation; YYH, YiqiYangyinHuoxue, referring to the combination of Shenmai injection and Danshen injection; Rh123, Rhodamine123; LY, LY294002.

Figure 5. Protective effect of YYH (10 µl/ml) on cardiomyocytes subjected to H/R is partially inhibited by PD98059. (A) Cell viability was measured via an MTT assay. (B) Mitochondrial membrane potential. Values are presented as the mean ± standard deviation (n=6). ##P<0.01 vs. Control; **P<0.01 vs. H/R; ▲▲P<0.01 vs. combination group. H/R, hypoxia/reoxygenation; YYH, YiqiYangyinHuoxue, referring to the combination of Shenmai injection and Danshen injection; Rh123, Rhodamine123.

Compared with that in the H/R group, pretreatment with YYH (10 µl/ml) resulted in a marked increase in the ΔΨm (P<0.01; Fig. 4B and C, Fig. 5B). However, these effects were partially abolished by LY294002 (P<0.01) or PD98059 (P<0.01). LY294002 or PD98059 alone exerted no effect on cell viability and ΔΨm. The results indicate that the PI3K/Akt pathway and the ERK1/2 pathway may be involved in the protective effect of YYH.

Discussion

Blockage of cardiac blood flow deprives the heart of its oxygen supply, resulting in myocardial injury. Timely restoration of the blood flow effectively attenuates ischemic injury; however, subsequent reperfusion induces secondary damage to the ischemic myocardium, known as reperfusion injury (30). The mechanisms underlying reperfusion injury are complex and multifactorial, with ROS generation, Ca2+ overload, opening of the mPTP, endothelial dysfunction and pronounced inflammatory responses all implicated in causing the damage (31). Radix Ginseng Rubra, Radix Ophiopogonis and S. miltiorrhiza Bunge, which are included in YYH, have been investigated to determine their potential combined pharmaceutical properties, including anti-inflammatory, anti-oxidant, microcirculation promotion and cardioprotective abilities (32,33). In the clinic, it has been reported that YYH improved myocardial reperfusion injury following PCI in patients with acute myocardial infarction (18).

In the present study, cardiomyocytes were subjected to H/R- and H2O2-induced injury. YYH was administered at three concentrations (2.5, 5 and 10 µl/ml) to investigate its cardioprotective actions. The results of the present study indicated that H/R reduced cell viability and ΔΨm, suggesting disruption of mitochondrial integrity and function. Of note, pre-treatment with YYH inhibited these decreases. The protective effects of YYH were abolished by LY294002, a specific inhibitor of PI3K, and PD98059, a specific inhibitor of the Erk1/2 pathway, suggesting that the PI3K/Akt and Erk1/2 signaling pathways are involved in the cardioprotective effects of YYH. A previous study by our group demonstrated that ginsenoside Rb1, a principal active component of SMI, directly inhibited mPTP opening on mitochondria isolated from rat hearts in vitro (34). Its effect on mPTP opening is mediated via phosphorylation of Akt and glycogen synthase kinase-3β, as demonstrated in a Langendorff-perfused rat heart model and in experiments using a H/R induced cardiomyocyte injury model that was subjected to H/R (34). Increasing evidence suggests that the PI3K/Akt and Erk1/2 pathways are involved in signaling cascades in myocardial IPC and have important roles in cardioprotection following myocardial I/R injury (35,36). Activation of the PI3K/Akt and Erk1/2 signaling stimulates mPTPs downstream, enhances cardiomyocyte survival and reduces morbidity and mortality following I/R injury (37).

mPTP is a non-selective conductance pore located in the inner mitochondrial membrane. mPTP opening contributes to the transition from reversible to irreversible myocardial I/R injury (38) by inducing mitochondrial swelling and outer membrane rupture, subsequently facilitating activation of caspases and release of pro-apoptotic proteins, and ultimately contributing to the induction of apoptosis. This process is initiated shortly after ischemia and amplified by reperfusion. ROS and Ca2+ are also elevated during myocardial I/R, following activation of mPTP opening, particularly during reperfusion. I/R injury induces mitochondria to produce high level of ROS, including superoxide anion (O2−·), hydroxyl radical (OH−·) and hydrogen peroxide H2O2 (39). Excessive amounts of ROS cause damage to mitochondria, inducing mPTP opening and mitochondrial depolarization (40). Furthermore, an increased concentration of cytosolic Ca2+ activates a variety of cell death-associated processes following I/R. Ca2+ is then transported into the mitochondria of cardiomyocytes via mitochondrial Ca2+ uniporters. Once mitochondrial Ca2+ overloading occurs, the mPTP response is triggered. Therefore, numerous cardioprotective processes primarily function via direct inhibition of mPTP opening or upstream factors (23,41).

The results of the present study revealed that YYH protects cardiomyocytes by blocking H/R- and H2O2-induced CK and LDH release, inhibiting ROS production, reducing oxidative stress-induced cytosolic and mitochondrial Ca2+ overload, and subsequently suppressing mPTP opening. Inhibition of mPTP opening may be a key event in mediating myocardial protection against I/R injury. This beneficial effect is associated with activation of the PI3K/Akt and Erk1/2 signaling pathways (19). However, it has not been established whether the mechanisms that mediate the effects of YYH in vitro are also involved in the therapeutic effects in vivo, and further studies are required to support this. The mitochondrial protection mechanism of YYH therapy also requires validation using animal models. Further research is also required to explore mechanisms of potential mPTP-targeting strategies and other mechanisms that may be involved in the effects of SMI and DSI combination using in vitro and in vivo analyses.

In summary, the present study demonstrated that YYH protects cardiomyocytes against H/R- and H2O2-induced injury through activation of the PI3K/Akt and Erk1/2 signaling pathway and inhibition of mPTP opening resulting from ROS generation and calcium overload (Fig. 6). The in vitro molecular mechanisms of action of YYH therapy and their protective effects against myocardial I/R in vivo require further exploration.

Figure 6. Summary scheme of the mechanism underlying inhibition of SMI plus DSI combination on H/R and H2O2-induced mPTP opening. H/R and H2O2 treatment induces accumulation of ROS, Ca2+m overload and an increase of mitochondrial inner membrane permeability. SMI plus DSI combination therapy activates the PI3K/Akt and Erk1/2 signaling pathways, therefore inhibiting mPTP opening and cardiomyocyte injury. H/R, hypoxia/reoxygenation; Ca2+c, cytosolic calcium; Ca2+m, mitochondrial calcium; ROS, reactive oxygen species; ΔΨm, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; PI3K, phosphoinositide 3-kinase; Erk1/2, extracellular signal-regulated kinase; SMI, Shenmai injection; DSI, Danshen injection.

Acknowledgements

Not applicable.

Funding

The present study was supported by funds from the National Natural Science Foundation of China (grant nos. 81774017 and 81202779) and the Scientific Research Project of Tianjin Education Commission (grant no. ٢٠١٧KJ١٤٠).

Availability of data and materials

The datasets generated and/or analyzed during the present study are included in this published paper.

Authors' contributions

LL, ZS and YW performed the experiments and wrote the paper. ZD revised the paper. DY, JL and HW performed the experiments. YL designed the experiments. All of the authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was performed in strict accordance with the recommendations in the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China. The protocols were approved by the Laboratory Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (Tianjin, China; permit no. TCM-LAEC20160035).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References