The Ethyl Acetate Extract of Aquilaria Crassna Protect

Full Length Research Paper

Ethylacetate extract of Aquilaria crassna preserve actin cytoskeleton on simulated ischemia induced cardiac cell death

Panadda Jermsri and Sarawut Kumphune*

Biomedical Research Unit in Cardiovascular Sciences (BRUCS) and Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Thapoo, Meung Phitsanulok 65000, Thailand.

Accepted 28 May, 2012

This paper investigates the effect of ethylacetate extract of Aquilaria crassna Pierre ex Lecomte on simulated ischemia-induced cytoskeleton disorganization in cardiac myoblast cell line, so as to provide some evidence for its traditional use. Rat cardiac myoblast cell line (H9c2), were subjected to 2 h simulated ischemia and 24 h of reperfusion, in the presence and absence of the ethylacetate extract of A. crassna. The cellular viability was determined by the reduction of 3-(4,5-dimethyl-2-thiazol)-2,5- diphenyl-2H-tetrazolium bromide (MTT) by mitochondrial reductases. Cells were pre-treated with 5 mg/ml of A. crassna extract for 1 h before, or at the beginning of 40 min simulated ischemia. The F-actin cytoskeleton organization was assessed in cells treated with 5 mg/ml prior to simulated ischemia. Cells were fixed and stained for F-actin by phalloidin-conjugated tetramethylrhodamine isothiocyanate (TRITC) and observed under fluorescence microscopy. The results showed that 2 h of simulated ischemia significantly produced cellular injury and cell death, which was significantly inhibited when treated with 5 mg/ml of the ethylacetate extract of A. crassna (p < 0.05). Treatment of the ethylacetate extract of A. crassna significantly preserved actin cytoskeleton organization. In conclusion, the ethylacetate extract of A. crassna reduced ischemia induced cell death preserve cytoskeleton organization.

Key words: Aquilaria crassna, ischemic heart disease, myocardial ischemia, actin cytoskeleton.

INTRODUCTION

Ischemic heart disease (IHD), or myocardial ischemia, is (Jennings and Reimer, 1991). It has been known that p38 a disease characterized by ischemia (reduced blood mitogen-activated protein kinase (p38 MAPK) is activated supply) of the heart muscle, usually due to coronary as a result of myocardial ischemia and reperfusion, which artery disease. It is the most common cause of death in predominantly lead to cellular injury and necrosis worldwide and it is predicted to be the majority cause of (Kumphune et al., 2010). Inhibition of p38 activation could motility in the near future (World Health Organization, reduce myocardial injury (Barancik et al., 2000; Clark et 2008). Myocardial ischemia occurs when the coronary al., 2007; Gorog et al., 2004; Kompa et al., 2008; flow is severely block, which results in the inadequacy of Kumphune et al., 2010; Martin et al., 2001; Sumida et al., the oxygen supply and the oxygen demands of the heart 2005; Tanno et al., 2003; Yada et al., 2004), suggesting tissue and finally progresses to cellular necrosis the therapeutic potential of p38 inhibitors in ischemic heart disease. The phosphorylation of p38 MAPK by upstream kinases results in the activation of p38 activity, which is capable of phosphorylating other downstream *Corresponding author. E-mail: [email protected]. Tel: kinases, such as heat shock protein 27 (HSP27) +66(0)83 484 1771. Fax: +66(0) 55 966 300. (Wachtel et al., 2002). Activation of HSP27, mediated by 4058 J. Med. Plants Res.

p38 MAPK phosphorylation during ischemia, seems too 24 to 48 h or until it reached 80% confluence, before performing important in controlling the cellular oxidation-reduction experiments. state, and also associated with actin cytoskeleton organization (Wachtel et al., 2002). Simulated ischemia (sI) Aquilaria crassna Pierre ex Lecomte or agarwood, the heartwood of tropical tree, belongs to the family Simulated ischemia was induced by incubating H9c2 cell with Thymelaeaceae and class Magnoliosida (Dash et al., specified modified Krebs-Henseleit buffer (137 mM NaCl, 3.8 mM 2008), which can be found in many countries in oriental KCl, 0.49 mM MgCl2, 0.9 mM CaCl2, and 4.0 mM HEPES) with 20 mM 2-deoxyglucose, 20 mM sodium lactate, and 1 mM sodium region (Dash et al., 2008; Kim et al., 1997; Miniyar et al., dithionite at pH 6.5. Control buffer composed of Krebs-Henseleit 2008). It has been used as folk medical treatment for buffer (137 mM NaCl, 3.8 mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2, many inflammatory diseases and also found to be used in and 4.0 mM HEPES), supplemented with 20 mM D-glucose and 1 treatment of cardiac disorders (Miniyar et al., 2008). mM sodium pyruvate. After simulate ischemia was achieved, the Interestingly, in Thailand, A. crassna extract known as ischemic buffer or control buffer were removed and the cells were one of the composition in Ya-hom, a traditional Thai subjected to reperfusion by the addition of 2 ml complete medium before further incubating at 37°C, 5% CO2 for 24 h. herbal formulation for the treatment of fainting by increasing blood pressure (Suvitayavat et al., 2004). Recently, our previous experiments on the ethylacetate Measurement of cell viability assay extract of A. crassna suggested the potent anti- inflammatory effect inhibiting tumor necrosis factor alpha The measurement of H9c2 cardiomyoblast viability was performed by the reduction of by mitochondrial reductases. At the end of (TNF-) expression by attenuating p38 MAPK activation reperfusion period, the medium was collected for lactate (Kumphune et al., 2011). Therefore, in the present study, dehydrogenase (LDH) activity. Cells were incubated with 0.01 g/ml we aim to investigate the effect of crude extract of this MTT for 2 h at 37C. Then, 1 ml of 0.04 M HCl in isopropanol was medicinal plant on ischemic-induced cytoskeleton added to each well. The converted dye was collected and the disorganization in cardiac myoblast cell line. optical density was determined spectrophotometrically at  570 nm with background subtraction at  650 nm (A570-650). The percentage of cell viability was calculated by comparing theA570-650 MATERIALS AND METHODS of treated samples with theA570-650 of untreated control group (100% viability). Plant and extraction

The heartwood of A. crassna Pierre ex Lecomte used in this Measurements of cellular injury experiment was supplied from Mr. Choosak Rerngrattanabhume. The plant was identified by Dr. Pranee Nangngam, of the Measurements of cellular injury of H9c2 cardiomyoblast cell line Department of Biology, Faculty of Science, Naresuan University. was measured based on the extracellular release of LDH, The specimen voucher number (002540) was kept at the according to the loss of plasma membrane integrity. The enzyme- Department of Biology Herbarium, Faculty of Science, Naresuan kinetic measurement of LDH activity (U/l) was performed in the University. The heartwood was sliced into small pieces. The dried supernatant of collected culture medium, after simulated plant (1 kg) was consecutively extracted with ethylacetate (EtOAc) ischemia/reperfusion, using a commercially available kit. Ten (800 ml reflux) for two days each. The resulting EtOAc solution was microlitter of collected culture medium was added to 1 ml of concentrated under reduced pressure to yield EtOAc extract (950 reaction reagent, and incubated at 37C for 1 min. The absorbance mg) (Kumphune et al., 2011). was measured at 340 nm wavelength, exactly after 1, 2, and 3 min. The mean absorbance change per minute (A/min) was used to calculate LDH activity. Chemicals and reagents

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum Actin cytoskeleton organization (Gibco BRL; Life Technologies Inc., New York, NY, USA) and 5 trypsin-EDTA, lactate dehydrogenase (LDH) liquid-UV test (Human, 1 × 10 of H9c2 cells were grown on the sterile coverslips, which Wiesbaden, Germany), 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H- was placed in 6 wells plate, and cultured in complete medium at tetrazolium bromide (MTT) (Ameresco, Solon, Ohio, USA), total-p38 37°C, 5% CO2 at sub-confluent densities. Cells were incubated with (T-p38) and diphospho-p38 (P-p38), (Santa cruz biotech, California, simulated ischemic buffer for 2 h, in the presence or absence of USA). Other chemicals were purchased from Sigma. ethylacetate extract of A. crassna, before being subjected to fluorescence staining procedure. The coverslips were washed twice with 2 ml of phosphate buffered saline (PBS) before fixing with 2 ml Cell culture of fixative agent (2% formaldehyde, 0.05% glutaraldehyde) at room temperature for 30 min. The cells on coverslips were permeabilized The rat cardiac myoblast cell line, H9c2 cell line (ATCC number with 2 ml of 0.5% Triton-X 100 in PBS at room temperature for 20 CRL-1446™), was maintained in DMEM supplemented with 10% min. The cells on coverslips were stained with 50 µg/ml of heat-inactivated fetal bovine serum, 100 units/ml of penicillin and tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin 100 µg/ml of streptomycin. Cell was maintained at 37°C, 5% CO2 at at room temperature for 40 min in dark moist box. Subsequently, sub-confluent densities. Sub-confluent cells (70 to 80%) were the coverslips were washed with 2 ml of PBS twice, before being subcultured 1:3. The cells were trypsinized, plated (1 × 105 cells/ml) nuclearly stained with 0.01 µg/ml 4',6-diamidino-2-phenylindole in 6-wells plate and were incubated in humidified CO2 incubator for (DAPI) at room temperature for 20 min. The coverslips were Jermsri and Kumphune. 4059

Table 1. Percentage of cell viability by MTT assay and released LDH activity (U/L).

Cell viability Release cellular LDH activity Treatment (Mean ± SD) (U/l) (Mean ± SD) Control 100 ± 1.824 29.56 ± 5.004 sI 13.36 ± 3.513 164.7 ± 9.069 sI +1 mg/ml A. crassna 17.58 ± 2.040 155.2 ± 16.97 sI +2 mg/ml A. crassna 39.81 ± 3.140 * 131.4 ± 18.58 sI +3 mg/ml A. crassna 56.40 ± 6.093* 142.2 ± 25.91 sI +4 mg/ml A. crassna 61.97 ± 5.516* 133.7 ±28.52 sI +5 mg/ml A. crassna 77.50 ± 1.128*# 52.24 ± 4.403*# sI +6 mg/ml A. crassna 75.90 ± 2.777*# 86.10 ± 4.971*# sI +7 mg/ml A. crassna 45.98 ± 5.422* 100.9 ± 4.334* sI +8 mg/ml A. crassna 53.17 ± 4.922* 100.7 ± 6.422*

*p < 0.05 compared to sI; # p < 0.05 compared to A. crassna treated group.

Table 2. Toxicity test of ethylacetate extract of A. crassna on cardiac myoblast cell viability.

Cell viability (%) Release cellular LDH Treatment (Mean ± S.D.) activity (U/l) (Mean ± S.D.) Control 100 ± 2.060 27.055 ± 2.676 Vehicle control (0.001% DMSO) 98.28 ± 5.178 29.023 ± 8.984 1 mg/ml A. crassna 100.4 ± 2.972 28.957 ± 2.053 2 mg/ml A. crassna 100.2 ± 2.737 29.022 ± 3.278 3 mg/ml A. crassna 98.65 ± 2.972 29.894 ± 2.938 4 mg/ml A. crassna 99.86 ± 2.737 30.002 ± 3.495 5 mg/ml A. crassna 99.80 ± 3.429 30.086 ± 3.049 6 mg/ml A. crassna 95.98 ± 2.737 29.580 ± 3.437 7 mg/ml A. crassna 96.53 ± 3.079 32.266 ± 3.827 8 mg/ml A. crassna 96.58 ± 3.129 33.890 ± 5.977

mounted by adding 20 µl of 50% glycerol on glass slide and sealed to 8 mg/ml of the ethylacetate extract of A. crassna prior the edges with nail varnish. The actin cytoskeleton was visualized to simulated ischemia. Pre-treatment of 2 to 8 mg/ml A. under fluorescence microscopy. The percentages of intact crassna extract before being subjected to simulated cytoskeleton were measured by number of cells with intact stress fiber in hundred total cells. ischemia significantly increased cell viability, when compared with simulated ischemic cell alone, in dose dependent manner (Table 1). In addition, the results Statistical analysis showed that 5 mg/ml of the ethylacetate extract of A. crassna gave highest percentage of cell viability up to Results are expressed as Mean ± standard deviation (SD). Data sets were analyzed by one-way analysis of variance followed by approximately 80%. However, the effect of A. crassna Tukey’s post hoc test. A value of p < 0.05 was considered extract on release LDH activity, reflecting cellular injury, statistically significant. showed that only A. crassna extract at 5 and 6 mg/ml could reduce LDH releasing. The higher concentration of A. crassna extract, although, reduced cell death, but RESULTS failed to reduce cellular injury. It was then tested if the A. crassna extract caused any harmful effect to the cardiac Effect of the ethyl acetate extract of A. crassna on cell. Cells were exposed to A. crassna extract at cardiac cell death concentration 1 to 8 mg/ml, and also vehicle control (0.01% dimethyl sulfoxide (DMSO)). The cell viability and The effect of ethyl acetate extract of A. crassna on cell cellular injury were measured as described earlier. The viability and cellular injury in H9c2, subjected to 2 h of results showed that in basal condition, treatment of H9c2 simulated ischemia and 24 h of reperfusion, was cells with A. crassna extract did not reduce cell viability performed by pre-incubation of various concentrations, 1 and cellular injury (Table 2). 4060 J. Med. Plants Res.

A. crassna pretreat-sl

A. crassna treat at sI

A. crassna pretreat + at sI

Figure 1. Effect of Aquilaria extract on ischemia-induced cytoskeleton disorganization. Cells were subjected to 2 h simulated ischemia, in the presence and absence of 5 mg/ml Aquilaria extract. After the end of the experiments, cells were fixed and stained for F- actin by phalloidin-conjugated TRITC, and nuclear staining by DAPI. Visualization of cytoskeleton organization was observed under fluorescence microscopy.

Effect of the ethylacetate extract of A. crassna on F- were stained with TRITC-conjugated phalloidin, as actin organization during simulate ischemia described in materials and methods. The results showed that cardiac cells subjected to simulated ischemia had In this experiment, it was hypothesized if the reduction of less stress fibers, or fragile F-actin network, and the cell death by treatment with A. crassna extract could stress fibers were rather disrupted (as shown in break, stabilize F-actin, and prevent remodeling of the diffuse, unconnected-dash line F-actin) (Figure 1). cytoskeleton. The H9c2 cells were subjected to simulated However, treatment of 5 mg/ml of A. crassna resulted in ischemia, in the presence and absence of 5 mg/ml of the stress fibers preservation or intact cytoskeleton, A. crassna extract, either 1 h pre-treatment prior to 40 especially when the treatment of A. crassna extract was min simulated ischemia, at the onset of 40 min simulated performed at the onset of simulated ischemia and also ischemia, and for the whole period of the experiment treatment before and during ischemia (Figure 1J to O). (pre-treatment + during simulated ischemia). Then, cells The results, which were represented as percentage of

Jermsri and Kumphune. 4061

A. Control Pretreat Pretreat DMSO A. DMSO sI crassna DMSO A. at sI crassna pretreat crassna at sI + at sI pretreat + at sI

Figure 2. The percentages of intact stress fiber. Cells were subjected to 2 h simulated ischemia, in the presence and absence of 5 mg/ml A. crassna extract. After the end of the experiments, cells were fixed and stained for F-actin by phalloidin-conjugated TRITC. The percentages of intact stress fiber were measured in separate 5 observed fields. * p < 0.05 versus sI and vehicle control of each group (ANOVA, n=3).

cells with intact cytoskeletal organization, showed that actin cytoskeleton organization (Rouse et al., 1994). It simulated ischemia had low number of cells with intact has been reported that activation of heat HSP27, which is cytoskeleton. Treatment of 5 mg/ml A. crassna extract downstream substrate of p38 MAPK, was found to be the significantly increased the percentage of cell with stress crucial mechanism attenuating cellular apoptosis and F- fiber or intact cytoskeleton (Figure 2). actin remodeling facilitation (Okada et al., 2005). Therefore, we tested the effect of A. crassna extract on actin cytoskeleton organization in cell subjected to DISCUSSION simulated ischemia. Phalloidin conjugated TRITC staining for actin cytoskeleton showed a well-organized stress A. crassna has been used in many traditional therapeutic fiber of actin filaments. The loss of stress fibers, purposes, such as treatment of inflammations, cancer, appeared as dash line of broken filaments caused by and was found to be one of the major compositions in simulated ischemia, was consistent with the previous traditional Thai herbal formulation targeting cardio- finding (Wachtel et al., 2002). Pre-treatment of the vascular system (Kumphune et al., 2011; Suvitayavat et ethylacetate extract of A. crassna could partially preserve al., 2004). Recently, the underline mechanism of anti- the cytoskeleton, which was observed by the increased inflammatory effect of the ethylacetate extract of A. percentage of intact stress fibers, when compared with crassna that inhibited tumor necrosis factor-alpha (TNF- that of simulated ischemia alone. Interestingly, treatment ) expression by attenuating p38 MAPK activation was of the ethylacetate extract of A. crassna at the onset of demonstrated (Kumphune et al., 2011). The phosphor- simulated ischemia gave higher percentages of intact rylation of p38 MAPK during myocardial ischemia stress fibers, which suggested the more efficiency to resulted in phosphorylating other downstream kinases, preserve the cytoskeleton organization. However, such as HSP27 (Rouse et al., 1994). Activation of treatment of the ethylacetate extract of A. crassna, both HSP27, mediated by p38 MAPK phosphorylation during pre-treat or during ischemia did not give higher number of ischemia, seems to be important in controlling the cellular cells with intact cytoskeleton. These results could oxidation-reduction state, and it is also associated with possibly be due to the anti-oxidative effect of the 4062 J. Med. Plants Res.

ethylacetate extract of A. crassna reported by Miniyar et Jennings RB, Reimer KA (1991). The cell biology of acute myocardial al. (2008), which when administered at the same time of ischemia. Annu. Rev. Med., pp. 225-246. Kim YC, Lee EH, Lee YM, Kim HK, Song BK, Lee EJ, Kim HM (1997). simulated ischemia could rapidly diminish or capture the Effect of the aqueous extract of Aquilaria agallocha stems on the oxidative radicals. immediate hypersensitivity reactions. J. Ethnopharmacol., 1: 31-38. In our hands, this is the first evidence showing the in Kompa AR, See F, Lewis DA, Adrahtas A, Cantwell DM, Wang BH, vitro anti-ischemic effect of this plant extract, on cardiac Krum H (2008). Long-term but not short-term p38 mitogen-activated protein kinase inhibition improves cardiac function and reduces myoblast cell line. However, the experiments in this cell cardiac remodeling post-myocardial infarction. J. Pharmacol. Exp. line have some limitations and weak points, as it may not Ther., 3:741-750. closely be related to real physiological settings in the Kumphune S, Bassi R, Jacquet S, Sicard P, Clark JE, Verma S, Avkiran intact heart. Therefore, more relevant models, such as an M, O'Keefe SJ, Marber MS (2010). A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation in vitro experiment in primary culture of isolated aggravates myocardial infarction and is prevented by the direct ventricular myocytes, an ex vivo experiment in isolated binding of SB203580. J. Biol. Chem., 5: 2968-2975. heart perfusion, or an in vivo experiment in animal model, Kumphune S, Prompun E, Phaebuaw K, Sriudwong P, Pankla R, will provide some functional data, which is close to the Thongyoo P (2011). Anti-inflammatory effects of the ethyl acetate extract of Aquilaria crassna inhibit LPS-induced tumour necrosis real physiological event in the heart and could lead to factor-alpha production by attenuating P38 MAPK activation. Int. J. more reliable interpretation. Moreover, this report was Green Pharm. A.D, 1: 43-48. performed using the crude extract, so identification of Martin JL, Avkiran M, Quinlan RA, Cohen P, Marber MS (2001). active compounds, together with its therapeutic Antiischemic effects of SB203580 are mediated through the inhibition of p38alpha mitogen-activated protein kinase: Evidence from ectopic applications, is a challenge needs to be further expression of an inhibition-resistant kinase. Circ. Res., 9: 750-752. investigated. Miniyar PB, Chitre TS, Karve SS, Deuskar HJ, Jain KS (2008). Anti- oxidant activity of ethyl acetate extract of Aquilaria agallocha on nitrite-induced methemoglobin formation. Int. J. Green Pharm., 1: 43- 44. Conclusion Okada T, Otani H, Wu Y, Kyoi S, Enoki C, Fujiwara H, Sumida T, Hattori R, Imamura H (2005). Role of F-actin organization in p38 Conclusively, treatment of the ethylacetate extract of A. MAP kinase-mediated apoptosis and necrosis in neonatal rat crassna protect cellular injury and death, and was also cardiomyocytes subjected to simulated ischemia and reoxygenation. Am. J. Physiol. Heart Circ. Physiol., 6: H2310-H2318. found to preserve cytoskeleton disorganization in cardiac Rouse J, Cohen P, Trigon S, Morange M, onso-Llamazares A, cell subjected to simulated ischemia model. Zamanillo D, Hunt T, Nebreda AR (1994). A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell, 6: 1027- 1037. ACKNOWLEDGEMENTS Sumida T, Otani H, Kyoi S, Okada T, Fujiwara H, Nakao Y, Kido M, Imamura H (2005). Temporary blockade of contractility during This work was supported by Naresuan University reperfusion elicits a cardioprotective effect of the p38 MAP kinase Research Endorsement Foundation R2554C045 (to SK) inhibitor SB-203580. Am. J. Physiol. Heart Circ. Physiol., 6: H2726- H2734. and Faculty of Allied Health Sciences Postgraduate Suvitayavat W, Kodchawongs J, Thirawarapan SS, Bunyapraphatsara Research Endorsement Foundation (to PJ). The authors N (2004). Effects of Ya-hom on the gastric secretion in rats. J. would like to express their sincere gratitude to Dr. Ethnopharmacol., 2-3: 331-338. Panumart Thongyoo, Department of Chemistry, Faculty Tanno M, Bassi R, Gorog DA, Saurin AT, Jiang J, Heads RJ, Martin JL, Davis RJ, Flavell RA, Marber MS (2003). Diverse mechanisms of Science and technology, Thammasart University, myocardial p38 mitogen-activated protein kinase activation: evidence Thailand for preparing the crude extract. for MKK-independent activation by a TAB1- associated mechanism contributing to injury during myocardial ischemia. Circ. Res., 3: 254- 261. Wachtel M, Frei K, Ehler E, Bauer C, Gassmann M, Gloor SM (2002). REFERENCES Extracellular signal-regulated protein kinase activation during

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