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Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of acute and long-term neurodegenerative diseases. This study was undertaken to examine the efficacy of Pogostemon cablin, a well-known herb in Korean traditional medicine, on ROS-induced brain cell injury. Pogostemon cablin effectively protected human neuroglioma cell line A172 against both the necrotic and apoptotic cell death induced by hydrogen peroxide (H2O2). The effect of Pogostemon cablin was dose dependent at concentrations ranging from 0.2 to 5mg ml−1. Pogostemon cablin significantly prevented depletion of cellular ATP and activation of poly ADP-ribose polymerase induced by H2O2. The preservation of functional integrity of mitochondria upon the treatment of Pogostemon cablin was also confirmed by 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Furthermore, Pogostemon cablin significantly prevented H2O2-induced release of cytochrome c into cytosol. Determination of intracellular ROS showed that Pogostemon cablin might exert its role as a powerful scavenger of intracellular ROS. The present study suggests the beneficial effect of Pogostemon cablin on ROS-induced neuroglial cell injury. The action of Pogostemon cablin as a ROS-scavenger might underlie the mechanism.
Ischemia or ischemia/reperfusion-induced cell injury is associated with a variety of life threatening conditions such as myocardial infarction, cerebral stroke and renal failure. Tissue injury resulting from ischemia or ischemia/reperfusion is mediated, in part, by the generation of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical and singlet oxygen (1). In the brain, they have been implicated in the pathogenesis of a wide range of acute and long-term neurodegenerative diseases including acute cerebral stroke, Parkinson's disease, Huntington's disease, ischemic trauma and seizures (2,3).
Brain cell membranes have a high content of polyunsaturated fatty acids that are particularly susceptible to the peroxidative attack by ROS. In addition, iron, which promotes cytotoxic radical formation, is accumulated in specific brain regions, such as the globus pallidus and substantia nigra. On the other hand, anti-oxidative defense mechanisms are relatively deficient in brain cells. The brain cell contains almost no catalase and has low concentrations of glutathione, glutathione peroxidase and vitamin E (4). The brain is, therefore, particularly vulnerable to the ROS attack.
We previously reported the effect of ROS on cell death and proliferation, which showed that the concentration of ROS would be very critical for cellular response (5). Cytoprotection against ROS is provided by a multilevel defense system, which comprises anti-oxidants (vitamin A, C, E and reduced glutathione) and anti-oxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Various constituents of natural products such as fruits, wine, tea and green vegetables are known to be a great source of effective anti-oxidants (6). Some Oriental herb medicines also have been expected to exert their effects as anti-oxidants, but little information is available yet.
Pogostemon cablin (P. cablin) is a well-known herb in Korean traditional medicine. It has effects on regulating vital energy and eliminating phlegm, and is a typical prescription used in treatments of apoplexy, syncope of Qi, phlegm syncope and syncope with eating and drinking etc. (7–10). It has been proven to be beneficial for patients with cerebral stroke (11). Several studies have been carried out on the composition of P. cablin and the presence of patchouli alcohol, pogostone, eugenol, α-bulnesene, rosmarinic acid, etc. has been revealed (11). Essential oils constitute about 1.5% of P. cablin and among them >50% is patchouli alcohol. In Korea, water extract of P. cablin was used for the treatment of fever caused by heart stroke, poor appetite, nausea, etc. The patchouli oil obtained by steam distillation is an important natural material in the perfumery and food industry and used to repel insect. Volatile composition of P. cablin obtained by steam distillation was extensively studied but non-volatile composition was not. Water extract was used for the treatment of apoplexy for a long time; however, the target site and mechanism responsible for the clinical efficacies are not clear yet. In 2006, Wang et al. (12) reported that two traditional Chinese medicine formulae reduced the oxidative stress induced by psychological stress.
There are many plants used for medicinal treatment in Asia. Generally, volatile essential oils are used in western country. But in Eastern Asia, plants for medication were generally used in water-extract and this prescription has been very effective for an age. From this point, we can expect that aqueous constituents as well as volatile ones might contain pharmacological activity.
Thus, the present study was carried out to determine whether P. cablin exerts beneficial effects against brain cell injury induced by hydrogen peroxide (H2O2), an oxidant on A172 cells, a human neuroglioma cell line.
P. cablin was purchased from Gwangmyung Natural Pharmaceutical Co. (Busan, Korea). Forty grams of dried P. cablin was boiled with 1000ml distilled water at 100°C for 3h and total extract was evaporated under reduced pressure for 24h to give 9.5g.
A172 cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained by serial passages in 75cm2 culture flasks (Costar, Cambridge, MA, USA). Cells were grown in Dulbeccos’ modified Eagles medium (DMEM, Gibco-BRL, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (Gibco-BRL, Grand Island, NY, USA) at 37°C in humidified 95% air/5% CO2 incubator. When the cultures reached confluence, subculture was prepared using a 0.02% EDTA–0.05% trypsin solution.
The HPLC system consisted of a Waters 1525 pump, Waters 2487 detector, Waters 717 autosampler (Waters, Milford, MA, USA) and a Waters Symmetry C-18 column (4.6×150mm2, 5μm). The mobile phase consisted of acetonitrile (mobile phase A) and water containing 1% acetic acid (mobile phase B). The mobile phase flow rate was 1ml min−1 and the split ratio to the HPLC was 3:7 and the detector wavelength was 320nm.
Rosmarinic acid from Aldrich (St Louis, MO, USA) was used as standard. A total of 8.4mg of rosmarinic acid was solubilized in 10.0ml methanol. The methanol stock solution of rosmarinic acid was diluted to different concentrations. Aliquot (10μl) of each diluted solution was analyzed by HPLC and standard curve was derived by plotting the peak area ratios of rosmarinic acid as a function of concentration.
One milligram of dried extract was dissolved into 1.0ml of distilled water and then filtered using 0.45μm syringe filter. Ten microliters was used for HPLC analysis and concentration was calculated from the following equation:
Cells were grown on microscopic coverglass in 12-well culture plates and all the experiments were carried out 2 days after seeding. After washout of the culture media, cells were exposed to the indicated concentration of H2O2 in Hank's balanced salt solution (HBSS) for 3h at 37°C. After exposure, each cell group was stained with 2μl mixture of 270μM acridine orange and 254μM ethidium bromide (1:1), and observed under reflected fluorescence microscopy. Injured cells appeared as orange color, whereas intact cells appeared as green.
Measurement of cell viability by trypan blue exclusion experiment is based on the fact that injured cells lose the ability to exclude the dye (13). After exposure to chemicals, cells were detached and harvested using 0.05% trypsin–0.53mM EDTA, incubated with 4% trypan blue solution and were counted by using a hemocytometer under light microscopy. Cells failed to exclude the dye were considered non-viable.
After exposure of cells to P. cablin on 12-well culture plates, media bathing the cells were collected separately, and cells on the plates were lyzed by sonication in 0.2% Triton X-100. Lactate dehydrogenase (LDH) activity in the cellular extracts and incubation media were determined by using an LDH assay kit. LDH release was calculated as the percentage of LDH activity in incubation media over the total activity.
Cells were exposed to 0.1mM H2O2 for 3h and, after washout of floating dead cells by necrotic injury, returned to H2O2-free culture media containing 10% fetal bovine serum. After further incubation for 18h, cells were assayed for apoptosis by analysis of DNA fragmentation and TUNEL staining. For analysis of DNA fragmentation, low molecular weight genomic DNA was extracted as follows. Cells (2×106) were scraped from the culture dish, washed in phosphate-buffered saline and lyzed in 0.5ml of extraction buffer (10mM Tris, pH 7.4, 0.5% Triton X-100 and 10mM EDTA) for 20min on ice. The lysates were then centrifuged at 15000g for 10min at 4°C. DNA in supernatants were extracted with phenol–chloroform, precipitated in ethanol, resuspended in Tris/EDTA, pH8.0, containing 20μg ml−1 RNase A and incubated for 1h at 37°C to digest RNA. Recovered DNA fragments were separated by electrophoresis in 2% agarose gel and visualized by staining with ethidium bromide.
For in situ detection of apoptotic cells, TUNEL assay was performed using ApoTag peroxidase in situ apoptosis detection kit (Intergen, Purchase, NY, USA). In brief, cells cultured on coverglasses were exposed to chemicals and fixed in 1% paraformaldehyde for 10min. The fixed cells were incubated with digoxigenin-conjugated dUTP in a TdT-catalyzed reaction for 60min at 37°C and were then immersed in stop/wash buffer for 10min at room temperature. The cells were then incubated with anti-digoxigenin antibody conjugated with peroxidase for 30min. The DNA fragments were stained using 3,3′-diaminobenzidine (DAB) as a substrate.
ATP levels were measured on cells with a luciferin–luciferase assay (14). After an exposure to oxidant stress, the cells were solubilized with 500μl of 0.5% Triton X-100 and acidified with 100μl of 0.6M perchloric acid and placed on ice. Cell suspension was then diluted with 10mM potassium glutamate buffer containing 4mM MgSO4 (pH7.4). One hundred microliters of 20mg ml−1 luciferin–luciferase was added to 10μl of diluted sample. Light emission was recorded at 20s with a luminometer (MicroLumat LB96P, Berthold, Germany) and normalized with total protein content.
Cells were treated with H2O2 for 10min in a buffer containing 28mM NaCl, 28mM KCl, 2mM MgCl2, 56mM HEPES (pH 7.5), 0.01% digitonin and 125mM NAD (containing 0.25 μCi [3H]NAD). Permeabilized cells were incubated for 5min at 37°C, and the protein ribosylated with [3H]NAD was precipitated with 200μl of 50% (w/v) trichloroacetic acid. After washing twice with trichloroacetic acid, the protein pellet was solubilized with 200μl of 2% (w/v) sodium dodecyl sulfate in 0.1M NaOH and incubated at 37°C for overnight, and the radioactivity was determined by scintillation counting.
Intact mitochondria reduce 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) to formazan (15). Therefore, the ability of cells to form formazan from MTT is a good indicator of mitochondrial function. A total 5μl of MTT was added to each well (final concentration of 62.5μg ml−1). After exposure to chemicals, the supernatant was removed and the formed formazan crystals in viable cells were solubilized in DMSO. The concentration of formazan was determined by reading the absorbance at 570nm using a spectrophotometer.
After exposure to chemicals, cells were washed twice with ice-cold phosphate-buffered saline at 4°C and resuspended in 1ml of extraction buffer (50mM PIPES, pH 7.0, 50mM KCl, 5mM MgCl2, 5mM ethylene glycol tetraacetic acid (EGTA), 1mM phenylmethylsulfonyl fluoride, 10 μg ml−1 leupeptin and 10μg ml−1 pepstatin A). Cells were lyzed by five cycles of freezing in liquid nitrogen and thawing at 37°C. After verifying >90% of cells were lyzed by microscopic examination, the lysates were centrifuged at 100000g for 1h at 4°C. The resulting supernatant mainly consisted of cytosolic fraction was separated from the pellet containing the cellular membrane and organelles, and was analyzed for cytochrome c by western blots using anti-cytochrome c antibody.
Intracellular level of ROS was determined by measuring 2,7-dichlorofluorescein (DCF) fluorescence (16). The non-fluorescent ester dye 2,7-dichlorofluorescein diacetate (DCFH-DA) penetrates into the cells and is hydrolyzed to DCFH by the intracellular esterases. The probe is rapidly oxidized to the highly fluorescent compound DCF in the presence of cellular peroxidase and ROS, such as H2O2. Cells were pre-incubated at 37°C for 10min in a fluorescent cuvette containing 3ml of glucose-free buffer with 20μM DCFH-DA (from a stock solution of 20mM DCFH-DA in ethanol). After pre-incubation, cells were treated with H2O2 and incubated up to 60min. During the incubation process, fluorescent intensity was monitored on a spectrophotometer with excitation wavelength at 485nm and emission wavelength at 530nm.
Acridine orange and 2,7-dichlorofluorescein diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR, USA). Other chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA).
Data are presented as means±SE When necessary, data were analyzed by analysis of variance (ANOVA) followed by Duncan's multiple comparison test. A value of P<0.05 was considered statistically significant.
To check whether the effective contents in P. cablin were present after extraction, we carried out HPLC analysis. As described in introduction, rosmarinic acid is one of the effective ingredients in P. cablin so we used commercial rosmarinic acid as standard. Extraction was carried out after boiling for 30min, 1, 2 and 4h each and then the extracts were applied to HPLC analysis. As shown in Table 1, rosmarinic acid was detected in extracts of P. cablin and the concentration of rosmarinic acid was increased as the extraction time became longer. It implies that the effective constituents in P. cablin could be present in extracts after even >4h boiling.
The fluorescence microscopic analysis in Fig. 1 visualizes the typical image of A172 cells injured by 0.5mM H2O2. Injured cells were stained with acridine orange. While non-viable cells fluoresced orange or red, viable cells fluoresced green. Preserved contour of the nuclei indicated that the cell injury was necrotic rather than apoptotic. Protective effect of P. cablin against H2O2-induced injury is shown in the right panel of the Fig. 1.
In Fig. 2, P. cablin protected cell injury induced by H2O2 in a dose-dependent manner. Trypan blue exclusion ability and LDH release were adopted as indexes of cell injury. Measurement of cell viability by trypan blue exclusion ability was based on the fact that injured cells lose the ability to exclude the dye (13). LDH is an intracellular enzyme and its release is a useful marker of membrane damage, a characteristic of necrotic cell death. When cells were treated with 0.5mM H2O2 for 3h, 58.4% of cells lost their ability to exclude the dye and 19.6% of the LDH activity was found in incubation media. P. cablin treatment decreased both indexes of cell injury in a concentration-dependent manner. At the concentration of 1mgml−1, the levels of trypan blue stain and LDH release were decreased 58.2 and 63.4%, respectively.
In Fig. 3, the protective effects of P. cablin upon other types of oxidant-induced cell injuries were examined. Cells were exposed to chemical hypoxia or chemical oxidants, t-butyl hydroperoxide (t-BHP) and menadione. Chemical hypoxia was induced by combination of glucose deprivation and the treatment of antimycin A (20μM), mitochondrial electron transport inhibitor. All these chemicals induced cell injury or death as determined by trypan blue exclusion and LDH release assays. In the presence of P. cablin (1mgml−1), cellular injury was significantly decreased (Fig. 3). This result suggests that P. cablin might have protective effect against various types of oxidant-induced injuries.
The protective effect of P. cablin against oxidative stress-induced apoptosis was examined with human neuroglioma cells. DNA fragmentation is one of the most characteristic biochemical features of apoptosis. A172 cells were first exposed to 0.1mM H2O2 for 3h and cell viability was checked with trypan blue exclusion assay. Less than 15% of cells resulted in necrotic cell death (data not shown). After removing the floating dead cells, cells were returned to H2O2-free culture media. After further incubation for 18h, cells were analyzed for apoptosis by DNA fragmentation and TUNEL assay. Treatment of cells with 0.1mM H2O2 induced DNA fragmentation as shown by typical laddering pattern in the agarose gel electrophoresis (Fig. 4A). Apoptotic cells were visualized by TUNEL staining in the micrographs shown in Fig. 4B. Nearly half (47.1%) of cells in H2O2-treated cells were TUNEL-positive (mid panel of Fig. 4B). In the presence of P. cablin, the numbers of TUNEL-positive cells were significantly reduced (right panel of Fig. 4B). The dosage-dependent effects of P. cablin on H2O2-induced apoptosis are summarized in Fig. 5.
In general, ROS-induced tissue injury is accompanied by decrease in intracellular ATP concentration (1). To examine whether P. cablin could prevent H2O2-induced ATP depletion, time-dependent changes of intracellular ATP content of H2O2-treated cells in the absence or presence of P. cablin (1mg ml−1) were checked (Fig. 6). ATP concentration in H2O2-treated cells decreased to <10% of its control value within 3h. However, in the presence of P. cablin, H2O2-induced ATP depletion was significantly attenuated.
It has been reported that activation of PARP is implicated in H2O2-induced ATP depletion and neurotoxicity (17). As shown in Fig. 7, exposure of cells to H2O2 resulted in a large increase in PARP activity. However, this increase in PARP activity was significantly inhibited by P. cablin.
In addition to PARP activation, deterioration of mitochondrial function is a major determinant to deplete intracellular ATP. Exposure of cells to 0.5mM H2O2 produced a significant loss of mitochondrial function as evidenced by the decrease in MTT reduction level to 42.3% of that of control cells (Fig. 8). In the presence of P. cablin, the decease in MTT reduction was significantly prevented. These data suggest that P. cablin prevents mitochondrial dysfunction induced by H2O2.
It is well established that release of cytochrome c from mitochondrial inner membrane into cytosol is a key event to relate mitochondrial damage with apoptotic cell death (18). As shown in Fig. 9, treatment of cells with H2O2 induced release of cytochrome c and this effect was remarkably prevented by P. cablin.
There is increasing evidence that tissue injury resulting from ischemia/reperfusion or chemical oxidant is mediated by intracellular generation of ROS (1). As shown in Fig. 10, H2O2 induced a significant increase of intracellular ROS. In the presence of P. cablin, intracellular ROS was remarkably reduced. The effectiveness of P. cablin as intracellular ROS reducer at the concentration of 1mg ml−1 was comparable to that of well-known ROS scavengers such as superoxide dismutase (200Uml−1), catalase (200Uml−1) and dimethylthiourea (10mM). This result suggests that P. cablin might exert its effect as an effective ROS scavenger.
All aerobic cells generate ROS including superoxide, H2O2 and hydroxyl radicals, enzymatically or non-enzymatically. The mitochondrial electron transport chain is the principal site of cellular production of ROS (19,20). ROS can also be generated during the process of phagocytosis, ischemia/reperfusion and metabolism of many drugs and other xenobiotic chemicals (1). At the same time, the abundant anti-oxidant defense systems of most cells, both enzymatically and non-enzymatically, prevent ROS from causing cell injury. Nevertheless, there are a number of conditions in which the rate of formation of ROS is increased and/or anti-oxidant defenses of cells are weakened. Therefore, the targeted development of new anti-oxidant drugs has been required.
The data in the present study clearly suggested that P. cablin might act as an effective cytoprotectant against neuroglial cell injury induced by ROS. It was effective to protect apoptotic cell death as well as necrotic cell death by various types of oxidants.
Several lines of evidence suggest that the effect of P. cablin to preserve mitochondrial function during oxidative stress might be a key event responsible for its protective effect against cell death. Furthermore, it significantly attenuated H2O2-induced ATP depletion. The effect of P. cablin to prevent PARP activation as shown in Fig. 7 could provide another clue to explain the mechanism of ATP preservation.
Since PARP catalyzes the transfer of ADP-ribose from NAD to protein with the concomitant release of nicotinamide, the activation of this enzyme results in depletion of NAD and a consequent reduction of ATP, which may be involved in the pathogenesis of oxidant-induced injury. PARP has been demonstrated to be involved in the cell death caused by H2O2 in neuronal cells (15,21) and various non-neuronal cell systems (22,23). However, the role of PARP in oxidant-induced glial cell death has not been established. Although, present study clearly demonstrated that PARP is activated by H2O2 and P. cablin prevents the activation of PARP stimulated by H2O2, it is unclear whether ATP depletion resulting from PARP activation is a mediator of H2O2-induced cell death. While ATP depletion has been reported to induce the oxidant-induced cell death (24), other studies have revealed that the oxidant-induced injury is disassociated with ATP depletion (25,26).
It is interesting to note that P. cablin prevents H2O2-induced cytochrome c release (Fig. 9). Onset of ischemia leads to depletion of oxygen in the tissue resulting in impaired mitochondrial oxidative phosphorylation within seconds. ATP concentration almost amounts to about 10% of normal within 5min. This failure of energy production results in MPT and subsequent release of cytochrome c. Released cytochrome c activates caspase cascades, which are critical for the execution phase of apoptosis, and generates ROS. Generated ROS initiates another cycle of apoptosis described earlier and propagates ischemic cell death (18,27). P. cablin-induced down-regulation of ROS blocks the propagation of injury. From this result, we can postulate that P. cablin exerts its pharmacologic effect through ROS scavenging.
The conclusive results from Fig. 10 clearly show that P. cablin would be an effective scavenger of ROS. The overall protective effects observed in this study might be associated with the role of P. cablin as a ROS-scavenger. It was not measured in this study, which component of P. cablin is responsible for the ROS-scavenging effect. It may be a natural component(s) included in P. cablin or a newly formed compound(s) through extraction process. Further analytical study should be needed to elucidate this.
The standardization of herbal medicine has been managed through the government agency. The Korea Food and Drug Administration (KFDA) guides this by the Korean Herbal Pharmacopoeia (28), which suggests criteria for oriental medicine. The extraction method used in this study was same as the method in clinical application.
In conclusion, the present study provides clear evidence for the anti-oxidant effect of P. cablin on neuroglial cell line. ROS scavenging activity of P. cablin might underlie the mechanism. However, the exact component and mechanism responsible for the cytoprotection against ROS should be further identified.