Part 1 | Herba Cistanche Extract Enhances Mitochondrial ATP Generation in Rat Hearts And H9c2 Cells

Contact: emily.li@wecistanche.com

Hoi Yan Leung and Kam Ming Ko

Department of Biochemistry, Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong SAR, China

Keywords: ATP, Cistanche deserticola, H9c2 cells, heart, mitochondria

Abstract

To investigate the pharmacological basis of the “Yang- invigorating” action of Herbal Cistanche [the dried whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae)], in Chinese medicine, the effects of the methanol extract of Herba Cistanche on mitochondrial ATP generation capacity were examined using an ex vivo rat heart model and an in situ H9c2 cell assay. Treatment with Herba Cistanche extract increased the myocardial mitochondrial ATP generation capacity in a dose-dependent manner in rats, as assessed by in vitro measurement. The stimulation of ATP generation capacity was associated with a parallel enhancement in mitochondrial electron transport supported by pyruvate but not succinate. Herba Cistanche treatment also increased the myocardial mitochondrial complex I and complex III activities, with the extent of stimulation on complex I activity being larger. Herba Cistanche treatment produced a dose- and time-dependent increase in mitochondrial ATP generation capacity in H9c2 cells. The results indicate that Herba Cistanche treatment can increase mitochondrial ATP generation in rat hearts ex vivo and H9c2 cells in situ, possibly through enhancing oxidative phosphorylation.

Cistanche can improve heart function, lower blood pressure, and lower blood lipids

Introduction

“Yang invigoration,” which embodies the generalized enhancement of body function in Chinese medicine, involves the use of energy in driving various biochemical processes.

In this regard, ATP, which is the universal currency of energy in fueling cellular function, is mainly generated through oxidative phosphorylation in mitochondria. We have postulated that “Yang invigorating” herbs possess the ability to increase mitochondrial ATP generation capacity (Ko et al., 2004). This is supported by our recent finding that “Yang- invigorating” but not “Yin-nourishing” Chinese tonifying herbs could enhance the myocardial ATP generation capacity in mouse hearts ex vivo (Ko et al., 2006). Herba Cistanche, the dried whole parasitic plant (excluding the flower) of Cistanche deserticola Y.C. Ma (Orobanchaceae), is classified as a ‘Yang-invigorating’ tonifying herb in traditional Chinese medicine (Chen, 1998), and it appears as the most popular ingredient in a number of Chinese patents for- mulations used for Yang-invigoration’. In China and Japan, Herba Cistanche is widely used for the treatment of a host of Yang deficiency symptoms. Pharmacological studies indicated that Herba Cistanche could scavenge free radicals (Xiong et al., 1996), produce sedative (Lu, 1998), antiaging (Lee et al., 1990), antinociceptive and anti-inflammatory effects (Lin et al., 2002), and enhance immune function (Wu et al., 2005). The main active ingredient of Herba Cistanche is phenylethanoid glycosides (Ouyang et al., 2003), which have been found to have antibacterial, antistress, and antioxidative properties (Xiong et al., 1998), as well as anti-apoptotic effects on cultured neurons (Tian & Pu, 2005). In the current study, in order to investigate the pharmacological basis of the Yang-invigorating action of Herba Cistanche, the effects of Herba Cistanche treatment on the ATP generation capacity in mitochondria isolated from rat hearts ex vivo and cultured H9c2 cardiomyocytes in situ were examined. To explore the biochemical mechanism underlying the ATP generation–enhancing action, the effects of Herba Cistanche treatment on mitochondrial electron transport and complex I–IV activities were also examined in rat hearts.

The effective ingredients of Cistanche can scavenge various active oxygen free radicals

Materials and Methods

Chemicals, cell culture, and herbal materials

ATP, ADP, and 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl- tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Luciferase solution (ATPlite) was obtained from PerkinElmer (Boston, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL Life Technologies (Grand Island, NY, USA). H9c2 cell line was purchased from ATTC (Rockville, MD, USA). Herba Cistanche was supplied by a local herbal dealer (Lee Hoong Kee). The herb was authenticated by the supplier, and a voucher specimen (HKUSTY00301) was deposited in the Department of Biochemistry, Hong Kong University of Science & Technology (HKUST).

Herbal extraction

Herba Cistanche (100g) was cut into small pieces and then extracted by heating under reflux in 300 mL methanol at 65◦C for 2 h. The procedure was repeated twice. The pooled extract was dried by evaporating the solvent under reduced pressure, and the methanol extract of Herba Cistanche was obtained at a yield of 39% (w/w). Chemical analysis indicated that the methanol ex- tract of Herba Cistanche contained total saponins, total flavonoids, total lignans, and polysaccharides at concentrations of 1.62% (w/w), 0.08%, 0.15%, and 75.6%, respectively.

Animal care

Adult male Sprague-Dawley rats (8–10 weeks; 250–300g) were maintained under a 12-h dark/ light cycle in an air/humidity-controlled room at about 22◦C and allowed food and water ad libitum in the Animal Care Facilities at HKUST. Experimental protocols were approved by the Research Practice Committee at HKUST.

Drug treatment

Animals were randomly divided into groups, with five to six animals in each. In the treatment groups, rats were intragastrically administered with the methanol extract of Herba Cistanche (dissolved/suspended in water) at increasing daily doses (0.031–0.5 g/kg) for 3 days. Control animals received water only. Twenty-four hours after the last dosing, the heart was obtained from phenobarbital-anesthetized animals and subjected to biochemical analysis.

Preparation of mitochondrial fractions and measurement of ATP generation capacity (ATP-GC) ex vivo

Heart left ventricular tissue samples were excised and rinsed with ice-cold isotonic buffer (0.32 M sucrose, 1 mM EDTA, 50 mM Tris/HCl, pH 7.4). Cardiac mitochondrial fractions were prepared by differential centrifugation in isotonic buffer at 4◦C. A 10% (w/v) cardiac homogenate was prepared by homogenizing the minced ventricular tissue with a Teflon-glass homogenizer at 4000 rpm for 20–30 complete strokes. The homogenate was centrifuged at 600 × g for 10 min to remove nuclei and cell debris. The supernatant was then centrifuged at 8000 × g for 30 min to sediment the mitochondria (Evan, 1992). The pellets were resuspended in 1 mL of the isotonic buffer and reconstituted the mitochondrial fractions. The mitochondrial ATP-GC of untreated animals was measured by the method of Leung et al. (2005)

Cistanche polysaccharides can delay the physiological degeneration of organs and the degeneration of cell morphology

Measurement of mitochondrial electron transport

The measurement of electron transport in isolated mitochondria, which is based on the reduction of MTT, as per[1]formed as described by Cohen et al. (1997). Mitochondria were prepared at a protein concentration of 1 mg/mL with an incubation buffer (250 mM sucrose, 50 mM HEPES, 10 mM KH2PO4, 2 mM MgCl2, 1 mM EGTA, pH 7.4). An aliquot (40 µL) of the mitochondria was mixed with 100 µL each of 15 mM pyruvate or 0.5 mM succinate and 0.42 mg/mL MTT. The reaction mixture was incubated at 37◦C for 10 min with gentle shaking. After the incubation, the reaction mixture was terminated by the addition of 100 µL lysis buffer (10%, w/v, sodium dodecyl sulfate, and 45% dimethylformamide, adjusted to pH 4.7 with glacial acetic acid). After standing for 5 min, absorbance readings of the reaction mixture were taken with a microtiter plate reader (Bio-Rad, Hercules, CA, USA) and reported as the difference between 570 nm and 630 nm. Data were expressed as a percentage of the mean value of the control (i.e., Herba Cistanche–untreated) group.

Cell culture

H9c2 cells, a permanent cell line derived from cardiac myoblasts (Hescheler et al., 1991), were cultured as monolayers in DMEM supplemented with 10% (v/v) FBS. The medium contained glucose (4.5 g/L) and glutamine (4.5 mM), supplemented with NaHCO3 (17 mM), penicillin (100 IU/mL), and streptomycin (100µg/mL). All cells were grown under an atmosphere of 5% (v/v) CO2 in air at 37◦C. The medium was replaced by fresh medium every 2 or 3 days. A stock of cells was grown in a 75 cm2 culture flask and split before confluence at a subcultivation ratio of 1:10. For ATP-GC assay, H9c2 cells were seeded at a density of 2.5 × 104 cells/well into a 24-well plate. After the cell attachment, Herba Cistanche extract (dissolved in phosphate-buffered saline; PBS) was applied in the medium, and the cells were incubated for increasing periods (2–16 h) of time. Control (untreated) cells were given the PBS only.

Measurement of ATP-GC in situ

After the indicated periods of incubation with Herba Cistanche extract at increasing concentrations (50–300 µg/mL), the ATP-GC assay was performed. The medium was aspirated and cells were treated with digitonin (50 µg/mL) in an incubation buffer (120 mM KCl, 5 mM KH2PO4, 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl2, 0.5% BSA, pH 7.4) for 3 min at 37◦C. After aspirating the digitonin, glutamate (5 mM), malate (5 mM), and ADP (60 µM) were added to the cells for mitochondrial ATP generation, which was monitored at increasing time intervals ranging from 0 to 15 min. The reaction was terminated by the addition of 60 µL of perchloric acid (30%, w/v), and the reaction mixtures were then centrifuged at 600 × g for 10 min at 4◦C. An aliquot (120 µL) of the supernatant was mixed with 90 µL of 1.4 M KHCO3 for neutralization. The mixtures were centrifuged again at 600 × g at 4◦C, and the supernatants were measured for ATP content as described earlier. The ATP-GC of the untreated cells was estimated by computing the area under the curve of the graph (AUC1) plotting ATP generated (nmol/mg protein) against time (0–15 min) and expressed in arbitrary units. For Herba Cistanche–treated cells, AUC1 values of increasing incubation times (3, 5, 7, 10, and 15 min) were normalized to a respective mean control value from untreated samples and expressed as percent control. Then, the area under the curve (AUC2) of the graph plotting percent control values against incubation time (3–15 min) was computed and expressed in arbitrary units. Data of Herba Cistanche–treated groups were expressed as a percentage of the control from the equation:

[AUC2(HerbaCistanche − treated)/AUC2(untreated)] ×100%

Measurements of complex I–IV and citrate synthase activities

Mitochondrial electron transport chain (ETC) complex I– III activities were measured by spectrophotometric methods using complex-specific substrates (NADH for complex I, succinate for complex II, and decylubiquinol for complex III) as described (Grad & Lemire, 2004; Hsu et al., 2005; Mark et al., 2001). Complex IV activity was measured using a cytochrome c oxidase assay kit from Sigma. Mitochondrial citrate synthase activity was measured by the method of Srere (1969).

Protein assay

Protein concentrations of mitochondrial fractions and cell lysates were determined using a BioRad protein assay kit using bovine serum albumin as standard (0.038–0.600 mg/mL).

Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM). They were analyzed by one-way analysis of variance (ANOVA). Post hoc multiple comparisons were done with LSD. P values <0.05 were regarded as statistically significant.

Results

Effects of Herba Cistanche treatment on myocardial mitochondrial ATP-GC in rats

As shown in Figure 1, treatment with Herba Cistanche extract at doses up to 0.5 g/kg increased myocardial mitochondrial ATP-GC in a dose-dependent manner in rats, with the degree of stimulation being 89% at a dose of 0.5 g/kg.

Effects of Herba Cistanche treatment on mitochondrial electron transport in rat hearts

The extent of electron transport in isolated mitochondria was measured by monitoring the reduction of MTT. The substrate used for the assay was either pyruvate or succinate.

Figure 1.The effect of Herba Cistanche treatment on mitochondrial ATP generation capacity in rat hearts ex vivo

Each bar represents the mean ± SEM, with n = 5. Animals were orally treated with Herba Cistanche at the indicated daily doses for 3 days. ATP generation capacity was measured as described in “Materials and Methods.” Data were expressed in percent control with respect to values (AUC2) of the respective untreated control group. ∗Significantly different from the respective untreated control group; significantly different from the Herba Cistanche–treated group at 0.5 g/kg.

Figure 2. Effects of Herba Cistanche treatment on mitochondrial electron transport in rat hearts

Each bar represents the mean ± SEM, with n = 5. (a) Pyruvate-supported (b) succinate-supported mitochondrial electron transport was measured by MTT reduction, as described in “Materials and Methods.” Data were expressed in the percentage of untreated control values [pyruvate-supported: OD570nm− OD630nm = 0.0206 ± 0.0001 (SEM), n = 5; succinate-supported: male, 0.255 ± 0.015, n = 5]. ∗Significantly different from the respective untreated control group; a significantly different from 0.5 g/kg group.

As shown in Figure 2a, Herba Cistanche treatment dose-dependently increased the extent of mitochondrial electron transport supported by pyruvate in rat hearts, with the optimal stimulation observable at doses of 0.5 g/kg. However, no significant changes in the extent of mitochondrial electron transport supported by succinate were detected in Herba Cistanche–treated rat hearts (Fig. 2b).

For Part 2, please click here.