Phytomedicine
Volume 19, Issue 2 , Pages 99-110, 15 January 2012

Danshen–Gegen decoction protects against hypoxia/reoxygenation-induced apoptosis by inhibiting mitochondrial permeability transition via the redox-sensitive ERK/Nrf2 and PKCɛ/mKATP pathways in H9c2 cardiomyocytes

  • Po Yee Chiu

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • ,
  • Hoi Yan Leung

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • ,
  • Pou Kuan Leong

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • ,
  • Na Chen

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • ,
  • Limin Zhou

      Affiliations

    • School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
    • ,
  • Zhong Zuo

      Affiliations

    • School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
    • ,
  • Philip Y. Lam

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • ,
  • Kam Ming Ko

      Affiliations

    • Division of Life Science, The Hong Kong University of Science & Technology, Clear Water Bay, China
    • Corresponding Author InformationCorresponding author.

published online 07 September 2011.

Article Outline

Abstract 

Danshen–Gegen (DG) Decoction, an herbal formulation containing Radix Salviae miltiorrhizae and Radix Puerariae lobatae, has been used for the treatment of coronary artery disease in Chinese medicine. In the present study, the involvement of ERK- and PKCɛ-mediated pathways in the cytoprotection against apoptosis afforded by DG pretreatment was investigated in H9c2 cardiomyocytes. Pretreatment with a methanol extract of aqueous DG decoction protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated the enhancement of cellular reduced glutathione and a reduced sensitivity to Ca2+-induced mitochondrial permeability transition. DG extract increased the production of cytochrome P-450 (CYP)-dependent reactive oxygen species (ROS) in H9c2 cardiomyocytes, which was accompanied by the concomitant activation of ERK1/2 and PKCɛ. The DG-induced ERK1/2 activation was followed by the translocation of Nrf2 from the cytosol to the mitochondria accompanied by an increase in the expression of glutathione-related antioxidant proteins. In addition, the increased expression of hemeoxygenase-1 was associated with the activation of Akt and BAD, indicative of anti-apoptotic activity. In conclusion, DG treatment activated both ERK/Nrf2 and PKCɛ pathways, presumably by ROS arising from CYP-catalyzed processes, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.

Abbreviations: ABT, 1-aminotriazole, Akt, alpha-serine threonine kinase, BAD, Bcl-2 associated death promoter, CYP, cytochrome P-450, ERK, extracellular signal regulated kinase, DCFDA, 2′-7′-dichlorofluorescein diacetate, DG, Danshen–Gegen, DMTU, dimethylthiouracil, GCL, γ-glutamyl cysteine ligase, GCLm, modulatory subunit of GCL, G6DPH, glucose 6-phosphate dehydrogenase, GR, glutathione reductase, GSH, reduced glutathione, 5-HD, 5-hydroxydecanoate, HO-1, hemeoxygenase 1, mKATP, mitochondrial ATP-dependent potassium channel, MPT, mitochondrial permeability transition, Nrf2, nuclear factor eryhtroid 2-related factor 2, PBS, phosphate-buffered saline, PI, propidium iodide, PKCɛ, protein kinase C-epsilon, ROS, Reactive oxygen species

Keywords: Salviae miltiorrhizae, Puerariae lobatae, Extracellular signal-regulated protein kinase (ERK), Protein kinase C-epsilon (PKCɛ), Mitochondrial permeability transition, Apoptosis, H9c2

 

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Introduction 

Danshen and Gegen, which are roots of Salviae miltiorrhizae and Puerariae lobatae, respectively, are traditional Chinese medicinal herbs that have been widely used in China, Japan, Korea and Taiwan for the treatment of angina pectoris (Ji et al., 2000) and myocardial infarction (Adams et al., 2006). The herbal formulation made of Danshen and Gegen (Danshen–Gegen decoction) has long been used for the treatment of coronary artery disease (Xie et al., 2003). Raw herbs of Danshen and Gegen, and their isolated compounds, were reported to produce beneficial effects on cardiovascular function in human subjects (Tam et al., 2009), rodents (Ji et al., 2003) and cultured human endothelial cells (Sieveking et al., 2005). Recent studies in our laboratory have demonstrated that both acute and long-term treatment with an aqueous extract prepared from Danshen and Gegen in a ratio of 7:3 (w/w) protected the myocardium against ischemia/reperfusion injury in rats ex vivo (Chiu et al., 2011b, Chiu et al., 2011c). In addition, the treatment with this Danshen–Gegen (DG) aqueous extract immediately after the intraperitoneal administration of isoproterenol was found to reduce the extent of myocardial damage in rats (Wong et al., 2011). The cardioprotection afforded by DG pre-/post-treatment may at least in part be attributed to the reactive oxygen species (ROS)-mediated activation of protein kinase C-epsilon (PKCɛ), which in turn opens a mitochondrial ATP-dependent potassium channel (mKATP), causing inhibition of a mitochondrial permeability transition (MPT) (Chiu et al., 2011b, Wong et al., 2011). The finding that the cardioprotection afforded by long-term DG pretreatment was paralleled by enhancements in mitochondrial glutathione antioxidant status suggests the activation of a redox-sensitive extracellular signal-regulated protein kinase (ERK) pathway in eliciting the glutathione antioxidant response.

PKCɛ and ERK play an essential role in regulating cell survival and death through ROS-mediated processes (Kabir et al., 2006, Slupsky et al., 2007). In a short time-course within minutes, ROS can activate both PKCɛ and ERK, with the former translocating to the mitochondrion and causing opening of the mKATP (Costa and Garlid, 2008). In a more protracted time-course within hours, the activated ERK can phosphorylate the nuclear factor erythroid 2-related factor 2 (Nrf2), which then translocates from the cytosol to the nucleus, with subsequent enhancement in the expression of antioxidant defensive genes such as the subunits of γ-glutamyl cysteine ligase (GCL), thioredoxin, glutathione S-transferases and NAD(P)H-quinone oxidoreductase (Jeyapaul and Jaiswal, 2000, Kensler et al., 2007, Liu et al., 2007, Zhang et al., 2006). In the present study, we aimed to investigate the involvement of ERK- and PKCɛ-mediated pathways in the cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by DG pretreatment in H9c2 cardiomyocytes, a clonal cell line derived from embryonic rat heart tissue (Hescheler et al., 1991).

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Materials and methods 

Preparation of DG extract 

The Danshen–Gegen aqueous extract with an optimal crude herb ratio of Danshen to Gegen (7:3, w/w), as assessed by cardioprotection against ischemia/reperfusion injury (Chiu and Ko, 2004), was prepared in large-scale for experimental and clinical investigations, as described in Chiu et al. (2011b). To remove the sugars (monosaccharides and disaccharides) that could affect the cultured cells, the DG aqueous extract (100g) was extracted by heating under reflux in 100ml of methanol. The procedure was repeated twice. The pooled extract was dried by evaporation under reduced pressure to obtain the DG extract at a yield of 40%, and the DG extract was stored at 4°C prior to use. In addition to monosaccharides and dissaccharides, polysaccharides, which are unlikely to produce cardioprotective action, were also removed by methanol extraction. As a result, DG extract produced cytoprotective action similar to that of the aqueous DG extract in H9c2 cardiomyocytes, but with a lower effective concentration (data not shown).

Chemical analysis of the DG extract 

DG extract were performed as described in our previous study with minor modifications relating to the instrument and chromatographic conditions (Chang et al., 2008). HPLC analysis indicated that the DG extract contained the following marker compounds (μg/100mg; mean±SD, n=3): Danshensu (2406.7±100.5), Salvianolic acid B (1407.3±40.0), Protocatechuic aldehyde (132.7±5.9), Puerarin (2821.4±82.4), Daidzein 8-C-apiosyl-glucoside (644.5±31.4), Daidzin (260.9±7.1) and Daidzein (260.7±13.9). The methanol DG extract had higher concentrations of these marker compounds than those in the Danshen–Gegen aqueous extract (Chiu et al., 2011b), but both preparations showed similar relative concentrations of the marker compounds. HPLC fingerprint analysis of the methanol extract prepared from two batches of DG aqueous extract showed substantial variations (2–150%) in the contents of marker compounds (Fig. 1 and Table 1). The two DG extracts inhibited cell apoptosis to varied extents which correlated with the concentrations of daidzein 8-C-apiosyl-glucoside and daidzin in the extracts (Table 2). These two markers may be used for chemical standardization of DG preparation.

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  • Fig. 1. 

    HPLC fingerprint analysis of the methanol extract of two batches of DG aqueous preparation. Contents of chemical markers of the methanol extract of DG aqueous preparation (Batched A is the sample used in the experiment). Peaks were detected at 280nm (1: Danshensu; 2: Protocatechuic aldehyde; 3: Puerarin; 4: Daidzein 8-C-apiosyl-glucoside; 5: Daidzin; 6: Salvianolic acid B; 7: Daidzein; 8: Cryptotanshinone; 9: Tanshinone I; 10: Tanshinone IIA).

Table 1. Content of chemical markers in the methanol extract of two batches of DG.
AnalytesContent (μg/100mg) (n=3)
Batch ASDBatch BSDMeanInter-batch variation (%)
Danshensu2406.7100.5655.245.91530.9114
PCA132.75.918.93.075.8150
Puerarin2821.482.42761.1128.92791.32.2
DAG664.531.4865.636.2765.126
Daidzin260.97.1394.025.4327.541
SAB1407.340.04997.5172.93202.4112
Daidzein260.713.9210.213.5235.521

Note: PCA, Protocatechuic aldehyde; DAG, Daidzein 8-C-apiosyl-glucoside; SAB, Salvianolic acid B.

Table 2. Bioassay on the methanol extract of 2 batches of DG preparation: effect on hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes.
Yield of methanol extraction% Protection against apoptosis
100μg/ml300μg/ml
Batch A532255
Batch B602165

H9c2 cardiomyocytes were treated with the methanol extracts of DG (batch A and B) at the indicated equivalent doses (with respective to the dry aqueous DG extracts) for 24h. The cells were then subjected to 2h of hypoxia followed by 16h of reoxygenation. The extent of hypoxia/reoxygenation-induced apoptosis was estimated by flow cytometry. Data were expressed as percent protection with respect to the untreated control.

Cell culture 

H9c2 cardiomyocytes were maintained in DMEM medium supplemented with 10% (v/v) fetal calf serum, 100IU/ml penicillin and 100μg/ml streptomycin. All cells were grown under an atmosphere of 5% (v/v) CO2 in air at 37°C. Cells were grown to 80% confluence prior to the exposure to drugs in the various experiments.

In vitro hypoxia/reoxygenation challenge 

After the incubation with DG extract at 30 or 100μg/ml for 24h, cells were then subjected to 2h of hypoxia and followed by 16h of reoxygenation, as previously described (Chiu et al., 2008).

Annexin V and PI staining 

Apoptotic cell death was measured by Annexin V-FITC-Fluos/propidium iodide (PI) staining followed by flow cytometry analysis using a COULTER® EPICS® XL™ Flow Cytometer (Beckman Coulter, Fullerton, CA), as described previously (Chiu et al., 2008).

Measurement of mitochondrial cytochrome c release 

Mitochondrial cytochrome c release was assessed by measuring cytosolic cytochrome c levels by Western blot analysis, as described in Chiu et al. (2008).

Measurement of cellular reduced glutathione 

Cell lysates were prepared as described in Chiu et al. (2006). Reduced glutathione (GSH) levels in cell lysates were measured using an enzymatic method of Griffith (1980).

Measurement of mitochondrial permeability transition (MPT) pore opening with Calcein AM 

The opening of the MPT pore was measured by adopting the method of MitoProbe™ Transition Pore Assay Kit (Molecular Probes, Eugene, OR) with minor modifications using Calcein AM and CoCl2, as described in Chiu et al. (2008).

Cytochrome P-450 (CYP)-dependent ROS production 

The extent of ROS production was measured by using 2′-7′-dichlorofluorescein diacetate (DCFDA) in H9c2 cardiomyocytes, with a modification of the procedure described by Bansal et al. (2010). For the time-course study, cells were incubated with the DG extract at concentrations ranging from 30 to 300μg/ml (dissolved in phosphate-buffered saline, PBS) and DCFDA for increasing periods of time (1–4h). For the CYP inhibitor and antioxidant study, cells were pre-incubated with 1-aminobenzotriazole (ABT, a wide-spectrum CYP inhibitor; 10mM final concentration, 2h) or dimethylthiouracil (DMTU, a thiol-containing antioxidant; 20mM final concentration, 1h) prior to the addition of DG extract at 300μg/ml. Vehicle (i.e. PBS) was added for the appropriate control. Then cells were co-incubated with ABT/DMTU and the DG extract, and the fluorescence intensity of each well was measured at an excitation wavelength of 485nm and an emission wavelength of 530nm by Victor V2 Multi-label Counter (Perkin Elmer, Turku, Finland) every 15min for 6h at 37°C. The fluorescence intensity of DG-treated cells was normalized with reference to the respective time-matched vehicle control. The extent of DG-induced ROS production was estimated by computing the area under the curve (AUC) plotting normalized fluorescence intensity against time (min), and expressed in arbitrary units.

Western blot analysis of ERK1/2 

Cells were incubated with the DG extract (300μg/ml) or vehicle for increasing periods of time (1–24h). Cells for ERK1/2 analysis were first incubated with serum-free medium for a 24-h period of starvation, which was followed by incubation with drug-containing serum-free medium. Total and phosphorylated-ERK1/2 levels were measured by Western blot analysis, as described in Chiu et al. (2011a).

Preparation of nuclear extracts and Western blot analysis of Nrf2 

Cells were first treated with the DG extract for 3h, followed by the incubation with fresh medium (without drug) for increasing periods of time (2–18h). Cells were harvested by trypsinization at the indicated period of time, and the nuclear fraction was prepared as described (Langston et al., 2008). Nuclear Nrf2 level was measured by Western blot analysis, as described in Chiu et al. (2011a).

Western blot analysis of glutathione-related antioxidant proteins 

Cells were treated with the DG extract as described in the Nrf2 experiment and measured for levels of GSH and glutathione-related antioxidant proteins at increasing time intervals. Cellular GSH levels were measured as described previously. Glutathione antioxidant response was assessed by measurements of cellular glutathione antioxidant protein levels, as described in Chiu et al. (2011a).

Western blot analysis of HO-1, Akt and BAD 

Cells were treated with the DG extract at 300μg/ml for increasing periods of time (2–18h). Equal amounts of cell lysates (50μg protein) were subjected to 10% SDS-PAGE followed by Western blotting using antibodies against inducible hemeoxygenase (HO-1), alpha-serine threonine kinase (Akt) and Bcl-2 associated death promoter (BAD), which are components involved in HO-1/Akt pathway. All antibodies were rabbit polyclonal antibodies, which are specific for the HO-1 (1:500; Santa Cruz Biotechnology), phosphorylated and non-phosphorylated antibodies of Akt (Ser 473) (1:1000; Cell Signal Technology) and BAD (Ser 155) (1:1000; Cell Signal Technology). All immuno-stained protein bands were analyzed by densitometry, and the extents of Akt and BAD activation were estimated by computing the ratio of phosphorylated protein level to non-phosphorylated protein level in DG-treated cells with respect to the time-matched control, and the amounts (arbitrary units) of HO-1 were normalized with reference to β-actin (1:1000; Cell Signal Technology) levels (arbitrary units) in the sample.

Western blot analysis of PKCɛ 

Cells were treated with the DG extract at 300μg/ml or vehicle for increasing periods of time (1–8h). After the incubation, cells were harvested with 2ml of 0.1mM PMSF in PBS and then centrifuged at 400×g for 10min at 4°C. The cells were then resuspended in 400μl of lysis buffer, and the lysates were further processed by passing through a syringe needle (21G1/2) repetitively for 10 times. The mitochondrial and cytosolic fractions were prepared by differential centrifugation (Wang et al., 2001). In essence, the homogenates were centrifuged at 1000×g for 10min at 4°C. The resultant supernatants were centrifuged at 100,000×g for 1h at 4°C. The pellet was washed twice with the lysis buffer and then resuspended into 100μl of ice-cold of lysis buffer supplemented with 1% Triton X-100. After measuring the protein concentrations, the cytosolic and mitochondrial fractions were mixed with 6 volumes of SDS sample loading buffer. Mitochondrial and cytosolic proteins (40μg) were resolved using 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. An equal loading of proteins was confirmed by staining with Ponceau-S solution [0.5% (w/v) Ponceau S in 1% (v/v) acetic acid] (Bannur et al., 1999). After blocking with 5% skim milk, the membranes were incubated with anti-PKCɛ antibody (Upstate, Temecula, CA) at 4°C overnight. The membrane was subsequently washed and incubated with peroxidase-conjugated secondary antibody (1:2000; Cell Signaling Technology) for 2h at room temperature. Immuno-stained bands were quantitatively analyzed. The intensity of immuno-stained PKCɛ band was normalized to that of the sum of Ponceau S-stained bands in the sample. The ratio of mitochondrial (m)PKCɛ level to cytosolic (c)PKCɛ level indicated the extent of PKCɛ translocation.

Effect of ABT and DMTU on ERK 1/2 activation and PKCɛ translocation 

Cells were pre-incubated with ABT (10mM final concentration, 2h) or DMTU (20mM final concentration, 1h) prior to the addition of DG extract at 300μg/ml. Cells were then co-incubated with ABT/DMTU and the DG extract for 4h. After the incubation, cells were lysed with 300μl of lysis buffer. Equal amounts of cell lysate (10μg) were subjected to 12% SDS-PAGE and Western blot analysis of phosphorylated and total ERK 1/2 were performed.

For the study on PKCɛ translocation, cells were pre-incubated with ABT/DMTU and then co-incubated with ABT/DMTU and the DG extract for 3h. The cells were harvested, and the cytosolic and mitochondrial fractions were prepared as described above. Equal amounts of cytosolic and mitochondrial proteins (40μg) were subjected to 10% SDS-PAGE, respectively, and Western blot analysis using PKCɛ antibody was performed. The extent of PKCɛ translocation was indicated by the ratio of mPKCɛ level to cPKCɛ level.

Effects of PKCɛ and mKATP inhibition on cytoprotection against apoptosis 

Cells were first treated with the PKCɛ translocation inhibitor (Peptide EAVSLKPT, Calbiochem, Darmstadt, Germany; 10μM, 1h,) or mKATP inhibitor [5-hydroxydecanoate (5-HD), Sigma Chemical Co.; 100μM, 1h] prior to the DG treatment at 300μg/ml of DG for 3h, and the cells were then subjected the hypoxia/reoxygenation challenge. The cell lysates were prepared for apoptotic cell death analysis by Annexin V/PI staining and cellular GSH levels were measured following the procedures as described above.

Effect of post-drug exposure time on cytoprotection 

Cells were treated with the DG extract (300μg/ml) for 3h without or with subsequent incubation with fresh medium for 12h. The cells were then subjected to hypoxia/reoxygenation challenge and measurements of apoptotic cell death and cellular GSH level were performed.

Statistical analysis 

Data were analyzed by one-way Analysis of Variance. Post hoc multiple comparisons were performed using Least Significant Difference test. P values<0.05 were regarded as a statistically significant difference.

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Results 

Effects of DG pretreatment on hypoxia/reoxygenation-induced apoptosis and associated changes in cellular GSH and mitochondrial permeability transition 

DG treatment (30 and 100μg/ml) for 24h decreased the extent of apoptosis in cultured H9c2 cardiomyocytes, with the extent of inhibition being 19 and 23%, respectively, when compared with the DG-untreated control (Fig. 2a). A two hour-period of hypoxia followed by 16h of reoxygenation caused a significant increase in the extent of apoptosis in H9c2 cardiomyocytes, with the extent of apoptotic cell death being increased by 1.1-fold, when compared with the unchallenged control. DG pretreatment (30 and 100μg/ml) concentration-dependently inhibited the hypoxia/reoxygenation-induced apoptosis, with the degree of protection being 38 and 60%, respectively, when compared with that of the DG-untreated and challenged control.

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  • Fig. 2. 

    Effects of DG exposure on hypoxia/reoxygenation-induced apoptosis and associated changes in cellular GSH and sensitivity to Ca2+-induced mitochondrial permeability transition in H9c2 cardiomyocytes. Cultured H9c2 cardiomyocytes were incubated with a methanol extract of Danshen–Gegen decoction (DG) at 30 or 100μg/ml for 24h and then subjected to hypoxia/reoxygenation (Hypox/Reoxy) challenge (2h hypoxia followed by 16h reoxygenation). The cells were harvested for measurements of (a) apoptotic cell death, (b) cellular reduced glutathione (GSH) level, (c) Ca2+-induced mitochondrial permeability transition (MPT), and (d) mitochondrial cytochrome c release, as described in the Section “Materials and methods”. Values given are means±SD, with data obtained from triplicate samples. *Significantly different from the Non-Hypo/Reoxy control (CON) group; # significantly different from the Hypo/Reoxy group.

While the DG treatments caused concentration-dependent increases (14 and 34%) in cellular GSH levels, hypoxia/reoxygenation challenge decreased cellular GSH levels in H9c2 cardiomyocytes (Fig. 2b). The cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by DG pretreatment was associated with increases in cellular GSH levels (5 and 27%), as compared to that of the DG-untreated and challenged control.

DG treatment caused a reduction in the extent of Ca2+-induced MPT pore opening (Fig. 2c), but hypoxia/reoxygenation caused a significant increase (61%) in the extent of MPT pore opening in H9c2 cardiomyocytes. The cytoprotective effect of DG pretreatment was associated with decreases in the extent of Ca2+-induced MPT pore opening (9 and 33%), when compared with that of DG-untreated and challenged control.

To further investigate the effect of DG treatment on MPT pore opening which can eventually leads to cytochrome c release from the mitochondria, hypoxia/reoxygenation-induced mitochondrial cytochrome c release was also measured (Fig. 2d). DG pretreatment suppressed the hypoxia/reoxygenation-induced release of cytochrome c (by 33 and 44%) in H9c2 cardiomyocytes, relative to that of the DG-untreated and challenged control.

DG induced a CYP-mediated ROS production 

To explore whether DG can trigger a redox-sensitive signaling pathway, DG-induced ROS production in H9c2 cardiomyocytes was examined. DG caused a time- (1–4h) and concentration- (30–300μg/ml) dependent increase in ROS production in H9c2 cardiomyocytes under the present experimental conditions (Fig. 3a). When the amount of ROS produced was estimated by computing the area under the curve plotting the percent increase in fluorescence (with respect to the appropriate time-matched PBS control) against incubation time (up to 4h), DG treatment at 300μg/ml for 3h increased ROS production by 35% (Fig. 3b). Pre-/co-incubation with ABT (10mM) or DMTU (20mM) abrogated the DG-induced ROS production in H9c2 cardiomyocytes, with ABT producing a greater inhibitory effect than that of DMTU (Fig. 3b).

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  • Fig. 3. 

    DG-induced and CYP-mediated ROS production in H9c2 cardiomyocytes. (a) Reactive oxygen species (ROS) production in H9c2 cardiomyocytes. Cells were incubated with the DG extract (30–300μg/ml) or vehicle (i.e. phosphate-buffered saline, PBS) and 2′-7′-dichlorofluorescein diacetate. The fluorescence intensity of cultured cells was monitored every hour for 6h, as described in the Section “Materials and methods”. Data, which were normalized with time-matched controls, are expressed as a percentage of value at time zero. *Significantly different from the 30μg/ml DG group at the respective incubation time; †significantly different from the 100μg/ml DG group at the respective incubation time. (b) Effects of aminobenzotriazole (ABT) and dimethylthiouracil (DMTU) on DG-induced ROS production in H9c2 cardiomyocytes. Cells were pre- and co-treated with ABT (10mM) or DMTU (20mM) prior to and during incubation with DG extract at 300μg/ml for 3h. The extent of ROS production was estimated and expressed as a percentage of control (no DG treatment), as described in the Section “Materials and methods”. Values given are the mean±SD, with data obtained from triplicate samples. *Significantly different from the respective control group.

DG caused a time-dependent ERK activation via CYP-mediated ROS production 

Fig. 4a shows that DG treatment at 300μg/ml caused time-dependent increases in the ratio of phospho-kinase to total kinase level of ERK1/2, indicative of ERK activation. The activation of ERK was maximal at 3h, with the extent of ERK1/2 phosphorylation being increased by 2.5- and 3.17-fold, respectively, when compared with the time-matched vehicle (i.e. PBS) control.

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  • Fig. 4. 

    CYP-catalyzed ROS production mediated the DG-induced ERK1/2 activation in H9c2 cardiomyocytes. (a) Time-course of DG-induced ERK1/2 activation in H9c2 cardiomyocytes. Cells were starved by incubation in serum-free medium overnight. Starved cells were then incubated with the DG extract at 300μg/ml or vehicle (PBS) in serum-free medium for increasing periods of time (1–24h). *Significantly different from the time-matched control. (b) Effect of ABT and DMTU on ERK1/2 activation. Cells were pre- and co-treated with ABT (10mM, 2h) or DMTU (20mM, 1h) prior to and during the incubation with DG extract at 300μg/ml for 3h. The level of ERK1/2 activation was expressed as the ratio of phospho-ERK1/2 level to the respective total ERK1/2 level. Values given are means±SD, with data obtained from triplicate samples. *Significantly different from the CON group; †significantly different from the respective CON group with inhibitor.

To investigate the involvement of CYP and ROS in DG-induced ERK activation, the effects of ABT and DMTU on ERK1/2 activation were examined in DG-treated H9c2 cardiomyocytes. As shown in Fig. 4b, pre- and co-treatment of ABT (10mM) or DMTU (20mM) completely abrogated the DG-induced ERK1/2 activation, and the resultant level of ERK activation was even lower than under un-stimulated (i.e. basal) conditions.

DG induced Nrf2 activation and a glutathione antioxidant response 

A 3-h treatment of the H9c2 cardiomyocytes with DG extract (300μg/ml) caused a time-dependent change in nuclear Nrf2 level, an indirect measure of Nrf2 activation, during the post-drug treatment period, with the degree of stimulation being maximal (46%, compared to the value obtained immediately after drug exposure, i.e. at time 0) at 2h post-DG exposure (Fig. 5a). The Nrf2 level had decreased gradually to the time 0 value by 12–18h following the exposure to DG. The increase in nuclear Nrf2 level was followed later by an increase in the expression of enzymes related to glutathione redox cycling and synthesis, namely, glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6DPH) and modulatory subunit of GCL (GCLm), with the maximum degree of stimulation occurring at 8–10h post-DG exposure for G6DPH (28%) and GCLm (45%) and GR (31%) (Fig. 5b). While G6PDH and GCLm declined from the peak to values obtained at time 0 following 10h of exposure to DG, the GR level decreased rapidly from the peak at 12h post-DG exposure (Fig. 5b). Time-dependent increases in cellular GSH levels were also observed after DG treatment, with the degree of stimulation being maximal (56%) at 12h post-DG treatment (Fig. 5b). GSH levels then declined gradually after reaching peak values.

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  • Fig. 5. 

    Time-course of DG-induced Nrf2 activation and glutathione antioxidant response in H9c2 cardiomyocytes. Cells were incubated with the DG extract for 3h followed by incubation with a culture medium without DG extract for 2–18h. Levels of (a) nuclear Nrf2 and (b) cellular glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6DPH), γ-glutamyl cysteine ligase modulatory subunit (GCLm) and GSH were measured and expressed as % control [with respect to the value immediately after post-DG exposure (i.e., time 0)]. Values given are means±SD, with data obtained from triplicate samples. *Significantly different from the respective value at time 0.

DG caused a time-dependent induction of HO-1 and activation of Akt/BAD 

The activation of Nrf2 was associated with an enhanced expression of HO-1, with the degree of stimulation being maximal (31%) at 4h post-DG exposure (Fig. 6). The increase in the expression of HO-1 was followed by a later increase in the ratio of phospho-kinase to total-kinase of Akt and BAD, indicative of Akt and BAD activation, respectively, with the degrees of activation of Akt (1.26-fold) and BAD (1.05-fold) being maximal at 6 and 8h post-DG treatment, respectively. The activation of both Akt and BAD then subsided, with the ratio of phosho-kinase to total kinas falling below the time 0 value at 10h post-DG exposure (Fig. 6).

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  • Fig. 6. 

    Time-course of DG-induced activation of the Akt/BAD pathway in H9c2 cardiomyocytes. Cells were incubated with DG extract for 3h followed by incubation with the culture medium devoid of DG for 2–18h. Levels of HO-1, Akt and BAD were measured and expressed as % control [with respect to the value immediately after post-DG exposure (i.e., time 0)]. Values given are means±SD, with data obtained from triplicate samples. *Significantly different from the respective value at time 0.

DG induced a time-dependent PKCɛ activation via CYP-mediated ROS production 

To investigate the signaling pathway triggered by ROS produced in DG-treated cells, the translocation of PKCɛ from the cytosol to the mitochondria was examined. As shown in Fig. 7a, exposure to DG (300μg/ml) caused a time-dependent increase in the ratio of mitochondrial to cytosolic PKCɛ, indicative of PKCɛ activation. The maximal degree of PKCɛ translocation was observed at 3h post-DG treatment, at which point the ratio mPKCɛ to cPKCɛ was increased 2.8-fold, when compared with the respective time-matched and DG-untreated control.

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  • Fig. 7. 

    DG induced PKCɛ activation via CYP-mediated ROS production in H9c2 cardiomyocytes. (a) Time-course of DG-induced PKCɛ translocation. Cells were incubated with DG at 300μg/ml for increasing periods of time (1–8h). *Significantly different from the time-matched control. (b) Effect of ABT and DMTU on PKCɛ translocation. Cells were pre- and co-incubated with ABT (10mM, 1h) or DMTU (20mM, 1h) prior to and during the incubation with DG at 300μg/ml for 3h. Mitochondrial and cytosolic fractions were prepared and measured for PKCɛ levels by Western blot analysis, as described in the Section “Materials and methods”. Values given are means±SD, with data obtained from triplicate samples. *Significantly different from the respective time-matched control group; †significantly different from the respective control group with inhibitor.

To confirm the involvement of CYP and ROS in the activation of PKCɛ, the effects of ABT and DMTU on DG-induced PKCɛ translocation were examined. As shown in Fig. 7b, pre- and co-incubation with ABT (10mM) or DMTU (20mM) largely suppressed DG-induced PKCɛ translocation, with PKCɛ translocation being inhibited by 54 or 46%, respectively, when compared with the respective DG-treated control group.

PKCɛ and mKATP inhibition suppressed DG-induced protection against apoptosis in hypoxic/reoxygenated cardiomyocytes 

To investigate the possible involvement of a PKCɛ-mediated signaling pathway in the cardioprotective action of DG, inhibitors of PKCɛ translocation (Peptide EAVSLKPT) and mKATP (5-HD) were used in the study. While the susceptibility of H9c2 cardiomyocytes to hypoxia/reoxygenation-induced apoptotic cell death was increased by the inhibition of PKCɛ or mKATP, preincubation with DG (300μg/ml for 3h) did not protect against hypoxia/reoxygenation induced-apoptosis in the presence of PKCɛ or mKATP inhibition (Fig. 8a). The abolition of DG-induced cardioprotection corresponded with the failure in reversing the hypoxia/reoxygenation-induced depletion in cellular GSH (Fig. 8b).

  • View full-size image.
  • Fig. 8. 

    Effects of PKCɛ and mKATP inhibition on DG afforded protection against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. Cells were preincubated with PKCɛ translocation inhibitor (Peptide EAVSLKPT) (10μM) and mKATP inhibitor (5-hydroxy decanoate, 5-HD) (100μM) for 1h prior to the DG treatment (300μg/ml) for 3h. The cells were then subjected to hypoxia/reoxygenation challenge, as described in Fig. 1. Cells were harvested for measurements of (a) apoptotic cell death and (b) cellular GSH level. Values given are mean±SD, with data obtained from triplicate samples. *Significantly different from the non-Hypo/Reoxy CON group; #significantly different from the Hypo/Reoxy CON group; §significantly different from the respective Hypo/Reoxy CON group with inhibitor.

DG caused both acute and long-term protection against hypoxia/reoxygenation-induced apoptosis 

To investigate the relative contribution of PKCɛ/mKATP (early onset) and ERK/Nrf2 (late onset) pathways to DG-induced cytoprotection, the effect of DG treatment on hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes was examined immediately after a 3-h incubation with DG (300μg/ml) and at 12-h post-DG exposure. As shown in Fig. 9a, the extent of protection against apoptosis assessed at 12-h post-DG exposure was greater than that observed at time zero, i.e. immediately after the addition of DG (47% versus 28%). The increased extent of cytoprotection was associated with a corresponding increase in the levels of cellular GSH in both unchallenged (45% versus 23%) and challenged (31% versus 17%) cardiomyocytes, when compared with the respective DG-untreated control (Fig. 9b).

  • View full-size image.
  • Fig. 9. 

    Acute and long-term effects of a 3-h DG exposure on hypoxia/reoxygeantion-induced apoptosis in H9c2 cardiomyocytes. Cells were preincubated with DG extract at 300mg/ml for 3h, without (0h) and with (12h) post-DG exposure incubation with the normal medium treatment, as described in Fig. 6, and then subjected to hypoxia/reoxygenation challenge. Cells were harvested and prepared for measurements of (a) apoptosis and (b) cellular GSH level. Values given are the mean±SD, with data obtained from triplicate samples. *Significantly different from the non-Hypo/Reoxy CON group; #significantly different from the Hypo/Reoxy CON group; §significantly different from the 3h+0h DG group in respective non-Hypo/Reoxy or Hypo/Reoxy group.

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Discussion 

Re-introduction of oxygen to previously hypoxic cardiomyocytes can cause necrotic and apoptotic cell death (Robin et al., 2007). In the present study, hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes was found to be suppressed by incubation with DG extract. The hypoxia/reoxygenation-induced cell apoptosis was associated with a depletion of cellular GSH and an increased sensitivity to Ca2+-induced MPT pore opening, both of which were partially reversed by prior incubation with DG. Consistent with this, recent in vivo studies in rats have shown that both acute and long-term exposure to an aqueous DG extract afforded protection against myocardial ischemia/reperfusion injury which was associated with increased mitochondrial GSH levels and decreased the sensitivity of mitochondria to Ca2+-induced permeability transition (Chiu et al., 2011b, Chiu et al., 2011c).

The DG-induced enhancement of cellular GSH and inhibition of MPT are likely mediated by the redox-sensitive ERK/Nrf2 and/or PKCɛ/mKATP signaling pathway (Jaburek et al., 2006, Trachootham et al., 2008). Consistent with this, DG extract was found to stimulate ROS production in H9c2 cardiomyocytes, with a concomitant activation of ERK1/2 and PKCɛ. The inhibition of DG-induced ROS production as well as ERK/1/2 and PKCɛ activation by ABT and/or DMTU support the involvement of CYP-catalyzed, oxidant-generating reactions in the process. The DG-induced activation of ERK1/2 was followed by a later activation of Nrf2 during the post-DG exposure period, with subsequent increases in the expression of glutathione-related antioxidant proteins and HO-1. While GCLm is responsible for increasing the de novo synthesis of GSH, GR and G6DPH likely play a crucial role in the cellular glutathione redox cycling, and both processes are important for GSH recovery following oxidant challenge (Franklin et al., 2009, Han et al., 2006, Jain et al., 2003). The increased expression of these enzymes is therefore likely to be instrumental in preventing cell apoptosis – a process mediated by oxidants that can cause acute glutathione redox imbalance and thereby trigger the apoptotic signaling (Circu and Aw, 2010). The increased expression of HO-1 induced by the DG extract was followed by the activation of Akt/BAD pathway. It has been shown that BAD phosphorylation by Akt causes the formation of the BAD protein homodimer and thus leaves Bcl-2 free to inhibit the BAX-triggered apoptosis (Datta et al., 2000, Tan et al., 2000). The DG-induced phosphorylation of BAD is therefore anti-apoptotic.

The cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by the incubation with the DG extract for 3h was completely abrogated by the inhibition of PKCɛ translocation or mKATP activation. This observation suggests a crucial role of the PKCɛ/mKATP pathway in eliciting the DG-induced cytoprotection in H9c2 cardiomyocytes. Activation of PKCɛ prior to ischemia has been demonstrated to afford cardioprotection by mimicking ischemic preconditioning by brief episodes of ischemia and reperfusion (Dorn et al., 1999). While the role of mKATP in ischemic preconditioning is not well understood (Halestrap et al., 2007), the prolongation of onset time for MPT by ischemic preconditioning was found to be accompanied by the translocation of PKCɛ from the cytoplasm to mitochondria in isolated adult rat cardiomyocytes (Juhaszova et al., 2004). Conceivably, ROS generated from the biotransformation of DG-derived components in cardiomyocytes may activate both ERK/Nrf2 and PKCɛ pathways. Both cellular responses elicited by DG extract pretreatment render cardiomyocytes more resistant to hypoxia/reoxygenation-induced apoptosis. In this connection, mitochondrial ERK and PKCɛ may form a functional signaling module, which in turn leads to the phosphorylation and thus inactivation of BAD (Baines et al., 2002). It should, however, be noted that while many chemical constituents in the DG extract likely produce ROS through CYP-catalyzed biotransformation resulting in cytoprotection, it is also possible that components such as puerarin and diadzein can act directly by opening the Ca2+-activated potassium channel and activating PKCɛ or inhibiting nuclear factor-kappa B activation, respectively (Gao et al., 2006, Kim et al., 2009).

Interestingly, the DG-induced increase in cellular GSH was also observed after 3-h of DG exposure, and this effect was largely or completely suppressed by PKCɛ or mKATP inhibition, respectively, in non-hypoxic/reoxygenated H9c2 cardiomyocytes. While the mechanism underlying the GSH stimulatory effect produced by DG in such a short time interval remains to be investigated, it is unlikely to be due to an increase in GSH synthesis because the Nrf2-mediated increase in GCLm expression in H9c2 cardiomyocytes requires a longer lag time after the DG exposure. Alternatively, the stimulatory action on GSH, which was completely inhibited by 5-HD, may be related to the opening of mKATP, presumably through increasing intra-mitochondrial ROS production (Garlid et al., 2009). The finding that the 3-h DG exposure followed by a 12-h incubation in the absence of DG caused a larger extent of protection against cell apoptosis than that of a 3-h drug exposure per se suggests an important role of the ERK1/2/Nrf2 signaling pathway in affording cytoprotection over an extended time interval following the exposure to DG extract.

In conclusion, preincubation with the DG extract protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. This cytoprotection was associated an enhancement of cellular GSH levels and a reduced sensitivity to Ca2+-induced MPT. DG activated both ERK/Nrf2 and PKCɛ pathways, presumably by ROS arising from CYP-catalyzed reactions, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.

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Conflict of interest 

All authors have no conflict of interest to disclose.

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Acknowledgement 

The work described in this paper was partially supported by a grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project No. AoE/B-10/01).

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PII: S0944-7113(11)00261-3

doi:10.1016/j.phymed.2011.07.002

Phytomedicine
Volume 19, Issue 2 , Pages 99-110, 15 January 2012

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