Protective action of the ginsenoside Rh3 in a rat myocardial ischemia-reperfusion injury model by inhibition of apoptosis induced via p38 mitogen-activated protein kinase/caspase-3 signaling.

OBJECTIVE: To investigate the protective effects of the ginsenoside Rh3 on rats subjected to myocardial ischemia-reperfusion (MIR) via its impact on caspase-3 and the p38 mitogen-activated protein kinase (MAPK) pathway.
METHODS: Fifteen male Sprague-Dawley rats were randomly categorized into the MIR group (MY group, n = 5), sham surgery group (SS group, n = 5), and ginsenoside Rh3 group (GR group, n = 5).
RESULTS: The MY group exhibited the largest myocardial infarctions compared with the GR and SS groups. The GR group exhibited significantly higher cell viability of cardiomyocytes and significantly decreased apoptosis compared with the MY group. Fibrils of infarcted tissue in the GR group were disordered but less swollen, with a more organized fibril orientation than those in the MY group. The GR group showed reduced p-p38 MAPK protein and caspase-3 mRNA expression levels compared with the MY and SS groups.
CONCLUSIONS: Rh3 significantly improved myocardial necrosis and caspase-3 levels in myocardial tissues by suppressing the p38 MAPK pathway, thereby inhibiting caspase-3 involvement in apoptosis. Thus, Rh3 was effective in inhibiting the escalated apoptotic pathway in myocardial infarction and can potentially serve as a useful therapeutic agent to rescue myocardial infarction.

Introduction

Ginsenoside is an active ingredient extracted from the Araliaceae family of plants (ginseng, American ginseng, Panax notoginseng) by Japanese scientists in the 1960s. Subsequently, many scientific studies have found that ginsenosides have rich pharmacological effects, including vasodilation, anti-inflammatory, anti-oxidant, anti-cancer, blood sugar regulation, and nerve protection effects. The anti-cancer effect of ginsenosides has attracted the attention of many scientists, making ginsenosides a focus of research for natural anti-cancer medicines. After more than half a century of research, scientists have discovered more than 100 ginsenosides. Among them, Rg3 is the earliest discovered and most studied ginsenoside.

Myocardial ischemia-reperfusion (MIR) injury (MIRI) is common in patients with cardiac disease. It is caused by damage to the myocardial tissues during the sudden restoration of blood supply to ischemic myocardial tissues.[1,2] Its incidence is higher in middle-aged and elderly individuals, particularly in those with coronary artery disease and disorders that lead to heart failure, cardiac arrest, and sudden death.[3] Myocardial ischemia is a common complication of heart disease, and although specific therapies improve the blood flow to the injured myocardial tissues and can reduce mortality resulting from myocardial ischemia, reperfusion can cause other disorders such as reperfusion arrhythmias.[4] In traditional Chinese medicine, ginseng is considered to be the most important of all herbs and has great medical value.[5]

Contemporary studies have confirmed ginsenosides to be the principal components of ginseng, with a variety of medicinal properties, including prevention of apoptosis and aging. Ginsenosides have also shown great value in the treatment of chronic diseases and MIRI.[6] The p38 mitogen-activated protein kinase (MAPK) signaling pathway is involved in extracellular signal regulation. Research has shown that p38 MAPK exists widely in various animal cells and has essential regulatory effects on gene expression and the cell cycle.[7-9] This study was designed to investigate the differences in infarction size in a rat MIR model in untreated tissues and after Rh3 treatment. Furthermore, alterations in p-p38 MAPK protein expression levels, caspase-3 mRNA expression, cell viability, and apoptosis were evaluated in cells treated with and without Rh3. The aim of this study was to gain insight into a plausible role of the ginsenoside Rh3 as an anti-apoptotic agent via its protective effect on MIRI and demonstrate its effects on caspase-3 through the p38 MAPK signaling pathway.

Methods

General data

Fifteen male Sprague-Dawley (SD) rats weighing 250 to 300 g and six neonatal SD rats aged 1 to 3 days were purchased from the Laboratory Animal Center. All rats were initially housed and fed for 1 week to allow acclimatization. This research was approved by the Animal Ethics Committee of the First People’s Hospital of Wenling, China.

Rat MIR model and experimental grouping

The rats were randomly divided into three groups: 1) MIR group (MY group, n = 5), in which the rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), wherein only a chest incision was made in rats without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5), in which the rats were used to establish MIR models and received Rh3 treatment by intragastric administration for 24 hours.

After fasting for 12 hours, rats were injected in the abdomen with an anesthetic. Once the rat was unresponsive, it was laid flat with the face positioned upward, and the left side of the chest was shaved. After disinfection with ethyl alcohol, an incision was made to open the intercostal space between the fourth and fifth ribs. A retractor was placed, and the heart was located and gently removed. SS group rats were threaded below the left ventricle and left atrium without ligation. In MY and GR group rats, the left coronary artery was threaded and ligated. An electrocardiogram showing elevation of ventricular depolarization and initiation of ventricular repolarization (ST-segment) and the cardiac muscles under the ligature becoming pale were indicators of successful ischemia. The heart was ligated for 30 minutes, and the ligature was then removed to allow myocardial reperfusion for 120 minutes. ST-segment reduction indicated successful reperfusion. The heart was replaced, and the incision was sutured, followed by cardiopulmonary resuscitation until the rat was able to breathe spontaneously. The rats were housed under typical conditions, eating and drinking regularly. Rats in the GR group were intragastrically administered the ginsenoside Rh3 (Sichuan Victory Biological Technology Co., Ltd., China) (0.5 mg/kg/day) once a day for 2 weeks. Rats in the SS group were intragastrically administered saline of the same volume. All rats were housed under the same environment. At the end of the experiment, all rats were sacrificed to collect myocardial cells.

Isolation of fresh cardiomyocytes

The hearts were quickly removed from the neonatal rats under sterile conditions, washed with phosphate-buffered saline, and cut into pieces in serum-free Dulbecco’s modified Eagle’s medium (DMEM). Collagenase I (1 mg/mL) and 0.25% trypsin in a ratio of 1:1 were used for repeated digestion at 37°C, four times, 15 minutes each. The cell suspension was collected and added to DMEM with 10% serum to terminate the digestion. After centrifugation for 10 minutes at 300 ×g, the cell pellet was incubated for 2 hours before removing the fibroblasts and purifying the myocardial cells. Two hours later, non-adherent myocardial cells were transferred into DMEM, seeded onto a culture plate at a density of 5 × 105 cells/well, and incubated for 24 hours. Cultured cells were transferred into a sugar- and serum-free medium. Ischemia was simulated with hypoxia and reperfusion in a hypoxia incubator for 2 hours, and the cells were then transferred into a standard medium in a reoxygenation incubator for 2 hours. During reoxygenation, the cells were divided into two groups: one for normal cultivation (MY group) and the other treated with 0.5 mg/mL of the ginsenoside Rh3 (GR group); the control group was provided (SS group) with a regular oxygen supply. We employed this model to investigate the effect of Rh3 on cardioprotection against MIRI in vitro. Cell proliferation, apoptosis, and protein and mRNA expression were analyzed after 22 hours of reoxygenation.

Detection of myocardial infarction size using triphenyl tetrazolium chloride dye

Cardiac tissues from the three groups were transversely cut into four or five sections of 2-mm thickness and immersed in 1% triphenyl tetrazolium chloride (TTC, Sigma-Aldrich, St Louis, MO, USA) buffer for incubation at room temperature for 10 minutes to stain the viable tissue surrounding the infarct. The surviving tissue exhibited a color that was different from that of nonviable tissue. Images were acquired with a Zeiss imaging system (Gottingen, Germany), and assessment of the TTC dye was carried out using Image Pro software (Media Cybernetics, Rockville, MD, USA) to determine the infarct size, which was calculated using the formula: MIS% = (infarcted size/total area) × 100%.

Pathological changes in myocardial tissues

The myocardial tissues of the three groups were fixed for 15 minutes, embedded in paraffin, and cut into 5-µm sections. Morphological observation was conducted after goat serum (Shanghai Xinfan Biotechnology Co. Ltd., China) treatment and hematoxylin–eosin (H&E, Shenzhen Ziker Biological Technology Co. Ltd., China) staining for 6 and 8 minutes, respectively.

Detection of myocardial apoptosis by flow cytometry

Myocardial apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC) kit (Biovision Inc., Mountain View, CA, USA). Annexin V-FITC, propidium iodide (PI), and HEPES buffer were mixed in a ratio of 1:2:50. Approximately 1 × 106 cells were resuspended in 100 µL of the dye liquor, shaken, left at room temperature for 15 minutes, and mixed after another addition of 1 mL of HEPES buffer. Myocardial apoptosis was indicated by FITC and PI fluorescence using 525 nm and 620 nm bandpass filters, respectively, excited at 488 nm.

Detection of cardiomyocyte proliferation

After dilution to 1 × 105/mL and cultivation for 12 hours, three groups of myocardial cells were placed into a 96-well plate. Once the bottom of the petri dish was covered, 10 µL of Cell Counting Kit-8 (CCK-8) reagent (Beijing Jiehui Bogao Bio-Tech Co. Ltd., China) was added. After allowing the mixture to stand at room temperature for 30 minutes, the optical density (OD) at 450 nm was measured in a microplate reader.

p38 MAPK levels in rat myocardial cells

Myocardial cells were collected as described. For electrophoresis, a 12% separation gel and an aqueous layer of 1 cm were prepared. While the electrophoresis apparatus underwent gel aggregation for 1 hour, a spacer gel was prepared and applied. The samples were separately loaded at 50 µg/well for electrophoresis at 90 V. Next, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane for 5 to 10 s. The membrane was then separated from the tank and rinsed with buffered saline (TBST) twice for 10 minutes. The membrane was then placed in a blocking solution in a horizontal shaker for 2 hours. The cells were incubated with primary rabbit anti-mouse p38 MAPK (1:500, Abcam PLC, Cambridge, UK) and GADPH (1:1000, Abcam PLC) antibodies at 4°C overnight. The cells were then rinsed three times with TBST for 10 minutes each and incubated at room temperature with peroxidase-labeled goat anti-rabbit secondary antibody (1:2000, Bioss, Beijing, China) within 2 hours. The cells were again rinsed three times with TBST for 10 minutes each and then incubated with chemiluminescence reagents for another 1 minute before enhanced chemiluminescence development. A gel imaging system (Bio-Rad, Hercules, CA, USA) was used to acquire images. All of the immunoblotted bands were subjected to photodensitometry. The relative protein expression was determined as the ratio of the OD value of the target band and the internal control. The PVDF membrane was evaluated by an automatic chemiluminescence imaging system.

Caspase-3 mRNA expression in myocardial cells

Myocardial cells mixed with chloroform were shaken and centrifuged at 1000 ×g for 10 minutes until the solution turned milky white. Following centrifugation at 1000 ×g at 4°C for 30 minutes, addition of isopropanol, and after further centrifugation at 1000 ×g for 30 minutes, the pellet was mixed with 75% ethanol, centrifuged again at 1000 ×g for 30 minutes, dried, and stored at −80°C. Total RNA was extracted and reverse transcribed into cDNA following the instructions of the kits used (60°C, 10 minutes; 95°C, 72°C, and 30s each; and 95°C, 5 minutes; 40 cycles). GAPDH was used as the internal reference, and the experiments were conducted in triplicate. Caspase-3 mRNA expression was determined by relative quantification using the 2−ΔΔCT method as shown in Table 1.

Table 1.

Primer sequences.

Protein	Gene	Primer sequence
Caspase-3	F	5ʹ-TGCGGTAGCAGCACATAATGG-3′ʹ
	R	5ʹ-CCAGTGCAGGGTCCGAGGT-3′ʹ
GAPDH	F	5ʹ-TGAACGGGAAGCTCACTGG-3′ʹ
	R	5ʹ-TCCACCACCCTGTTGCTGTA-3′ʹ

Statistical analysis

SPSS version 22.0 software (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. The differences in infarction size, caspase-3 mRNA expression, cell apoptosis and proliferation, p-p38MAPK protein expression, and pathological changes in myocardial tissues in the MY group, SS group, and GR group were assessed by analysis of variance. T-tests were conducted within and between the groups. A value of P < 0.05 was considered statistically significant.

Results

Representative image of myocardial infarction

The white part in the upper left corner of the image shown in Figure 1 is the site of myocardial infarction.

Figure 1.

Representative image of myocardial infarction.

Myocardial infarction size in the three groups

The largest myocardial infarction (53.93 ± 4.14%) was observed in the MY group; in comparison, the GR group had a reduced infarction size (21.68 ± 6.17%), whereas no myocardial infarction was observed in the SS group (P < 0.05), as shown in Table 2.

Table 2.

Percentage of infarct size in the three groups of rats (%).

Group	Number of Rats	Area Percentage (%)	F	P
SS group	5	0	9.705	<0.001
GR group	5	21.68 ± 6.17%*
MY group	5	53.93 ± 4.14%*#

*: Compared with the SS group, P < 0.05, #: Compared with the GR group, P < 0.05.

Cardiomyocyte cell viability in the three groups

The OD values in the SS and GR groups were higher than those in the MY group (P < 0.05). The SS group showed the highest and the MY group showed the least cell viability. Cell viability in the GR group was significantly higher than that in the MY group (P < 0.05), as shown in Figure 2.

Figure 2.

Myocardial cell viability in the three groups. Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5).

*: Compared with the SS group, P < 0.05, #: Compared with the GR group, P < 0.05.

Cardiomyocyte apoptosis in the three groups

The MY group had the highest number of apoptotic cells (P < 0.05), with an apoptosis rate of 61.35 ± 6.38%. The SS group had the lowest number of apoptotic cells (P < 0.05), with a rate of 16.57 ± 2.74%. The apoptosis rate in the GR group (26.97 ± 3.75%) was significantly decreased compared with that in the MY group (P < 0.05, Figures 3 and 4).

Figure 3.

Cardiomyocyte apoptosis in the three groups. Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5)

(a) SS group, (b) GR group, (c) MY group.

Figure 4.

Apoptosis rate of cardiomyocytes in the three groups. Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5).

*: Compared with the SS group, P < 0.05, #: Compared with the GR group, P < 0.05.

Pathological changes in the myocardial tissues in the three groups

As observed in Figure 5, the orientation of fibrils in myocardial tissues in the SS group was orderly and not misaligned, unlike that in the MY group, in which the fibrils were fractured, and the large amount of cellular cytoplasm induced tissue swelling. Fibrils in myocardial tissues in the GR group were disordered but showed reduced swelling, having a reduced amount of myocardial cytoplasm. The fibril orientation was more organized in the SS group than in the MY group.

Figure 5.

Orientation of myocardial tissue lesions in the three groups (×200). Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5).

(a) SS group, (b) GR group, (c) MY group.

Determination of the p-p38MAPK level in myocardial cells by western blotting

Western blotting was used to determine the p-p38MAPK protein levels in the MY group, SS group, and GR group from the myocardial tissue in vivo. According to the grayscale image, the highest level of p-p38MAPK was found in the MY group (P < 0.05), and the lowest level was in the GR group (P < 0.05). Compared with the MY and SS groups, the GR group exhibited reduced protein expression (P < 0.05 for all), as shown in Figure 6.

Figure 6.

p38 MAPK protein content in myocardial tissue. Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5).

*: Compared with the SS group, P < 0.05, #: Compared with the GR group, P < 0.05.

(a) p-p38MAPK protein content in the three groups of cardiomyocytes.

(b) Comparison of protein content among the three groups.

Caspase-3 mRNA expression in the three groups

Quantitative real-time (qRT)-PCR analyses indicated that the GR group had the lowest caspase-3 mRNA expression level in myocardial tissues (P < 0.05), whereas the MY group had the highest level (P < 0.05). Compared with that in the MY and SS groups, the caspase-3 mRNA expression in the GR group was significantly reduced (all P < 0.05), as shown in Figure 7.

Figure 7.

Caspase-3 mRNA expression in the three groups. Myocardial ischemia-reperfusion (MIR) group (MY group, n = 5), rats were used to establish MIR models; 2) sham surgery group (SS group, n = 5), only a chest incision was made in rats, without cardiomyocyte injury; and 3) ginsenoside Rh3 group (GR group, n = 5).

*: Compared with the SS group, P < 0.05, #: Compared with the GR group, P < 0.05.

Discussion

Our study investigated the role of Rh3 in the recovery from MIRI and consisted of three experimental groups (MIRI, sham MIR, and the test MIRI group treated with Rh3) to compare tissue and cellular alterations associated with the pathology of MIRI and the potential role of Rh3 in rescuing MIRI. Additionally, cardiomyocytes were isolated from rats and used as an in vitro model of ischemia-reperfusion, whereby ischemia was simulated with hypoxia and reperfusion with reoxygenation. The three experimental groups were set up in parallel with the in vivo setup and employed to investigate the effects of Rh3 on MIRI by assessing cell viability using the CCK-8 assay, apoptosis using annexin V-FITC/PI flow cytometry, p-p38 MAPK protein expression using western blotting, and caspase-3 mRNA expression using PCR to assess the potential role of Rh3 in protecting cardiomyocytes from hypoxia/reoxygenation-induced cell death.

MIRI refers to the increased myocardial damage caused by the sudden restoration of blood flow to previously ischemic myocardial tissues.[10] It occurs in patients with myocardial ischemia caused by conditions such as myocardial infarction and coronary arteriosclerosis.[11] Currently, the incidence of ischemic heart disease in the elderly population remains high. Of the available therapies, including drug treatment, surgery is the most effective.[12] Nonetheless, some patients might have unsatisfactory outcomes because MIRI may occur after surgery, which is likely to increase the patient’s morbidity. Accordingly, effective methods to reduce MIRI are urgently needed.[13]

Our results showed that the largest myocardial infarction size occurred in the MY group; the GR group had a smaller infarction size, whereas no myocardial infarction was observed in the SS group. After anaerobic cultivation of myocardial cells, OD values in the SS and GR groups were higher than those in the MY group, and cell viability in the GR group was significantly increased compared with that in the MY group. Moreover, the MY group had the highest number of apoptotic cells, whereas the SS group had the lowest number of apoptotic cells. Apoptosis in the GR group was significantly decreased compared with that in the MY group. As shown in Figure 3, the orientation of fibrils in myocardial tissues in the SS group was orderly and not as misaligned as that in the MY group, in which the fibrils were swollen and disrupted. Fibrils in the GR group specimens were disordered but showed reduced swelling with a small amount of myocardial cytoplasm. The fibrils in the GR group were better aligned than those in the MY group. Finally, according to the grayscale image, the GR group exhibited a reduced p38 MAPK protein level compared with the MY and SS groups. qRT-PCR analysis showed that caspase-3 mRNA expression in the GR group was obviously reduced compared with that in the MY and SS groups. Studies have shown that the p38 MAPK signaling pathway is significantly associated with the occurrence of cardiac diseases, including MIRI, coronary artery disease, and myocardial infarction.

Hypertension is also caused by activation of the p38 MAPK signal.[14,15] A study on rats with cerebral infarction reported that the activation of the p38 MAPK signaling pathway increased as the infarct size increased, and a large number of neuronal cells became apoptotic, suggesting that the p38 MAPK signaling pathway is also involved in the occurrence of cerebral infarction.[16] Studies have found that the use of drugs to pretreat ischemic limbs resulted in a reduction in ischemic lesions, which may be related to the decreased activity of caspase-3. The caspase expression level was positively correlated with myocardial cell apoptosis, and the ginsenoside Rh3 was shown to reduce myocardial cell apoptosis by reducing the caspase-3 content.[17,18] Zhang et al.[19] confirmed that Rh3 improves cerebral ischemia by inhibiting P38 MAPK signaling. Rh3 has therapeutic effects on the cardiac tissues of rats subjected to MIR. Hence, it might suppress the formation of thrombus and reduce myocardial tissue apoptosis.[20-22]

During the modeling process, rats with other obvious complications were excluded from the experiment to prevent interference with the detection of signaling factors in the experiment. For several reasons, we could not perform some tests that would be useful in this study. Future experiments should involve the use of different methods to elucidate the mechanism by which Rh3 exerts its effects on MIRI rats and its effect on p38 MAPK and caspase-3 levels to provide more valuable evidence to support the treatment of clinical MIRI patients. In conclusion, the ginsenoside Rh3 improved myocardial necrosis and caspase-3 expression in myocardial tissues. Thus, Rh3 might be a potent suppressor of the p38 MAPK signaling pathway and has therapeutic potential.