Protective Effects of Six PPTs on Hypoxia/Reoxygenation Induced Cardiomyocyte Injury by Different Treatments

Tingbo CHEN, Dong HUANG, Ziming ZHOU, Gengting DONG

1. Zhongshan Torch Polytechnic, Zhongshan 528436, China; 2. Zhongshan Functional Cosmetics (Zhongshan Torch Polytechnic) Engineering Research Center, Zhongshan 528436, China; 3. Zhongshan Hospital of Traditional Chinese Medicine, Zhongshan 528400, China

Abstract [Objectives] To detect the protective effects of six protopanaxatriols (PPTs) on hypoxia/reoxygenation (H/R) induced cardiomyocyte injury by different treatments. [Methods] The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used for detecting the protective effects of six PPTs including ginsenoside Rg1, Re, Rf, Rg2, (R) Rh1 and (S) Rh1 on cell viability reduced by H/R in different treatments. And the adenosine triphosphate (ATP) content and mitochondrial membrane potential (MMP) were used for detecting the mitochondrial function change during PPTs treatment. [Results] Among six PPTs, ginsenoside Rg1, Re, Rf, Rg2 and (R) Rh1 at the concentration of 12.5 μM significantly increased the cell survival when treated before and during H/R. These five PPTs also significantly increased the ATP content and MMP reduced by H/R in the same manner. In comparison, only Rg1 significantly increased the cell viability compared with H/R group by pretreating and treating the cells during hypoxia process. [Conclusions] Different treatments affect the protective effects of PPTs. When treated before and during H/R, ginsenoside Rg1, Re, Rf, Rg2 and (R) Rh1 protect the cardiomyocyte against H/R injury mitochondrial function, and only ginsenoside Rg1 has protective effects when treated before and during hypoxia process.

Key words Protopanaxatriols (PPTs), Ginsenoside Rg1, Mitochondrial function, Treatment process

1 Introduction

According to the WHO report, the world’s biggest killer is ischaemic heart disease (IHD), responsible for 16% of the world’s total deaths[1]. Timely and effective reperfusion therapeutics such as thrombolytic therapy or primary percutaneous coronary intervention (PCI) could decrease the patient death. With the extension of the ischemia time, the reperfusion may induce additional damage to myocardium called "ischemia/reperfusion (I/R) injury". Mechanism studies show that mitochondria as the power house of cells play an important role during the cardiac I/R injury. Lack of O2lead cell acidification and Na+is exported into the cytosol via the Na+/Ca2+exchanger causing a rise in Ca2+concentration. The inhibited oxidative phosphorylation limits ATP generation, which further disables Ca2+exports by ATPases. With the ATP consumption during the hypoxia, the mitochondrial membrane potential (MMP) keeps at a low condition. At reperfusion, oxygen returns to cells results in reactive oxygen species (ROS) generation and mitochondrial Ca2+accumulation. The restoration of normal pH triggers PT pore opening and releases cell death-inducing proteins, such as cytochrome C, leading to the cell death in I/R injury[2-3].

PanaxginsengC.A.Meyer, a famous traditional Chinese herbal medicine, has been used for thousands of years in China. Ginsenosides, including protopanaxadiols (PPDs) and protopanaxatriols (PPTs), are considered as the major pharmacologically active ingredients, producing antioxidant, anti-inflammatory and antiapoptotic effects. Ginsenoside Rg1, Re, Rf, Rg2, (R) Rh1 and (S) Rh1 are active components of PPTs and had the protective effects in the cardiovascular disease. The mechanisms of Rg1 are proangiogenic and vascular remodeling[4], anti-fibrotic[5], antioxidant and intracellular calcium homeostasis[6]. Ginsenoside Re protects the cardiovascular system by regulating contractive and electromechanical alternans (EMA), producing antiarrhythmic, anti-ischemic, and angiogenic regeneration[7]. The mechanisms are related to intracellular calcium homeostasis, antioxidant, and anti-inflammation[8]. However, the mechanism studies of Rf, Rg2 and Rh1 on the cardiovascular diseases are limited.

Mitochondria plays an important role during the cardiac I/R injury and mitochondria has become a novel therapeutic target. Up to now, the TCM that focuses on maintaining the mitochondria against I/R in cardiomyocyte has not been clearly verified. The aim of current study is to find out the characteristic features of changes in the mitochondrial function induced by H/R in cardiac cells, and then to evaluate the protective effects of six PPTs on mitochondria against H/R injury by different treatments.

2 Experiment

2.1 Cell cultureRat H9c2 cardiomyocyte cell line was purchased from the American Type Culture Collection (CRL1446, ATCC, USA) and cultured with DMEM (Gibco, Big Cabin, Oklahoma, ME, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Big Cabin, Oklahoma, ME, USA) and 1%V/Vpenicillin/streptomycin (P/S) (Gibco, Big Cabin, Oklahoma, ME, USA) at 37 ℃, 5% CO2humidified atmosphere.

2.2 Hypoxia/reoxygenation (H/R) in cardiac cellsHypoxia in H9c2 cells was induced by replacing the normoxia medium with 5 kinds of mediums (DMEM (H) (4.5 g/L glucose), DMEM (H) +FBS, DMEM (L), DMEM (L) (1 g/L glucose) +FBS, Krebs-Ringer Bicarbonate (KRB) buffer (115 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 24 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM Hepes, 0.01%BSA) and placing the plates or dishes in a Billups-Rotenberg modular incubator chamber (Billups-Rotenberg, CA, USA) saturated with 99.99% nitrogen at 37 ℃ for different time. After hypoxia progress, the medium was replaced with DMEM complete medium, and the cells were incubated in the normoxia condition (95% air+5% CO2) for different time.

2.3 Treatment with six PPTsGinsenoside Rg1, Re, Rf, Rg2, R-Rh1, S-Rh1 were purchased from National Institutes for food and drug control. Six PPTs were dissolved in dimethylsulfoxide to produce a 100 mM stock solution and diluted with culture medium to indicated final concentrations before treatment. H9c2 cells were treated with six PPTs prior reperfusion or prior and during reperfusion:

For prior reperfusion manner: cells were pre-treated with six PPTs for 24 h and during hypoxia process. After seeding 24 h, cells were cultured with six PPTs diluted in DMEM complete medium for 24 h. Then cells were washed 3 times with KRB wash buffer and cultured with KRB culture buffer with six PPTs for 3 h of hypoxia. The cells were treated with reoxygenation procedure as previously described.

For prior and during reperfusion manner: cells were pre-treated with six PPTs for 24 h and during H/R processes. After seeding 24 h, cells were cultured with six PPTs diluted in DMEM for 24 h. Then cells were washed 3 times with KRB wash buffer and cultured with KRB culture buffer with six PPTs for 3 h of hypoxia. Cells were treated with reoxygenation using DMEM complete medium with six PPTs for 3 h.

2.4 MTT assayCell viability was determined by MTT assay. Cells were seeded onto the 96-well plate at a density of 1×104cells/well and cultured overnight for cell adhesion. After H/R treatment, cells in each well were incubated with 10 μl MTT (5 mg/mL, Sigma-Aldrich, St.Louis, MI, USA) at a final concentration of 500 μg/mL for 4 h at 37 ℃. 100 μL SDS-HCl was added to each well and the plate was incubated at 37 ℃ overnight. The absorbance was detected at 570/650 nm on an Infinite M200 pro microplate reader (Tecan, Switzerland). The cell viability is calculated as the percentages of the absorbance of treated cells divided by the absorbance of control cells.

2.5 MMP assay by JC-1 stainingMMP was determined by JC-1 mitochondrial membrane potential assay kit (Abcam, Cambridge, UK). The cells were loaded with 2 μg/mL JC-1 diluted in 1×supplemented buffer and incubated for 10 min at 37 ℃ in the dark. After staining, the cells were treated in accordance with the H/R processes. The red and green fluorescence were detected by an Infinite M200 pro microplate reader. JC-1 aggregate yielding a red fluorescence and JC-1 forms monomers that yields green fluorescence. MMP was calculated by the ratio of red to green fluorescence. The fluorescence of JC-1 was detected at wavelengths of 530 nm (green) and 590 nm (red), with an excitation wavelength of 470 nm by an Infinite M200 pro microplate reader (Tecan, Switzerland). MMP was calculated by the ratio of red to green fluorescence.

2.6 ATP content assayThe ATP level of H9c2 cells was measured using a luminescent ATP detection assay kit (Abcam, Cambridge, UK). Briefly, the cells were lysed by the detergent buffer and the microplate was shook for 5 min. Then reconstituted substrate solution was added to the wells and the microplate was shook for 5 min. The microplate was kept in dark for 10 min. The ATP concentration was analyzed using an Infinite M200 pro microplate reader (Tecan, Switzerland).

2.7 Statistical analysisAll data were expressed as the means±SEM, and each experiment was repeated at least three times. One-way analysis of variance (ANOVA) with multiple comparisons using Dunnett’s Test (GraphPadPrism, GraphPad Software Inc., San Diego, CA, US) was performed to analyze difference between different groups.P<0.05 was considered statistically significant.

3 Results and analysis

3.1 Effects of six PPTs on cell viability reduced by H/R

3.1.1Cells pre-treated with six PPTs. The cells were pre-treated 24 h and during hypoxia process with six PPTs. As shown in Fig.1, Rg1 increased cardiomyocyte survival after H/R injury in dose-dependent manner, while Re, Rf, Rg2, (R) Rh1 and (S) Rh1 did not increase the cell viability compared with H/R group. Rg1 at 12.5 μM significantly increased cell viability to 64.03%±1.11% in comparison with H/R. The cell viabilities of the 25 μM and 50 μM Rg1 groups were kept at high level, which indicates that Rg1 has the protective effect if pre-treated and treated the cells during hypoxia process.

Note: A. Rg1, B. Re, C. Rf, D. Rg2, E. (R) Rh1, F. (S) Rh1. The cell viability was detected by MTT assay and calculated as the percentages of the absorbance of treated cells divided by the absorbance of control cells. The values were the mean±SEM of three independent experiments. *P<0.05, and **P<0.01 compared with H/R group, ###P<0.001 compared with control group.

3.1.2Cells treated with six PPTs before and during H/R processes. Cells were pretreated 24 h and treated during the H/R processes with six PPTs. As shown in Fig.2, Rg1, Re, Rf, Rg2, and (R) Rh1 significantly increased the cell viability against the H/R injury. Rg1 increased cardiomyocyte survival after H/R injury in dose-dependent manner. Rg1 at 6.25 and 12.5 μM significantly increased cell viability to 62.36 %±1.91% and 66.86%±2.66% in comparison with H/R and the cell viability of 25 μM Rg1 group was kept at high level. The cell viability was increased after Re treatment at the concentration of 12.5 and 25 μM, which suggests that Re has protective effect on H/R model at the concentration higher than 12.5 μM during H/R process. Rf at 6.25, 12.5 and 25 μM also significantly increased cell viability compared with H/R group. Rg2 and (R) Rh1 at 12.5 and 25 μM separately increased cell viability against H/R injury, while (S) Rh1 did not increase the cell viability even at the 25 μM. All these results indicate that Rg1, Re, Rf, Rg2 and (R) Rh1 have the protective effects on the H/R model when treated the cells prior and during reperfusion. Rg1, Re, Rf, Rg2 and (R) Rh1 at the concentration of 12.5 μM significantly increased the cell survival against H/R injury, and the cell viabilities were slightly increased in the 25 μM treatment groups. So cells were treated with Rg1, Rf, Rg2 and (R) Rh1 at 12.5 μM in the further study.

Note: A. Rg1, B. Re, C. Rf, D. Rg2, E. (R) Rh1, F. (S) Rh1. The cell viability was detected by MTT assay and calculated as the percentages of the absorbance of treated cells divided by the absorbance of control cells. The values were the mean±SEM of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 compared with H/R group, ###P<0.001 compared with control group.

3.2 Effects of six PPTs on ATP content and MMP induced by H/RPrevious results showed that H/R leads to the mitochondrial dysfunction in H9c2 cells. The effects of six PPTs on ATP content was detected by the luminescent ATP detection assay kit. H9c2 cells were treated with Rg1, Re, Rf, Rg2, (R) Rh1, and (S) Rh1 at 12.5 μM following the therapeutic method. The results showed that Rg1, Re, Rf, Rg2 and (R) Rh1 significantly increased the ATP content reduced by H/R, which indicates that Rg1, Re, Rf, Rg2 and (R) Rh1 have the best protective effects on ATP content against H/R injury in H9c2 cells.

Note: Cells were pretreated 24 h and treated during H/R with Rg1, Re, Rf, Rg2, (R) Rh1, and (S) Rh1 at 12.5 μM. The ATP content was measured by a luminescent ATP detection assay kit. The ATP content in the H/R with or without six PPTs treatment groups were expressed as a percentage of the control group, which was set as 100%. The values were the mean±SEM of three independent experiments. *P<0.05, and **P<0.01 compared with H/R group, ###P<0.001 compared with control group.

MMP is another feature to evaluate mitochondrial function in H/R injury. Previous study showed that H/R significantly decrease the MMP and finally lead the cardiomyocyte death. So the effects of six PPTs on MMP were measured with JC-1 staining and the ratio of red to green florescence represented the MMP change. H9c2 cells were treated with Rg1, Re, Rf, Rg2, (R) Rh1, and (S) Rh1 at the concentration of 12.5 μM. As shown in Fig. 4, Rg1, Re, Rf, Rg2 and (R) Rh1 significantly inhibit the H/R induced MMP decreased, which indicates that Rg1, Re, Rf, Rg2 and (R) Rh1 have the best protective effects on the MMP against H/R injury in H9c2 cells.

Note: The cells were stained with JC-1 treated following the H/R processes with or without six PPTs treatment as the therapeutic method. The red and green fluorescence were detected by an Infinite M200 pro microplate reader. The values were the mean±SEM of three independent experiments. *P<0.05, and **P<0.01 compared with H/R group, ###P<0.001 compared with control group.

4 Discussion

As mitochondria are central in the cardiomyocyte survival that is instigated during myocardial infarction[2], we investigated mitochondrial dysfunction in H/R stress model. In the myocardial I/R, the ATP formation and MMP are decreased and in turn results in mitochondrial Ca2+overload and reactive oxygen species (ROS) generation[8]. With mitochondrial permeability transition pore opening, Bcl-2 activity is inhibited and Bax moves to the mitochondria leading to increased cytochrome C release to activate mitochondrial related apoptosis pathway[9-10]. In current study, H/R induced mitochondrial dysfunction by decreasing the mitochondrial respiration, MMP, ATP content. These indicators were used to evaluate the activity of six PPTs.

The results showed that the treatment of PPTs was before and during H/R, the cell viability in Rg1, Re, Rf, Rg2, and (R) Rh1 treatment groups were significantly increased compared with H/R group; while the treatment of PPTs was before and during hypoxia process, only ginsenoside Rg1 significantly increased the cell viability compared with H/R group. Therefore, the method of treatment plays an important role in the protective effects of PPTs.

Furthermore, Rg1, Re, Rf, Rg2, and (R) Rh1 significantly increased the ATP content and MMP reduced by H/R with the treatment before and during H/R. This suggested the protective effects of PPTs may be related to the mitochondrial function. The result further verified the mechanism of ginseng traditional used in cardiopathy.