Neuroprotective effects of Dendrobium alkaloids on rat cortical neurons injured by oxygen-glucose deprivation and reperfusion

ARTICLE IN PRESS Phytomedicine 17 (2010) 108–115

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Neuroprotective effects of Dendrobium alkaloids on rat cortical neurons injured by oxygen-glucose deprivation and reperfusion Q. Wang a,b, Q. Gong a, Q. Wu a, J. Shi a, a b

Department of Pharmacology, Zunyi Medical College, 201 Dalian Road, Zunyi 563000, China School of Pharmacy and Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China

a r t i c l e in f o

Keywords: Dendrobium alkaloids Oxygen-glucose deprivation/reperfusion Neuron Apoptosis

a b s t r a c t In this study we investigated the protective effects of alkaloids from Dendrobium spez. on cortical neurons injured by oxygen-glucose deprivation/reperfusion (OGD/RP) in vitro. Rat primary cultured cerebral cortical neurons were investigated at different time points of OGD/RP. The MTT assay and the lactate dehydrogenase (LDH) release were used to determine cell viability. The concentration of intracellular free calcium [Ca2+]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuron damage. Morphologic changes of neurons following OGD/RP were examined by electron microscope. To evaluate neuron apoptosis, flow cytometry was performed and the expressions of caspase-3 and caspase-12 mRNA were examined by real-time quantitative PCR during OGD 2 h/RP 12 h. Treatment with Dendrobium alkaloids (0.0252.5 mg/l) significantly attenuated neuronal damage, with evidence of increased cell viability, decreased cell apoptosis, and decreased cell morphologic impairment. Furthermore, Dendrobium alkaloids inhibited [Ca2+]i elevation, increased MMP and decreased the expressions of caspase-3 and caspase-12 in a concentration-dependent manner at OGD 2 h/RP 12 h. Dendrobium alkaloids have significantly protective effects on OGD/RP-induced neuronal damages in rat primary neuron cultures. The protection against OGD/RP-induced apoptosis appears to be mediated through blocking the decrease in MMP and increase in [Ca2+]i, as well as by down-regulating mRNA expression of caspase-3 and caspase-12. & 2009 Published by Elsevier GmbH.

Introduction Ischemic brain injury in aged populations is a problem of enormous importance. Cerebral ischemia causes an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration (Roman 2004). Despite a large number of studies on promising neuroprotective agents, no successful therapy for ischemic brain injury has emerged. Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex (Mattson et al. 2000). Because of mitochondria dysfunction and altered intracellular calcium homeostasis in many different cell types, apoptotic death caused by hypoxia and hypoglycemia follow ischemia (Orrenius et al. 2003). Recently the existence of a novel apoptotic pathway in the endoplasmic reticulum (ER) has been demonstrated, and the activation of caspase-12 can lead to apoptosis. Since the overall process of ischemic brain injury is extremely complex, the protective effects of Chinese traditional medicines are receiving more attention in the effort to find agents for the treatment of ischemic brain vascular diseases.

Dendrobium, also known as ‘‘Shihu’’ or ‘‘Huangcao’’ in Chinese, is widely distributed in the South of China, and has been used in traditional Chinese medicine as a tonic and as a therapeutic agent for curing cataracts and throat inflammation (Bao et al. 2001). Research focused on its constituents and pharmacological activity has revealed several active ingredients, including alkaloids, stilbenoids, glycosides and polysaccharides (Zhang et al. 2005). Dendrobium extracts have been reported to possess immunostimulant activity (Jiangsu New Medical College 1986) and antioxidative effects (One 1995). Dendrobium alkaloids have high solubility in lipid and the potential ability to permeate the blood brain barrier (BBB). This study was aimed at addressing the effects of Dendrobium alkaloids on rat primary cultured neurons subjected to oxygen-glucose deprivation/reperfusion (OGD/RP), in an attempt to find a new multifunctional cytoprotective agent to treat ischemic brain vascular diseases.

Materials and methods Animals and drug

 Corresponding author. Tel.: +86 852 860 9528; fax: +86 852 860 9788.

E-mail addresses: [email protected], [email protected] (J. Shi). 0944-7113/$ - see front matter & 2009 Published by Elsevier GmbH. doi:10.1016/j.phymed.2009.05.010

Sprague-Dawley rats (Grade SPII, Certificate NO20030523) were purchased from the Laboratory Animal Center, Chongqing,

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Fig. 1. GC-analysis and chemical structure of Dendrobium alkaloids.

China. All animal studies were in compliance with Animal Care and Use Guidelines in China and were approved by the local Ethics Committee. Dendrobium was collected from Guizhou Province, P.R. of China in 2004. A specimen of this collection has been deposited at the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences. A finely-cut sample (1.2 kg) was soaked in MeOH (8 l) for 2 days; the extract was separated from the residue and the latter was then reextracted four times using boiling MeOH (each 5 l). The extracts were combined and concentrated to a blackish brown residue (148 g) under reduced pressure. The residue was extracted several times with 5% ap. HCl until the acid extract showed a negative reaction with Meyer’s reagent. The combined acid extracts were filtered, shaken thoroughly with Et2O to remove acidic and neutral substances and brought to pH 11.0 by addition of NH4OH under cooling with ice. The alkaline solution was extracted exhaustively

using Et2O. The Et2O extract was dried over anhydrate Na2SO4 and evaporated to leave 3.9 g bases, which contained as major active constituents, alkaloids at around 96.1%, and polysaccharides at around 1.2%. The alkaloid extracts were dissolved in CH2Cl2 and applied to a GC apparatus (Agilent 6890N) equipped with a hydrogen flame ionization detector. Capillary GC analysis was performed under the following conditions: capillary column HP-5 (0.32 mm i.d. and 0.25 mm film thickness) was from Agilent Technologies (City, CA, USA), detector temperature 280 1C, injector temperature 250 1C, carrier gas helium (0.7 ml/min), and injection volume 1 ml. GC oven temperature was kept at 150 1C for 5 min, programmed to 250 1C at a rate of 7 1C/min and kept constant at 250 1C for 5 min. Relative percentage contents of alkaloids were determined using the area under peaks from corresponding standard ion chromatography, using Agilent software. Dendrobine (C16H25O2N, 90.7%) was determined as the main alkaloid in this alkaloid

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extract. Dendramine (C16H25O3N, 2.31%), 3-Hydroxy-2-oxodendrobine (C16H23O4N, 1.29%), and Nobilonine (C17H27O3N, 4.47%) were also detected (Fig. 1). Chemical structures are shown in Fig. 1. The details of the alkaloid identification and purification will be reported elsewhere (manuscript in preparation by the Key Laboratory of Chemistry for Natural Products of Guizhou Province). Study design Cells were randomly divided into 7 groups: Control group; Vehicle group injured by OGD (1, 2, 4 h/RP 3, 12, 24 h, respectively); Treatment+OGD/RP group (administered Dendrobium alkaloids, final concentrations of 0.025 mg/l, 0.25 mg/l and 2.5 mg/l respectively). Cell culture and oxygen-glucose deprivation-induced neuronal cell injury Primary rat cortical neuronal cells were prepared from newborn Sprague-Dawley rats, as described by Zheng and Lin (2002). Briefly, cerebral cortices were collected in isotonic solution containing penicillin, streptomycin after being dissected free of meninges and blood vessels. Cells that dissociated from the cortex were pooled and resuspended in Dulbecco’s modified Eagle medium/F12 (DMEM/F12) which containing 15% fetal bovine serum, 5% horse serum (Sigma, St. Louis, MO), and 100 U/ml penicillin/streptomycin. The cells were plated at a density of 1 105/ml, seeded in poly L-lysine-coated culture dishes or 96well plates and maintained in a humidified incubator (37 1C with 5% CO2). On the third day, the cells were cultured with 1 mM cytosine arabinoside for 48 h. Cultures contain 495% neurons as routinely identified by neuron-specific enolase (NSE). The neuronal cell cultures were used from in vitro day 9 to 10, the culture medium of the Vehicle group was replaced with Earl’s balanced salt solution (EBSS) (in mg/l: 6800 NaCl, 400 KCl, 264 CaCl2  2H2O, 200 MgCl2  7H2O, 2200 NaHCO3, 140 NaH2PO4  H2O, and 1000 glucose, PH 7.2). Neurons were then washed three times with glucose-free EBSS medium and incubated for 1, 2 and 4 h in oxygen-free N2/CO2 (95%/5%) gas. The control group was incubated in EBSS with 10 mM glucose. Thereafter, the medium was replaced by standard culture medium. Assessment of cortical neuron viability and damage Cell viability was assessed by the measurement of the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to produce a dark blue formazan product (Hansen and Nielson, 1989). MTT was added to each well after OGD injury at a final concentration of 0.5 mg/ml. After 4 h incubation, the medium was removed, and cells were dissolved in DMSO. The formation of formazan was measured at 570 nm (TECAN infinite M200). Results of formazan formation were normalized to sham wash, and expressed as the percentage of viable cells. Neuronal damage in cortical cells was also quantitatively assessed by the measurement of lactate dehydrogenase (LDH), released from damaged or dead cells, in the extracellular fluid with microplate reader. A previous study has shown that the efflux of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed (Gwag et al., 1995). The amount of LDH released after cortical neuron lysis by 0.5% Triton 100 constitutes 100% cell death or ‘‘full kill’’. The extent of cell death was expressed as percentage of full kill.

Morphological change in the neurons was examined using a scanning electron microscope. Measurement of [Ca2+]i of cortical neuronal cells Measurement of [Ca2+]i of cortical neuronal cells was performed with the fluorescent Ca2+-chelator fura-2 as reported previously (Watanabe et al., 1996; Liu et al., 2007). After being washed with Hanks’ solution twice, cells were harvested by centrifugation at 1000 rpm for 5 min after trypsinization with 0.25% trypsin and then prepared for single cell suspension. Each group was harvested at a density of 106 cells/ ml. Only the cells with 95% viability as determined by Trypan blue staining were used. A final concentration of Fura-2/AM 5 mmol/l was added to the above isolated cell suspensions and incubated in darkness for 30 minutes at 37 1C, and then the cells were washed with Hank’s solution containing 0.2% bovine serum albumin to remove the residual Fura-2/AM. [Ca2+]i was measured with RF-5000 spectroflourometer. Calculation of intracellular calcium was made using the following equation: [Ca2+]i ¼ Kd[(R-Rmin)(Rmax-R)](Fmin/ Fmax). [Kd is the dissociation constant of Fura-2 for Ca2+ and was assumed to be 224 nmol/l at 37 1C (Graham and Burgoyne, 1994). Mitochondrial membrane potential (MMP) MMP was measured using a fluorescence method (Ikari et al., 2001). Using RF-5000 spectroflourometer at an excitation wavelength of 488 nm and an emission wavelength of 535 nm, we monitored the MMP by release of Rhodamine-123 from the mitochondria following OGD/RP. 5 mM Rhodamine-123 was added in 1 106 cells/ml for 15 min before fluorescence intensity was read. Apoptosis assays Neuronal apoptosis was assayed by flow cytometry with Annexin V/FITC kit, according to the methods of Grynkiewicz et al. (1985). Briefly, cells were collected after OGD for 2 h followed by RP for 12 h. Cells were washed twice in ice-cold PBS, and then resuspended in binding buffer (10 mM HEPES, PH 7.4, 0.14 M NaCl and 2.5 mM CaCl2) at a density of 1 106 cells/ml. Cells were incubated simultaneously with fluorescein-labeled annexin V and propidium iodide (PI) for 20 min at room temperature in darkness and analyzed by flow cytometry to evaluate neuronal apoptosis. Data were analyzed using Cell Quest software. Reverse transcription real-time quantitative PCR assay Expressions of caspase-3 and caspase-12 mRNA were detected using real-time quantitative PCR. Total RNA of primary cultured neurons was isolated by Trizol agent (Invitrogen, Carlbad, CA) and purified with RNeasy Mini Kit (Qiagen, Palo Alto, CA). RNA was spectrophotometrically quantified by measuring the optical density of samples at 260/280 nm. RNA was reversed transcribed with M-MLV. The nucleotide sequences of the primers used in this study were as follows: A caspase-3 (NM_012922): sense 50 -CAG AGC TGG ACT GCG GTA TTG A-30 , antisense 50 -AGC ATG GCG CAA AGT GAC TG-30 ; B caspase-12 (NM_130422): sense 50 -CTG GCC CTC ATC ATC TGC AA-30 , antisense 50 -TGG ACG GCC AGC AAA CTT-30 . C b-actin (NM_031144): sense 50 -TGA CAG GAT GCA GAA GGA GA-30 , antisense 50 -TAG AGC CAC CAA TCC ACA CA-30 . The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time quantitative PCR analysis (initial

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Fig. 2. Effects of Dendrobium alkaloids on cultured rat cortical neurons injured by OGD/RP, determined by MTT assay (A), and the extent of LDH release (B). Data are expressed as mean7SD from 6 separate experiments in quadruplicate. #po0.01 vs Control cells; *po0.05, **po0.01 vs corresponding OGD/RP+Vehicle groups. (Den L: Dendrobium alkaloids 0.025 mg/l; Den M: Dendrobium alkaloids 0.25 mg/l; Den H: Dendrobium alkaloids 2.5 mg/l).

template denaturation at 95 1C for 10 min, followed by 40 cycles of 95 1C for 15 s, 60 1C for 60 sec). The relative differences in expression among groups were expressed as cycle time (Ct). The Ct values of the interested genes were first normalized with bactin from the same sample, and then the relative difference between the Control group and each treatment group was calculated and expressed as a relative increase, with control set at 100%.

decreased MTT and increased LDH. The survival ratio and the percentage of LDH leakage are directly related to the time of exposure to OGD/RP (Fig. 2). Exposure to EBSS alone did not result in significant damage. Exposure to Dendrobium alkaloids significantly attenuated neuronal impairment induced by OGD/RP.

Statistical analysis

Compared to control group, the values of [Ca2+]i of cortical neurons at OGD 2 h/RP 12 h showed significant changes (nearly 3.5-fold vs control group), increased to (581725) nmol/l (n ¼ 6, po0.01, Fig. 3A). Compared to the vehicle group, the low, middle and high concentrations of Dendrobium alkaloids treatment after OGD 2 h/RP 12 h decreased [Ca2+]i in neuron cultures, and intracellular calcium concentrations were decreased from (581725) nmol/l to (518728) nmol/l, (441739) nmol/l and (313739) nmol/l (n ¼ 6, po0.01), after treatment with Dendrobium alkaloids at 0.025 mg/l (L), 0.25 mg/l (M) and 2.5 mg/l (H), respectively. Rhodamine-123 was used to evaluate loss of MMP in cortical cultures exposed to OGD/RP. MMP was assessed in neurons according to fluorescence ratios. Relative to normal neurons, MMP had decreased to (20.371.3) % (n ¼ 6, po0.01, Fig. 3B) in vehicle group, similar to the decrease of cell viability and increase of apoptosis ratio. Decreased MMP was returned to normal levels following the treatments with Dendrobium alkaloids to 3770.9%,

All data are presented as mean7SD. Statistical significance was determined using analysis of variance (ANOVA) with the SPSS 12.0 software package; po0.05 was considered statistically significant.

Results Effects of Dendrobium alkaloids on cell viability In the first series of experiments, the effects of Dendrobium alkaloids alone on cell viability were determined using the MTT assay. Treatment of cultured neurons with Dendrobium alkaloids at doses of 0.025 mg/l (L), 0.25 mg/l (M) and 2.5 mg/l (H) did not affect cell viability (data not shown). Exposure of cultured neurons to OGD/RP produced a significant increase in neuronal injury as compared to the control group, as evidenced by

Effects of Dendrobium alkaloids on [Ca2+]i and MMP

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Fig. 3. Effects of OGD/RP-induced alteration of [Ca2+]i and mitochondrial membrane potential (MMP) in rat cortical neurons. Intracellular concentrations of calcium (A); Mitochondrial membrane potential (B). Data are expressed as Mean7SD, n ¼ 6. #po0.01 OGD/RP+Vehicle group vs Control group; *po0.05, **po0.01 vs OGD/RP+Vehicle group.

Table 1 Effects of Dendrobium alkaloids on cell apoptosis in cultured neurons injured by oxygen-glucose deprivation 2 h/reperfusion with oxygen and glucose 12 h.

Control Vehicle Den L Den M Den H ##

n

Survival (%)

Apoptosis (%)

Death (%)

6 6 6 6 6

84.5670.26 42.0973.14## 54.3171.42 50.5470.96 55.3271.10

11.1170.17 49.1073.43## 33.1170.20 38.6670.53 34.6670.70

4.3370.23 8.8570.31 12.5871.28 10.7670.65 10.0270.67

po0.01 vs Control;

 po0.05,  po0.01 vs Vehicle.

4373.1%, and 6677.0%, at 0.025 mg/l (L), 0.25 mg/l (M) and 2.5 mg/l (H), respectively.

trees. Using a scanning electron microscope, we examined the ultra-structural changes of cortical neurons. The nuclei of neurons in the control group were big, round or oval and the euchromatin was distributed homogeneously. Structures of the mitochondria and rough endoplasmic reticulum were clear, and ribosomes were abundant (Fig. 5A). After cortical neurons were exposed to OGD 2 h/RP 12 h, the nucleus was irregular, chromatin mass was formed, mitochondria was swollen and vacuolar, cristae arrangement was disordered, mitochondria membrane was ruptured and endocytoplasmic reticulum was expanded (Fig. 5B). Treatment with Dendrobium alkaloids at 2.5 mg/l during the course of OGD/RP significantly reduced these neuronal damages, as compared with those of the vehicle group. Most of the nuclear chromatins were distributed homogeneously, swollen mitochondria were mitigated, lamellar cristae in mitochondria became clear, and ribosomes were more abundant than those in vehicle group (Fig. 5C).

Effects of Dendrobium alkaloids on cell apoptosis There was a low level (11.1%) of neuronal apoptosis in control group. The percentage of apoptosis increased significantly, to 49.1% in the vehicle group. Compared to the control group, there was a significant difference in apoptosis (po0.01). Treatment with Dendrobium alkaloids at 0.025 mg/l (L), 0.25 mg/l (M), and 2.5 mg/l (H) during OGD/RP decreased markedly the percentage of cell apoptosis from 49.1% to 38.6%, 34.7%, and 33.1%, respectively. The anti-apoptosis effect of the highest Dendrobium alkaloids concentration (2.5 mg/l) was the most dramatic (Table 1). Real-time quantitative PCR analysis of mRNA expression To determine whether Dendrobium alkaloids affect the regulation of apoptosis at the mRNA level, we carried out a real-time quantitative PCR analysis. Caspase-3 and caspase-12 mRNA expressions were significantly increased after OGD 2 h/RP 12 h (approximately 6-fold vs. normal condition). However, in the Dendrobium alkaloid-treated neurons, expressions of caspase-3 and caspase-12 mRNA were reduced significantly and in a concentration-dependent manner (Fig. 4). Morphological examination After exposure of neurons to OGD 2 h and RP 12 h at 37 1C, microscopy revealed the destruction of neurons, OGD/RP-induced neuronal loss and progressive degeneration, which were characterized by karyopyknosis or karyolysis and shortage of dendrite

Discussion Ischemic cerebrovascular disease has become one of the most devastating diseases, causing high morbidity, high disability rate, and high mortality in aged persons (Fein et al., 2000). To treat these diseases is a challenge. At present, more and more evidence indicates that Chinese herbal medicines have a beneficial role to play in the treatment of these disorders. Based on previous studies, we hypothesized that Dendrobium alkaloids have protective effects against neuronal damage and we confirmed the hypothesis in the present study. This is one of the first studies to investigate the effects of Dendrobium alkaloids on neuronal damage in the presence of OGD/RP. The system of cortical neuron cultures subjected to OGD/RP was used as a model of ischemic cerebrovascular disease, as described previously (Gifford et al., 1993), Dendrobium alkaloids were administrated at different concentrations in order to evaluate the protective role of Dendrobium alkaloids in OGD/RP damaged neurons. In this study, the degree of injury in cortical neuronal cell cultures exposed to OGD/RP was evaluated using MTT and LDH release. The results indicate that exposure of cultured neurons to OGD for 1, 2, or 4 hours elicited neuronal impairment, and that reoxygen and glucose for 3, 12, or 24 hours after OGD significantly reduced cell viability in a time-dependent manner. The OGD/RP injury was prevented by Dendrobium alkaloids at three different concentrations during the respective reperfusion period. These results indicated that Dendrobium alkaloids do have protective effects on OGD/RP in cortical neuronal cells. The neuroprotective

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Fig. 4. Effects of Dendrobium alkaloids on OGD/RP-induced expressions of caspase-3 and caspase-12 mRNA in rat cortical neurons as determined by real-time quantitative PCR. Data are Mean7SD of three separate experiments. # po0.01 OGD/RP+Vehicle group vs Control group; * po0.05, ** po0.01 vs OGD/RP+Vehicle group.

Fig. 5. The morphologic change of the primary cultured neurons. (A) Control group with electron microscope (  10000), Structure of mitochondria and rough endoplasmic reticulum were clear; (B) OGD/RP+Vehicle group with electron microscope (  10000): Mitochondria was swollen and vacuolar, cristae arrangement was disordered. Mitochondria membrane was ruptured; (C) OGD/RP+Dendrobium alkaloids 2.5 mg/l group with electron microscope (  10000): Swollen mitochondria were mitigated, lamellar cristae in mitochondria became clear.

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effects of Dendrobium alkaloids were most evident demonstrating the concentration-effect relationship at the time point of OGD 2 h/ RP 12 h. Thus we selected the time point of OGD 2 h/RP 12 h as the key point at which to study the protective mechanism of Dendrobium alkaloids. Ischemia or hypoxia can cause cell death by necrosis and apoptosis (MacManus and Linnik, 1997). In this study, we found that OGD/RP induced cortical neuron death, mainly through apoptosis. OGD/RP insult increased the apoptosis rate of neurons from 11.1% to 49.0% and Dendrobium alkaloids inhibited the apoptosis induced by OGD/RP, consistent with morphological changes examined with a scanning electron microscope. To further explore the anti-apoptotic mechanisms of Dendrobium alkaloids on OGD/RP injury, we performed an additional study of mitochondria function, [Ca2+]i overload, and mRNA expression of apoptotic enzyme genes. Calcium is an important second messenger involved in signal transduction and neurotransmitter release. Numerous studies have definitively indicated that the alteration of intracellular Ca2+ homeostasis plays a central role in initiating the apoptotic response (Orrenius et al., 2003). Elevation of [Ca2+]i leads to destabilization of the neuronal cyto-architecture and results in cell damage, and cell death. Our results showed Dendrobium alkaloids significantly reduced [Ca2+]i elevation at OGD 2 h/RP 12 h. Mitochondrial dysfunction is an early feature of nervous system ischemia. Rhodamine-123, the cell-permeable, cationic fluorescent dye, has been used successfully to monitor mitochondrial responses (Bindokas et al., 1998). Our data indicated that Dendrobium alkaloids stabilized MMP in a concentration-dependent manner. Because the activity-dependent release of rhodamine-123 from mitochondria is commonly considered as mitochondrial depolarization during increased mitochondrial Ca2+ uptake (Bindokas et al., 1998; Ward et al., 2000), we infer that Dendrobium alkaloids could alleviate calcium overload and maintain mitochondrial function, which assist the anti-apoptosis action. To further explore the anti-apoptotic effect of Dendrobium alkaloids at the mRNA level, we examined the expression of caspase-3. Caspase-3 action occurs downstream in the apoptotic pathway and involves in cleaving important substrates such as gelsolin, fodrin, actin, and poly ADP-ribose (Todor et al., 2002). The present study provided evidence that Dendrobium alkaloids prevented the expression of caspase-3 mRNA after OGD/RP injury. We also observed the expression of caspase-12 mRNA, another member of the caspase family. Caspase-12 is specifically localized on the cytoplasmic side (outer membrane) of the endocytoplasmic reticulum (ER) and is thought to play a role in ER stressmediated cell death. Caspase-12 has been shown to be activated by cytotoxic agents or ischemic insults (Lamkanfi et al., 2004; Oubrahim et al., 2002), and activated in models of neurodegenerative diseases (Larner et al., 2004), hypoxic insults (Shibata et al., 2003; Mouw et al., 2003). Hitomi et al. (2003) suggested that activation of ER-resident caspase-12 indirectly activates cytoplasmic caspase-3 and might be important in ER stress-induced neuronal apoptosis. Notably, our study also indicated that OGD/RP insult increased the expression of caspase-12 mRNA and the overexpression of caspase-3 mRNA simultaneously. Treatment with Dendrobium alkaloids could significantly reduce these effects in a concentration-dependent manner, parallel with its effects on [Ca2+]i elevation and apoptosis induced by OGD/RP. The fact that treatment with Dendrobium alkaloids prevents caspase-dependent cell apoptosis suggests that the molecular mechanisms of Dendrobium alkaloids’ effects may involve a caspase signaling cascade. Dendrobium alkaloids may act through signal transduction pathways to influence apoptosis.

Dendrobium is artificially cultivated in Zunyi and Cishui of Guizhou as a source of medicinal use, with over 3000 kg production/year. The active ingredients in Dendrobium and Dendrobium alkaloid derivatives are currently under investigation. In conclusion, we report a novel finding concerning the protective effect of Dendrobium alkaloids on OGD/RP neuronal injury in vitro. Our data indicate that Dendrobium alkaloids protect cells against OGD/RP by stabilizing MMP, inhibiting calcium overload, and lessening neuron apoptosis that is mediated, at least in part, by caspase-3-dependent and caspase-12-dependent pathway.

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