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MADP, a salidroside analog, protects hippocampal neurons from glutamate induced apoptosis
Life Sciences 103 (2014) 34–40
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MADP, a salidroside analog, protects hippocampal neurons from glutamate induced apoptosis Hua Xian a,1, Jing Zhao b,1, Yuan Zheng b, Meihong Wang b, Jun Huang c, Bingxin Wu c, Cheng Sun b,⁎, Yumin Yang b,⁎ a b c
Department of Pediatric Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, PR China Jiangsu Key Laboratory of Neuroregeneration, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China School of Medicine, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China
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Article history: Received 23 November 2013 Accepted 28 February 2014 Available online 11 March 2014 Keywords: Salidroside analog Hippocampal neurons Apoptosis Glutamate excitotoxicity Neurodegeneration
a b s t r a c t Aims: To investigate the anti-apoptotic effect of MADP, an analog of salidroside, against glutamate induced apoptosis in the cultured rat hippocampal neurons. Main methods: Cytotoxicity was determined by the MTT method and lactate dehydrogenase release to the medium. Cell apoptosis was evaluated by Hoechst 33342 staining, TUNEL assay and flow cytometric analysis. Western blotting was applied for detecting protein levels of cellular signaling molecules. Key findings: Our results showed that glutamate exposure significantly induces cell apoptosis, whereas the pretreatment of salidroside or MADP remarkably improves cell viability. Most importantly, the anti-apoptotic effect of MADP against glutamate insult is superior to salidroside. To explore the involved mechanisms, we measured some pro-apoptotic and anti-apoptotic protein levels, and several cell survival signaling pathways were analyzed as well. No visible alterations in Bcl-2 and Bax protein levels were observed by MADP or salidroside. Akt and JNK phosphorylation was robustly stimulated by MADP in the glutamate-treated neurons. Salidroside treatment results in a slight activation in Akt, while no significant alteration in JNK activity was observed. Significance: MADP exhibits higher capacity to attenuate glutamate induced cell apoptosis in the cultured rat hippocampal neurons, suggesting that MADP might be a better candidate than salidroside for developing novel drugs treating neuron loss associated disorders. © 2014 Elsevier Inc. All rights reserved.
Introduction Neurodegenerative diseases such as motor neuron disease and Alzheimer's disease affect 35 million people worldwide. They are chronic and progressive neurodegenerative illnesses characterized by memory deficits and cognitive decline owing to synaptic and neuronal loss in the hippocampus and cerebral cortex. Neurotoxicity plays a key role for the pathological progress of neurodegenerative diseases. Glutamate is an important neurotransmitter and a growing number of evidence shows that glutamate has been implicated in neurotoxicity (Arundine and Tymianski, 2004). Excessive glutamate will disturb the homeostasis of calcium in the cytoplasm, lead to mitochondrial membrane depolarization and eventually result in neuronal death (Atlante et al., 2001). Chemicals with ameliorating activities on glutamate excitotoxicity are receiving more and more attention since these ⁎ Corresponding authors. Tel./fax: +86 513 85511585. E-mail addresses: [email protected] (C. Sun), [email protected] (Y. Yang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.02.040 0024-3205/© 2014 Elsevier Inc. All rights reserved.
chemicals might be used as potential clinical drugs for treating neurodegenerative disorders. Salidroside, one of the most potent compounds in Rhodiola rosea L., has been shown to have multiple biological activities including antiaging (Mao et al., 2010), anti-oxidative stress (Yuan et al., 2013), antiinflammation (Diaz Lanza et al., 2001) and anti-cancer (Kucinskaite et al., 2004). Recently, it has been shown that salidroside exhibits potent anti-apoptotic effects in the various types of cells including neurons (Qu et al., 2012), cardiomyocytes (Zhong et al., 2010) and endothelia (Tan et al., 2009). Recently more and more salidroside was consumed especially in the Traditional Chinese Medicine. Extraction from Rhodiola plants is the main source of salidroside currently, although the content of salidroside in Rhodiola plants is quite low. Additionally, the hydrophilic property of salidroside affects its efficacity since hydrophilic molecules are hard to penetrate across the cytoplasmic membrane and blood brain barrier. All these limitations blockade the deep research and development of salidroside. For addressing these issues, we synthesized salidroside and its analogs and evaluated their anti-apoptotic activities in the PC12 cells (Meng et al., 2011). Of these, MADP possesses
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higher protective activity against apoptosis induced by hypoglycemia and serum limitation with comparison to salidroside. Salidroside has been shown to have a neuron-protective role in cultured rat hippocampal neurons (Chen et al., 2008). We speculate that, therefore, MADP also may have a stronger capacity against apoptosis induced by glutamate in hippocampal neurons. To test this speculation, we pretreated rat hippocampal neurons with MADP or salidroside and evaluated their anti-apoptotic effects against glutamate insult. The involved mechanisms responsible for the antiapoptotic effects of MADP and salidroside were also investigated.
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Cytotoxicity assay Hippocampal neurons were cultured in 96-well plates at a density of 5 × 105 cells per well. After treatment as aforementioned, the medium was discarded, and then 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was added and incubated at 37 °C for 4 h. Formazan crystals were dissolved in 100 μl of 20% SDS. The absorbance was measured at 570 nm by a microplate reader (Bio-Tek, Inc.). Lactate dehydrogenase (LDH) assay
Material and methods Reagents Salidroside (Fig. 1) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). MADP (Fig. 1) was synthesized as described previously (Meng et al., 2011). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), trypsin, poly-L-lysine, Neurobasal medium, B27, and Annexin V-FITC/PI kit were purchased from Invitrogen (Carlsbad, CA, USA). MTT, Hoechst 33342 and glutamate were purchased from Sigma-Aldrich (St. Louis, MO, USA). LDH test kit was purchased from Jiancheng (Nanjing, China). TUNEL cell death detection kit was from Roche (Mannheim, Germany). Rabbit anti-phospho-Erk, rabbit antiErk, rabbit anti-phospho-Akt (Ser 473), rabbit anti-phospho-p38, rabbit anti-Akt, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-Bax, rabbit anti-Bcl-2 and rabbit anti-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary hippocampal cell culture and treatments Hippocampal cell cultures were prepared from hippocampi of Sprague–Dawley rat embryos (E18–E19) (Chen et al., 2008). Briefly, the hippocampi were dissected under anatomy microscope and collected in Hank's balanced salt solution containing 137 mM NaCl, 5.36 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 4.16 mM NaHCO3, 5 mM glucose, 1 mM sodium pyruvate, and 10 mM HEPES (pH 7.4). Tissues were then digested in 0.125% trypsin solution for 5 min at 37 °C. The digestion was terminated by adding DMEM supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin. The digestion mixture was centrifuged at 1000 rpm for 4 min at 4 °C and the supernatant was discarded. The resulting pellet was re-suspended in DMEM and filtered with a 200 screen mesh. The filtrate containing primary hippocampal neurons were counted and seeded into cell culture plates coated with poly-L-lysine. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 for 4 h at 37 °C. Finally, the medium was changed with serum-free neurobasal medium supplemented with 2% B27, 2 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. The medium was replaced with fresh medium every 3–4 days. The cultured 8 day-old hippocampal neurons were pretreated with MADP or salidroside at the concentrations of 120 and 240 μM for 24 h, and then the neurons were exposure to 125 μM glutamate for 15 min. After excitotoxicity was induced, the cells were further incubated with fresh medium for 24 h.
The LDH activity was determined with a LDH assay kit according to the manufacturer's instructions. Briefly, hippocampal neurons were cultured in 24-well plates at a density of 5 × 104 cells/cm2. After treatments as mentioned above, 20 μl of cell culture supernatant was transfered to a new 96-well plate, and then 25 μl of substrate buffer was added. The mixture was incubated for 15 min at 37 °C. Blank and standard wells were carried out at the same time. In standard wells, 5 μl double distilled water, 20 μl 0.2 mM standard solution and 25 μl substrate buffer were added. In blank wells, only 25 μl double distilled water and 25 μl substrate buffer were added. Subsequently, 25 μl of 2, 4-dinitrobenzene hydrazine was added and incubated for 15 min at 37 °C. 250 μl of 0.4 M NaOH was added to stop the reactions. The absorbance of the mixture was measured at 450 nm with a microplate reader (Bio-Tek, Inc.). Hoechst 33342 staining Apoptotic neurons were characterized by Hoechst 33342 staining (Zhao et al., 2013). In brief, the hippocampal neurons were cultured in 24-well plates coated with poly- L -lysine at a density of 5 × 10 4 cells/cm 2 . After treatments the culture media were aspirated and the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. 10 μg/ml of Hoechst 33342 was added and the reaction was incubated for 15 min at room temperature. Apoptotic cells were observed by fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The Hoechst dye was excited at 340 nm and fluorescence emission was filtered with a 510 nm barrier filter. TUNEL assay The hippocampal neurons were grown on poly-L-lysine-coated cover slips at a density of 5 × 104 cells/cm2. After treatments, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, and then the fixed cells were washed three times with PBS. Subsequently, fluorescein 12-dUTP was added for detecting fragmented DNA with nicked ends in apoptotic cells. Slides were incubated for 1 h at 37 °C, and the reaction was terminated with 2 × SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.4). Apoptotic cells were detected as localized bright red cells (positive cells) in a red background by scanning laser confocal microscopy (Leica, Heidelberg, Germany). Cell nucleus was stained with DAPI. Data were expressed as the ratio of apoptotic neurons to total neurons.
Fig. 1. Chemical structures of salidroside and MADP.
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Flow cytometric analysis Apoptotic and necrotic cells were detected with an Annexin V-FITC/PI double staining kit. At the end of treatment, the cell culture media was transfered to a new centrifuge tube. The rest anchorage-dependent cells were washed in pre-cold PBS and added into the previously mentioned tube. After centrifuged at 1000 rpm for 5 min, the supernatant was discarded. The cell pellet was re-suspended in 195 μl Annexin V-FITC binding buffer. With incubation with 5 μl Annexin V-FITC for 10 min under dark, cells were treated as the last step and was re-suspended in 190 μl Annexin V-FITC binding buffer. At the end, neurons were stained with 10 μl PI in an ice bath. Viable cells (annexin V−PI−), apoptotic cells (annexin V+PI−), necrotic cells (annexin V+PI+) were analyzed immediately by FACScan flow cytometer. Total protein extraction and western blot analysis Total cell lysate preparation and Western blotting were described elsewhere with some modifications (Jiang et al., 2013). Cells were homogenized with a dounce homogenizer in an ice-cold tissue lysis buffer (25 mM Tris–HCl, pH 7.4; 10 mM NaF; 10 mM Na4P2O7; 2 mM Na3VO4; 1 mM EGTA; 1 mM EDTA; 1% NP-40; 10 μg/ml Leupeptin; 10 μg/ml Aproptonin; 2 mM PMSF and 20 nM Okadaic acid). After homogenization, lysates were rotated for 30 min at 4 °C and then subjected to centrifugation at 13,200 rpm for 20 min at 4 °C. Total protein concentration in the supernatants was determined by a protein assay kit (Thermo Scientific). After adding Laemmli buffer, samples were boiled at 100 °C for 5 min. The cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was then probed with rabbit anti-phospho-Erk, rabbit anti-Erk, rabbit antiphospho-Akt (Ser 473), rabbit anti-phospho-p38, rabbit anti-Akt, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-Bax and rabbit anti-Bcl-2. Rabbit anti-Tubulin was used as control for gel loading and transfer. Immune complexes were detected with appropriate second antibodies and chemiluminescence reagents and exposed to Kodak exposure films. The band density was quantified by the software of Quantity One (Bio-Rad). Statistical analysis Data were expressed as means ± SD. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) and subsequent Bartlett's test. Differences were considered significant at p b 0.05. Results Effects of MADP and salidroside on cell viability in the glutamate-injured hippocampal neurons In order to test whether MADP exhibits cytotoxicity in hippocampal neurons, cell viability was assayed by MTT method. In comparison with the control group, the pretreatment of MADP or salidroside at concentrations of 120 and 240 μM does not affect cell viability (Fig. 2A). Next, we examined the protective effects of MADP and salidroside in the cultured neurons against glutamate induced apoptosis. The cell viability was decreased to 60.9% with a 15-minute exposure of 125 μM glutamate. Salidroside significantly attenuates glutamate induced cell apoptosis. The cell viability was increased to 67.1% and 79.1%, respectively, by 120 and 240 μM of salidroside (Fig. 2B). Similarly, MADP also remarkably ameliorates cell apoptosis caused by glutamate exposure. 120 and 240 μM of MADP markedly restore the cell survival rate to 75.5% and 85.2%, respectively (Fig. 2B). It is worthy to note that, compared with salidroside, MADP has stronger protective effects against glutamate induced cell apoptosis in the cultured hippocampal neurons. Moreover, we also measured LDH release for evaluating the anti-
Fig. 2. MADP or salidroside attenuates cell apoptosis induced by glutamate in the cultured hippocampal neurons. (A) Effects of MADP or salidroside on cell viability. (B) Protective effects of MADP and salidroside against glutamate-induced cell apoptosis. (C) MADP or salidroside decreases LDH release in the glutamate-injured cells. Cells without any treatments were designed as the control group. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. Δp b 0.05 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD of three independent experiments.
apoptotic effects of MADP and salidroside. As shown in Fig. 2C, glutamate exposure significantly increases LDH release to 40.6% from 18.6%. The LDH release was decreased to 28.1% and 22.6% from 40.6%, respectively, by 120 and 240 μM of MADP. Meanwhile LDH release was reduced to 34.3% and 27.5%, respectively, by 120 and 240 μM of salidroside (Fig. 2C). These data clearly indicate that, compared with salidroside, MADP exhibits stronger neuron-protective ability for rescuing hippocampal neurons from glutamate induced apoptosis. Anti-apoptotic effects of MADP and salidroside on glutamate induced cell apoptosis Hoechst staining assay Hoechst 33342 is a type of blue-fluorescence dye. The condensed chromatin of apoptotic cells was brightly stained by Hoechst 33342, while the chromatin of non-apoptotic cells was more dimly stained.
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Fig. 3. Hoechst 33342 staining for apoptotic cell analysis in the cultured hippocampal neurons. The rat hippocampal neurons were cultured and treated as mentioned in Methods and materials. (A) Hoechst 33342 staining (a: control; b: glutamate; c: 120 μM MADP + glutamate; d: 240 μM MADP + glutamate; e: 120 μM salidroside + glutamate; f: 240 μM salidroside + glutamate). (B) The percentages of nuclear condensation in the cultured hippocampal neurons. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD. Scale bar = 20 μm.
As shown in Fig. 3A, most of the nuclei in the control cells were regular round shaped, with seldom observable condensation. In contrast, glutamate exposure markedly induces cell apoptosis as evidenced by lots of nuclei that were highly condensed. The statistical data showed that the apoptotic rate amounted to 73.6% compared to the control group (Fig. 3B). The nuclei condensation was significantly reduced by pretreatment with MADP or salidroside. The cell apoptotic rate was decreased to 64.2% and 52.5% from 73.6%, respectively, by 120 and 240 μM of salidroside. MADP treatment further improves cell survival under glutamate insult. The cell apoptotic rate was decreased to 59.2% and 42.7%, respectively, by 120 and 240 μM of MADP (Fig. 3B).
TUNEL assay To further ascertain the anti-apoptotic effect of MADP, we next examined cell apoptosis by the method of TUNEL assay. TUNEL staining recognizes the damaged DNA including double-stranded and singlestranded DNA breaks. The apoptotic hippocampal neurons were evaluated with TUNEL and the nucleus was visualized with DAPI. Glutamate exposure increases cell apoptosis as evidenced by an increment of TUNEL-positive cells. Pretreatment of MADP or salidroside significantly attenuates cell apoptosis (Fig. 4A,B). In comparison with salidroside, MADP exhibits stronger anti-apoptotic effect in the glutamate-injured neurons.
Flow cytometric analysis Furthermore, flow cytometric analysis was applied to distinguish necrotic from apoptotic cell death. The results showed that 55.1% of the hippocampal neurons underwent apoptosis by glutamate exposure, and 8.2% of the cells were at necrotic stage. Glutamate-induced apoptotic and necrotic cells were significantly suppressed by either MADP or salidroside, and MADP exhibits a stronger effect (Fig. 5A,B). For instance, the percentages of apoptotic and necrotic cells were account for 30.2% and 1.7%, respectively, by the pretreatment of MADP (240 μM), whereas these values were 38.1% and 5.2% in the cells pretreated with 240 μM of salidroside. The above data further indicate that, in comparison with salidroside, MADP has stronger neuron-protective effect against glutamate-induced cell apoptosis in the cultured hippocampal neurons.
Effects of MADP and salidroside on Bcl-2 and Bax protein levels To explore the molecular mechanisms responsible for the antiapoptotic roles of MADP and salidroside in the glutamate-injured neurons, we examined Bax and Bcl-2 protein levels. Bax is a major pro-apoptotic protein whereas Bcl-2 is a major anti-apoptotic protein. As shown in Fig. 6A, no significant alterations in Bcl-2 and Bax protein levels were observed by glutamate alone or glutamate plus salidroside or MADP. These results suggest that MADP, as well as salidroside, protects cultured hippocampal neurons from glutamate induced apoptosis and may not do so through regulation of pro-apoptotic and antiapoptotic protein levels. Effects of MADP and salidroside on Akt phosphorylation Akt pathway closely correlates with cell growth ability and thus Akt is considered as a main survival signal under stress conditions. Phosphorylated Akt (p-Akt) is an active form of Akt. Glutamate insult induces a significant decline in p-Akt protein levels as compared to the control cells. However, the pretreatment with MADP or salidroside remarkably improves p-Akt levels in the glutamate-injured neurons (Fig. 6B). The stimulating effect of MADP on p-Akt is much stronger than that of salidroside. Effects of MADP and salidroside on MAPK pathway In addition to Akt, mitogen-activated protein kinase (MAPK) signaling pathway was also analyzed. MAPK family comprises c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (Erk) and p38 MAPK. Phosphorylated JNK (p-JNK) protein level was dramatically decreased upon glutamate exposure. This decrease was partially restored by MADP, while no significant alteration occurred with salidroside (Fig. 6C). No visible difference in phosphorylated Erk1/2 (p-Erk1/2) protein level was observed among groups (Fig. 6D). Phosphorylated p38 (p-p38) protein level was too weak to be obtained (data not shown). Discussion Neurodegenerative diseases such as Huntington's disease, status epilepticus, Alzheimer's dementia and olivopontocerebellar atrophy are
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Fig. 5. Detection of apoptotic cells by flow cytometry. (A) The rat hippocampal neurons were cultured and treated as indicated in Methods and materials. Flow cytometric analysis was conducted using annexin-V-FITC and PI. (B) Percentages of necrotic and apoptotic neurons. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD.
Fig. 4. TUNEL assays for cell apoptosis and DNA breaks in the cultured hippocampal neurons. (A) TUNEL assay. (B) The quantitative analysis of the ratio of TUNEL positive cells under different experimental conditions. The DAPI-labeled nuclei are blue. The TUNEL-positive cells are red. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD. Scale bar = 50 μm.
involved in the neurotoxic nature of glutamate (Sahai, 1990). It has been demonstrated that the type of cell death encountered in neuronal cultures exposed to glutamate may depend on the intensity of the exposure and may involve two temporally distinct phases — necrosis and apoptosis. Neuron damage by 125 μM glutamate for 15 min mainly leads to cell apoptosis (Chen et al., 2008). The pathogenesis of these diseases includes overproduction of cytoplasmic Ca2 + and LDH release, up-regulation of apoptosis associated genes and proteins (Ankarcrona et al., 1995). To induce cell apoptosis in the cultured hippocampal neurons, we treated the cells with glutamate to establish a cell apoptosis model. A 15 min-exposure of 125 μM glutamate causes cell apoptosis according to the results of MTT, LDH release, Hoechst 33342 staining, TUNEL assay and flow cytometric analysis. This is consistent with
the previous report in which the same glutamate insult regimen was applied to induce hippocampal neuron apoptosis (Chen et al., 2008). It has been demonstrated that salidroside possesses neuronprotective activity in several kinds of neurons (Chen et al., 2008; Yu et al., 2010; Zhang et al., 2011b). Therefore, salidroside was considered as a potential lead compound for developing novel drugs treating some neuron loss associated disorders. As a clinical candidate, the hydrophilic property of salidroside impedes its penetration across the cytoplasmic membrane and blood brain barrier. For addressing this issue, we changed one hydroxyl group in salidroside into methoxyl group to increase lipophilic activity. Furthermore, accumulating evidence indicates that glucosamine-containing compounds may possess neuron-protective action (Hwang et al., 2010; Kisilevsky and Szarek, 2002). Thus, we also replaced another hydroxyl group with acetamido group to obtain the salidroside analog used in the current study, MADP. Herein we compared the neuron-protective effects of salidroside and its analog MADP. Our data showed that, with comparison to salidroside, MADP exhibits more potent ability for alleviating cell apoptosis induced by glutamate in the hippocampal neurons. One report also showed that MADP has stronger protective function against hypoglycemia and serum limitation induced cell apoptosis in PC12 cells (Meng et al., 2011). To explore the mechanisms responsible for the different anti-apoptotic ability between MADP and salidroside, we measured some pro-apoptotic and anti-apoptotic protein expressions. Cells that undergo survival or apoptosis are determined by the level of Bcl-2 family
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Fig. 6. Effects of MADP or salidroside on cell apoptotic and survival pathways. (A) The protein levels of Bcl-2 and Bax. (B) The protein levels of phosphorylated Akt (p-Akt) and total Akt. (C) The protein levels of phosphorylated JNK (p-JNK) and total JNK. (D) The protein levels of phosphorylated Erk1/2 (p-Erk1/2) and total Erk1/2. Tubulin was used as loading control. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD.
proteins (Kroemer, 1997; Meng et al., 2011; Yang and Korsmeyer, 1996). Among them, Bcl-2 and Bcl-XL are anti-apoptotic proteins, while Bax, BclXs, Bad, Bak and Bik are pro-apoptotic proteins (Chen et al., 2008). Upon physiological and/or pathological insults, the pro-apoptotic proteins belonging to Bcl-2 family will translocate into mitochondria and trigger the release of cytochrome c and the activation of terminal caspases which cause apoptosis. On the contrary, the anti-apoptotic proteins Bcl2 and Bcl-XL inhibit caspase activation. In the present study, the protein levels of Bcl-2 and Bax were not changed by MADP or salidroside, indicating that MADP and salidroside that attenuate cell apoptosis might not through stimulation of anti-apoptotic protein expression. This result is consistent with the previous report in which salidroside was found to have no effects on the protein levels of Bcl-2 and Bax in the glutamatetreated hippocampal neurons (Chen et al., 2008). PI3K/Akt pathway activation is closely related to cell survival in various cellular systems (Chen et al., 2008; Hsu et al., 2013; Zhang et al., 2011a). Akt phosphorylation was reduced by glutamate exposure in the hippocampal neurons, indicating that cell apoptosis was initiated by glutamate. The reduction in Akt phosphorylation was also observed in cells challenged with other insults including serum limitation (Liang et al., 2013), glucose deprivation (Xu et al., 2012) and hydrogen peroxide (Lee et al., 2013). MADP or salidroside markedly improves Akt phosphorylation compared with the cells treated with glutamate
alone. The data indicate that cell death induced by glutamate was partially rescued by MADP or salidroside. It is worthy to note that the protein level of p-Akt in the MADP-treated cells was much higher than that in the salidroside-treated group. These results indicate that MADP promotes cell survival under glutamate insult possibly through PI3K/Akt activation. This speculation still needs further ascertainment and pharmacological inhibitors of PI3K/Akt will be applied to address the issue. Unlike the PI3K/Akt pathway, MAPK pathway was generally considered as a cell death sign (Karin, 1998). MAPK family contains JNK, Erk and p38 kinase, which was activated by lots of stimuli including ultra violet (UV), osmotic stress, heat shock and proinflammatory cytokines (Johnson and Lapadat, 2002). On the contrary, MAPK also links cell survival other than cell apoptosis (Liu and Lin, 2005). Accumulating evidence suggests that JNK is involved in cell survival or anti-apoptosis process. There is an enhanced apoptosis in hindbrain and forebrain regions of mice deficient with both jnk1 and jnk2 at E10.5 (Kuan et al., 1999; Liu and Lin, 2005; Sabapathy et al., 1999). These observations suggest that JNK may link cell survival in these brain regions during development. Herein, we found that JNK activity was remarkably declined in the glutamate-injured hippocampal neurons. The decline was partially restored by MADP, while no significant alteration was found by salidroside. Therefore, we preclude that JNK is probably involved in cell survival other than cell apoptosis in the current cell model. Several
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reports have shown that Erk activation plays beneficial roles against cell apoptosis induced by a variety of environmental stimuli (Kuan et al., 1999; Lee et al., 2011; Liu and Lin, 2005; Sabapathy et al., 1999; Yu et al., 2010). However, our results showed that Erk activity was not affected by glutamate, salidroside or MADP. Taken together, we investigated the neuron-protective effects of salidroside and its new analog MADP against glutamate-induced cell apoptosis in the cultured rat hippocampal neurons. The protective effect of MADP is stronger than that of salidroside, suggesting that MADP might be a better candidate for developing novel agents treating neuron loss related disorders such as Alzheimer's disease and Huntington's disease. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by Grants from the National Natural Science Foundation of China (Nos. 81371687; 21242005; 81171457; 31271260), the Natural Science Foundation of Jiangsu Province (BK2011132) and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. References Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, et al. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 1995;15:961–73. Arundine M, Tymianski M. Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci 2004;61:657–68. Atlante A, Calissano P, Bobba A, Giannattasio S, Marra E, Passarella S. Glutamate neurotoxicity, oxidative stress and mitochondria. FEBS Lett 2001;497:1–5. Chen X, Liu J, Gu X, Ding F. Salidroside attenuates glutamate-induced apoptotic cell death in primary cultured hippocampal neurons of rats. Brain Res 2008;1238:189–98. Diaz Lanza AM, Abad Martinez MJ, Fernandez Matellano L, Recuero Carretero C, Villaescusa Castillo L, Silvan Sen AM, et al. Lignan and phenylpropanoid glycosides from Phillyrea latifolia and their in vitro anti-inflammatory activity. Planta Med 2001;67:219–23. Hsu YH, Chen TH, Chen YC, Cheng CY, Sue YM, Chen JR, et al. Urotensin II exerts antiapoptotic effect on NRK-52E cells through prostacyclin-mediated peroxisome proliferator-activated receptor alpha and Akt activation. Mol Cell Endocrinol 2013; 381:168–74. Hwang SY, Shin JH, Hwang JS, Kim SY, Shin JA, Oh ES. Glucosamine exerts a neuroprotective effect via suppression of inflammation in rat brain ischemia/reperfusion injury. Glia 2010;58:1881–92. Jiang M, Wang C, Meng Q, Li F, Li K, Luo L, Sun C. Cyclosporin A attenuates weight gain and improves glucose tolerance in diet-induced obese mice. Mol Cell Endocrinol 2013; 370:96–102. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911–2. Karin M. Mitogen-activated protein kinase cascades as regulators of stress responses. Ann N Y Acad Sci 1998;851:139–46.
Kisilevsky R, Szarek WA. Novel glycosaminoglycan precursors as anti-amyloid agents part II. J Mol Neurosci 2002;19:45–50. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Ned 1997;3:614–20. Kuan CY, Yang DD, Samanta Roy DR, Davis RJ, Rakic P, Flavell RA. The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron 1999;22:667–76. Kucinskaite A, Briedis V, Savickas A. Experimental analysis of therapeutic properties of Rhodiola rosea L. and its possible application in medicine. Medicina (Kaunas) 2004; 40:614–9. Lee JY, Oh TH, Yune TY. Ghrelin inhibits hydrogen peroxide-induced apoptotic cell death of oligodendrocytes via ERK and p38MAPK signaling. Endocrinology 2011;152: 2377–86. Lee KE, Kim EY, Kim CS, Choi JS, Bae EH, Ma SK, et al. Macrophage-stimulating protein attenuates hydrogen peroxide-induced apoptosis in human renal HK-2 cells. Eur J Pharmacol 2013;715:304–11. Liang QH, Liu Y, Wu SS, Cui RR, Yuan LQ, Liao EY. Ghrelin inhibits the apoptosis of MC3T3E1 cells through ERK and AKT signaling pathway. Toxicol Appl Pharmacol 2013;272: 591–7. Liu J, Lin A. Role of JNK activation in apoptosis: a double-edged sword. Cell Res 2005;15: 36–42. Mao G-X, Deng H-B, Yuan L-G, Li D-D, Yvonne Li Y-Y, Wang Z. Protective role of salidroside against aging in a mouse model induced by D-galactose. Biomed Environ Sci 2010;23:161–6. Meng Y, Guo Y, Ling Y, Zhao Y, Zhang Q, Zhou X, et al. Synthesis and protective effects of aralkyl alcoholic 2-acetamido-2-deoxy-beta-D-pyranosides on hypoglycemia and serum limitation induced apoptosis in PC12 cell. Bioorg Med Chem 2011;19: 5577–84. Qu ZQ, Zhou Y, Zeng YS, Lin YK, Li Y, Zhong ZQ, et al. Protective effects of a Rhodiola crenulata extract and salidroside on hippocampal neurogenesis against streptozotocininduced neural injury in the rat. PLoS One 2012;7:e29641. Sabapathy K, Jochum W, Hochedlinger K, Chang L, Karin M, Wagner EF. Defective neural tube morphogenesis and altered apoptosis in the absence of both JNK1 and JNK2. Mech Dev 1999;89:115–24. Sahai S. Glutamate in the mammalian CNS. Eur Arch Psychiatry Clin Neurosci 1990; 240(2):121–33. Tan CB, Gao M, Xu WR, Yang XY, Zhu XM, Du GH. Protective effects of salidroside on endothelial cell apoptosis induced by cobalt chloride. Biol Pharm Bull 2009;32: 1359–63. Xu C, Lv L, Zheng G, Li B, Gao L, Sun Y. Neuregulin1beta1 protects oligodendrocyte progenitor cells from oxygen glucose deprivation injury induced apoptosis via ErbB4-dependent activation of PI3-kinase/Akt. Brain Res 2012;1467:104–12. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996;88:386–401. Yu S, Shen Y, Liu J, Ding F. Involvement of ERK1/2 pathway in neuroprotection by salidroside against hydrogen peroxide-induced apoptotic cell death. J Mol Neurosci 2010;40:321–31. Yuan Y, Wu SJ, Liu X, Zhang LL. Antioxidant effect of salidroside and its protective effect against furan-induced hepatocyte damage in mice. Food Funct 2013;4:763–9. Zhang Q, Shen M, Ding M, Shen D, Ding F. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway. Toxicol Appl Pharmacol 2011a;252: 62–72. Zhang S, Chen X, Yang Y, Zhou X, Liu J, Ding F. Neuroprotection against cobalt chlorideinduced cell apoptosis of primary cultured cortical neurons by salidroside. Mol Cell Biochem 2011b;354:161–70. Zhao Y, Ling Y, Zhao J, Yuan Y, Guo Y, Liu Q, Wu B, Ding Z, Yang Y. Synthesis and protective effects of novel salidroside analogues on glucose and serum depletion induced apoptosis in PC12 cells. Arch Pharm 2013;346:300–7. Zhong H, Xin H, Wu LX, Zhu YZ. Salidroside attenuates apoptosis in ischemic cardiomyocytes: a mechanism through a mitochondria-dependent pathway. J Pharmacol Sci 2010;114:399–408.
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MADP, a salidroside analog, protects hippocampal neurons from glutamate induced apoptosis Hua Xian a,1, Jing Zhao b,1, Yuan Zheng b, Meihong Wang b, Jun Huang c, Bingxin Wu c, Cheng Sun b,⁎, Yumin Yang b,⁎ a b c
Department of Pediatric Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, PR China Jiangsu Key Laboratory of Neuroregeneration, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China School of Medicine, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, PR China
a r t i c l e
i n f o
Article history: Received 23 November 2013 Accepted 28 February 2014 Available online 11 March 2014 Keywords: Salidroside analog Hippocampal neurons Apoptosis Glutamate excitotoxicity Neurodegeneration
a b s t r a c t Aims: To investigate the anti-apoptotic effect of MADP, an analog of salidroside, against glutamate induced apoptosis in the cultured rat hippocampal neurons. Main methods: Cytotoxicity was determined by the MTT method and lactate dehydrogenase release to the medium. Cell apoptosis was evaluated by Hoechst 33342 staining, TUNEL assay and flow cytometric analysis. Western blotting was applied for detecting protein levels of cellular signaling molecules. Key findings: Our results showed that glutamate exposure significantly induces cell apoptosis, whereas the pretreatment of salidroside or MADP remarkably improves cell viability. Most importantly, the anti-apoptotic effect of MADP against glutamate insult is superior to salidroside. To explore the involved mechanisms, we measured some pro-apoptotic and anti-apoptotic protein levels, and several cell survival signaling pathways were analyzed as well. No visible alterations in Bcl-2 and Bax protein levels were observed by MADP or salidroside. Akt and JNK phosphorylation was robustly stimulated by MADP in the glutamate-treated neurons. Salidroside treatment results in a slight activation in Akt, while no significant alteration in JNK activity was observed. Significance: MADP exhibits higher capacity to attenuate glutamate induced cell apoptosis in the cultured rat hippocampal neurons, suggesting that MADP might be a better candidate than salidroside for developing novel drugs treating neuron loss associated disorders. © 2014 Elsevier Inc. All rights reserved.
Introduction Neurodegenerative diseases such as motor neuron disease and Alzheimer's disease affect 35 million people worldwide. They are chronic and progressive neurodegenerative illnesses characterized by memory deficits and cognitive decline owing to synaptic and neuronal loss in the hippocampus and cerebral cortex. Neurotoxicity plays a key role for the pathological progress of neurodegenerative diseases. Glutamate is an important neurotransmitter and a growing number of evidence shows that glutamate has been implicated in neurotoxicity (Arundine and Tymianski, 2004). Excessive glutamate will disturb the homeostasis of calcium in the cytoplasm, lead to mitochondrial membrane depolarization and eventually result in neuronal death (Atlante et al., 2001). Chemicals with ameliorating activities on glutamate excitotoxicity are receiving more and more attention since these ⁎ Corresponding authors. Tel./fax: +86 513 85511585. E-mail addresses: [email protected] (C. Sun), [email protected] (Y. Yang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.02.040 0024-3205/© 2014 Elsevier Inc. All rights reserved.
chemicals might be used as potential clinical drugs for treating neurodegenerative disorders. Salidroside, one of the most potent compounds in Rhodiola rosea L., has been shown to have multiple biological activities including antiaging (Mao et al., 2010), anti-oxidative stress (Yuan et al., 2013), antiinflammation (Diaz Lanza et al., 2001) and anti-cancer (Kucinskaite et al., 2004). Recently, it has been shown that salidroside exhibits potent anti-apoptotic effects in the various types of cells including neurons (Qu et al., 2012), cardiomyocytes (Zhong et al., 2010) and endothelia (Tan et al., 2009). Recently more and more salidroside was consumed especially in the Traditional Chinese Medicine. Extraction from Rhodiola plants is the main source of salidroside currently, although the content of salidroside in Rhodiola plants is quite low. Additionally, the hydrophilic property of salidroside affects its efficacity since hydrophilic molecules are hard to penetrate across the cytoplasmic membrane and blood brain barrier. All these limitations blockade the deep research and development of salidroside. For addressing these issues, we synthesized salidroside and its analogs and evaluated their anti-apoptotic activities in the PC12 cells (Meng et al., 2011). Of these, MADP possesses
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higher protective activity against apoptosis induced by hypoglycemia and serum limitation with comparison to salidroside. Salidroside has been shown to have a neuron-protective role in cultured rat hippocampal neurons (Chen et al., 2008). We speculate that, therefore, MADP also may have a stronger capacity against apoptosis induced by glutamate in hippocampal neurons. To test this speculation, we pretreated rat hippocampal neurons with MADP or salidroside and evaluated their anti-apoptotic effects against glutamate insult. The involved mechanisms responsible for the antiapoptotic effects of MADP and salidroside were also investigated.
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Cytotoxicity assay Hippocampal neurons were cultured in 96-well plates at a density of 5 × 105 cells per well. After treatment as aforementioned, the medium was discarded, and then 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was added and incubated at 37 °C for 4 h. Formazan crystals were dissolved in 100 μl of 20% SDS. The absorbance was measured at 570 nm by a microplate reader (Bio-Tek, Inc.). Lactate dehydrogenase (LDH) assay
Material and methods Reagents Salidroside (Fig. 1) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). MADP (Fig. 1) was synthesized as described previously (Meng et al., 2011). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), trypsin, poly-L-lysine, Neurobasal medium, B27, and Annexin V-FITC/PI kit were purchased from Invitrogen (Carlsbad, CA, USA). MTT, Hoechst 33342 and glutamate were purchased from Sigma-Aldrich (St. Louis, MO, USA). LDH test kit was purchased from Jiancheng (Nanjing, China). TUNEL cell death detection kit was from Roche (Mannheim, Germany). Rabbit anti-phospho-Erk, rabbit antiErk, rabbit anti-phospho-Akt (Ser 473), rabbit anti-phospho-p38, rabbit anti-Akt, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-Bax, rabbit anti-Bcl-2 and rabbit anti-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary hippocampal cell culture and treatments Hippocampal cell cultures were prepared from hippocampi of Sprague–Dawley rat embryos (E18–E19) (Chen et al., 2008). Briefly, the hippocampi were dissected under anatomy microscope and collected in Hank's balanced salt solution containing 137 mM NaCl, 5.36 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 4.16 mM NaHCO3, 5 mM glucose, 1 mM sodium pyruvate, and 10 mM HEPES (pH 7.4). Tissues were then digested in 0.125% trypsin solution for 5 min at 37 °C. The digestion was terminated by adding DMEM supplemented with 10% FBS and 100 U/ml penicillin and 100 U/ml streptomycin. The digestion mixture was centrifuged at 1000 rpm for 4 min at 4 °C and the supernatant was discarded. The resulting pellet was re-suspended in DMEM and filtered with a 200 screen mesh. The filtrate containing primary hippocampal neurons were counted and seeded into cell culture plates coated with poly-L-lysine. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 for 4 h at 37 °C. Finally, the medium was changed with serum-free neurobasal medium supplemented with 2% B27, 2 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin. The medium was replaced with fresh medium every 3–4 days. The cultured 8 day-old hippocampal neurons were pretreated with MADP or salidroside at the concentrations of 120 and 240 μM for 24 h, and then the neurons were exposure to 125 μM glutamate for 15 min. After excitotoxicity was induced, the cells were further incubated with fresh medium for 24 h.
The LDH activity was determined with a LDH assay kit according to the manufacturer's instructions. Briefly, hippocampal neurons were cultured in 24-well plates at a density of 5 × 104 cells/cm2. After treatments as mentioned above, 20 μl of cell culture supernatant was transfered to a new 96-well plate, and then 25 μl of substrate buffer was added. The mixture was incubated for 15 min at 37 °C. Blank and standard wells were carried out at the same time. In standard wells, 5 μl double distilled water, 20 μl 0.2 mM standard solution and 25 μl substrate buffer were added. In blank wells, only 25 μl double distilled water and 25 μl substrate buffer were added. Subsequently, 25 μl of 2, 4-dinitrobenzene hydrazine was added and incubated for 15 min at 37 °C. 250 μl of 0.4 M NaOH was added to stop the reactions. The absorbance of the mixture was measured at 450 nm with a microplate reader (Bio-Tek, Inc.). Hoechst 33342 staining Apoptotic neurons were characterized by Hoechst 33342 staining (Zhao et al., 2013). In brief, the hippocampal neurons were cultured in 24-well plates coated with poly- L -lysine at a density of 5 × 10 4 cells/cm 2 . After treatments the culture media were aspirated and the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. 10 μg/ml of Hoechst 33342 was added and the reaction was incubated for 15 min at room temperature. Apoptotic cells were observed by fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The Hoechst dye was excited at 340 nm and fluorescence emission was filtered with a 510 nm barrier filter. TUNEL assay The hippocampal neurons were grown on poly-L-lysine-coated cover slips at a density of 5 × 104 cells/cm2. After treatments, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, and then the fixed cells were washed three times with PBS. Subsequently, fluorescein 12-dUTP was added for detecting fragmented DNA with nicked ends in apoptotic cells. Slides were incubated for 1 h at 37 °C, and the reaction was terminated with 2 × SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.4). Apoptotic cells were detected as localized bright red cells (positive cells) in a red background by scanning laser confocal microscopy (Leica, Heidelberg, Germany). Cell nucleus was stained with DAPI. Data were expressed as the ratio of apoptotic neurons to total neurons.
Fig. 1. Chemical structures of salidroside and MADP.
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Flow cytometric analysis Apoptotic and necrotic cells were detected with an Annexin V-FITC/PI double staining kit. At the end of treatment, the cell culture media was transfered to a new centrifuge tube. The rest anchorage-dependent cells were washed in pre-cold PBS and added into the previously mentioned tube. After centrifuged at 1000 rpm for 5 min, the supernatant was discarded. The cell pellet was re-suspended in 195 μl Annexin V-FITC binding buffer. With incubation with 5 μl Annexin V-FITC for 10 min under dark, cells were treated as the last step and was re-suspended in 190 μl Annexin V-FITC binding buffer. At the end, neurons were stained with 10 μl PI in an ice bath. Viable cells (annexin V−PI−), apoptotic cells (annexin V+PI−), necrotic cells (annexin V+PI+) were analyzed immediately by FACScan flow cytometer. Total protein extraction and western blot analysis Total cell lysate preparation and Western blotting were described elsewhere with some modifications (Jiang et al., 2013). Cells were homogenized with a dounce homogenizer in an ice-cold tissue lysis buffer (25 mM Tris–HCl, pH 7.4; 10 mM NaF; 10 mM Na4P2O7; 2 mM Na3VO4; 1 mM EGTA; 1 mM EDTA; 1% NP-40; 10 μg/ml Leupeptin; 10 μg/ml Aproptonin; 2 mM PMSF and 20 nM Okadaic acid). After homogenization, lysates were rotated for 30 min at 4 °C and then subjected to centrifugation at 13,200 rpm for 20 min at 4 °C. Total protein concentration in the supernatants was determined by a protein assay kit (Thermo Scientific). After adding Laemmli buffer, samples were boiled at 100 °C for 5 min. The cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was then probed with rabbit anti-phospho-Erk, rabbit anti-Erk, rabbit antiphospho-Akt (Ser 473), rabbit anti-phospho-p38, rabbit anti-Akt, rabbit anti-phospho-JNK, rabbit anti-JNK, rabbit anti-Bax and rabbit anti-Bcl-2. Rabbit anti-Tubulin was used as control for gel loading and transfer. Immune complexes were detected with appropriate second antibodies and chemiluminescence reagents and exposed to Kodak exposure films. The band density was quantified by the software of Quantity One (Bio-Rad). Statistical analysis Data were expressed as means ± SD. Statistical differences between groups were analyzed by one-way analysis of variance (ANOVA) and subsequent Bartlett's test. Differences were considered significant at p b 0.05. Results Effects of MADP and salidroside on cell viability in the glutamate-injured hippocampal neurons In order to test whether MADP exhibits cytotoxicity in hippocampal neurons, cell viability was assayed by MTT method. In comparison with the control group, the pretreatment of MADP or salidroside at concentrations of 120 and 240 μM does not affect cell viability (Fig. 2A). Next, we examined the protective effects of MADP and salidroside in the cultured neurons against glutamate induced apoptosis. The cell viability was decreased to 60.9% with a 15-minute exposure of 125 μM glutamate. Salidroside significantly attenuates glutamate induced cell apoptosis. The cell viability was increased to 67.1% and 79.1%, respectively, by 120 and 240 μM of salidroside (Fig. 2B). Similarly, MADP also remarkably ameliorates cell apoptosis caused by glutamate exposure. 120 and 240 μM of MADP markedly restore the cell survival rate to 75.5% and 85.2%, respectively (Fig. 2B). It is worthy to note that, compared with salidroside, MADP has stronger protective effects against glutamate induced cell apoptosis in the cultured hippocampal neurons. Moreover, we also measured LDH release for evaluating the anti-
Fig. 2. MADP or salidroside attenuates cell apoptosis induced by glutamate in the cultured hippocampal neurons. (A) Effects of MADP or salidroside on cell viability. (B) Protective effects of MADP and salidroside against glutamate-induced cell apoptosis. (C) MADP or salidroside decreases LDH release in the glutamate-injured cells. Cells without any treatments were designed as the control group. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. Δp b 0.05 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD of three independent experiments.
apoptotic effects of MADP and salidroside. As shown in Fig. 2C, glutamate exposure significantly increases LDH release to 40.6% from 18.6%. The LDH release was decreased to 28.1% and 22.6% from 40.6%, respectively, by 120 and 240 μM of MADP. Meanwhile LDH release was reduced to 34.3% and 27.5%, respectively, by 120 and 240 μM of salidroside (Fig. 2C). These data clearly indicate that, compared with salidroside, MADP exhibits stronger neuron-protective ability for rescuing hippocampal neurons from glutamate induced apoptosis. Anti-apoptotic effects of MADP and salidroside on glutamate induced cell apoptosis Hoechst staining assay Hoechst 33342 is a type of blue-fluorescence dye. The condensed chromatin of apoptotic cells was brightly stained by Hoechst 33342, while the chromatin of non-apoptotic cells was more dimly stained.
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Fig. 3. Hoechst 33342 staining for apoptotic cell analysis in the cultured hippocampal neurons. The rat hippocampal neurons were cultured and treated as mentioned in Methods and materials. (A) Hoechst 33342 staining (a: control; b: glutamate; c: 120 μM MADP + glutamate; d: 240 μM MADP + glutamate; e: 120 μM salidroside + glutamate; f: 240 μM salidroside + glutamate). (B) The percentages of nuclear condensation in the cultured hippocampal neurons. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD. Scale bar = 20 μm.
As shown in Fig. 3A, most of the nuclei in the control cells were regular round shaped, with seldom observable condensation. In contrast, glutamate exposure markedly induces cell apoptosis as evidenced by lots of nuclei that were highly condensed. The statistical data showed that the apoptotic rate amounted to 73.6% compared to the control group (Fig. 3B). The nuclei condensation was significantly reduced by pretreatment with MADP or salidroside. The cell apoptotic rate was decreased to 64.2% and 52.5% from 73.6%, respectively, by 120 and 240 μM of salidroside. MADP treatment further improves cell survival under glutamate insult. The cell apoptotic rate was decreased to 59.2% and 42.7%, respectively, by 120 and 240 μM of MADP (Fig. 3B).
TUNEL assay To further ascertain the anti-apoptotic effect of MADP, we next examined cell apoptosis by the method of TUNEL assay. TUNEL staining recognizes the damaged DNA including double-stranded and singlestranded DNA breaks. The apoptotic hippocampal neurons were evaluated with TUNEL and the nucleus was visualized with DAPI. Glutamate exposure increases cell apoptosis as evidenced by an increment of TUNEL-positive cells. Pretreatment of MADP or salidroside significantly attenuates cell apoptosis (Fig. 4A,B). In comparison with salidroside, MADP exhibits stronger anti-apoptotic effect in the glutamate-injured neurons.
Flow cytometric analysis Furthermore, flow cytometric analysis was applied to distinguish necrotic from apoptotic cell death. The results showed that 55.1% of the hippocampal neurons underwent apoptosis by glutamate exposure, and 8.2% of the cells were at necrotic stage. Glutamate-induced apoptotic and necrotic cells were significantly suppressed by either MADP or salidroside, and MADP exhibits a stronger effect (Fig. 5A,B). For instance, the percentages of apoptotic and necrotic cells were account for 30.2% and 1.7%, respectively, by the pretreatment of MADP (240 μM), whereas these values were 38.1% and 5.2% in the cells pretreated with 240 μM of salidroside. The above data further indicate that, in comparison with salidroside, MADP has stronger neuron-protective effect against glutamate-induced cell apoptosis in the cultured hippocampal neurons.
Effects of MADP and salidroside on Bcl-2 and Bax protein levels To explore the molecular mechanisms responsible for the antiapoptotic roles of MADP and salidroside in the glutamate-injured neurons, we examined Bax and Bcl-2 protein levels. Bax is a major pro-apoptotic protein whereas Bcl-2 is a major anti-apoptotic protein. As shown in Fig. 6A, no significant alterations in Bcl-2 and Bax protein levels were observed by glutamate alone or glutamate plus salidroside or MADP. These results suggest that MADP, as well as salidroside, protects cultured hippocampal neurons from glutamate induced apoptosis and may not do so through regulation of pro-apoptotic and antiapoptotic protein levels. Effects of MADP and salidroside on Akt phosphorylation Akt pathway closely correlates with cell growth ability and thus Akt is considered as a main survival signal under stress conditions. Phosphorylated Akt (p-Akt) is an active form of Akt. Glutamate insult induces a significant decline in p-Akt protein levels as compared to the control cells. However, the pretreatment with MADP or salidroside remarkably improves p-Akt levels in the glutamate-injured neurons (Fig. 6B). The stimulating effect of MADP on p-Akt is much stronger than that of salidroside. Effects of MADP and salidroside on MAPK pathway In addition to Akt, mitogen-activated protein kinase (MAPK) signaling pathway was also analyzed. MAPK family comprises c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (Erk) and p38 MAPK. Phosphorylated JNK (p-JNK) protein level was dramatically decreased upon glutamate exposure. This decrease was partially restored by MADP, while no significant alteration occurred with salidroside (Fig. 6C). No visible difference in phosphorylated Erk1/2 (p-Erk1/2) protein level was observed among groups (Fig. 6D). Phosphorylated p38 (p-p38) protein level was too weak to be obtained (data not shown). Discussion Neurodegenerative diseases such as Huntington's disease, status epilepticus, Alzheimer's dementia and olivopontocerebellar atrophy are
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Fig. 5. Detection of apoptotic cells by flow cytometry. (A) The rat hippocampal neurons were cultured and treated as indicated in Methods and materials. Flow cytometric analysis was conducted using annexin-V-FITC and PI. (B) Percentages of necrotic and apoptotic neurons. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD.
Fig. 4. TUNEL assays for cell apoptosis and DNA breaks in the cultured hippocampal neurons. (A) TUNEL assay. (B) The quantitative analysis of the ratio of TUNEL positive cells under different experimental conditions. The DAPI-labeled nuclei are blue. The TUNEL-positive cells are red. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD. Scale bar = 50 μm.
involved in the neurotoxic nature of glutamate (Sahai, 1990). It has been demonstrated that the type of cell death encountered in neuronal cultures exposed to glutamate may depend on the intensity of the exposure and may involve two temporally distinct phases — necrosis and apoptosis. Neuron damage by 125 μM glutamate for 15 min mainly leads to cell apoptosis (Chen et al., 2008). The pathogenesis of these diseases includes overproduction of cytoplasmic Ca2 + and LDH release, up-regulation of apoptosis associated genes and proteins (Ankarcrona et al., 1995). To induce cell apoptosis in the cultured hippocampal neurons, we treated the cells with glutamate to establish a cell apoptosis model. A 15 min-exposure of 125 μM glutamate causes cell apoptosis according to the results of MTT, LDH release, Hoechst 33342 staining, TUNEL assay and flow cytometric analysis. This is consistent with
the previous report in which the same glutamate insult regimen was applied to induce hippocampal neuron apoptosis (Chen et al., 2008). It has been demonstrated that salidroside possesses neuronprotective activity in several kinds of neurons (Chen et al., 2008; Yu et al., 2010; Zhang et al., 2011b). Therefore, salidroside was considered as a potential lead compound for developing novel drugs treating some neuron loss associated disorders. As a clinical candidate, the hydrophilic property of salidroside impedes its penetration across the cytoplasmic membrane and blood brain barrier. For addressing this issue, we changed one hydroxyl group in salidroside into methoxyl group to increase lipophilic activity. Furthermore, accumulating evidence indicates that glucosamine-containing compounds may possess neuron-protective action (Hwang et al., 2010; Kisilevsky and Szarek, 2002). Thus, we also replaced another hydroxyl group with acetamido group to obtain the salidroside analog used in the current study, MADP. Herein we compared the neuron-protective effects of salidroside and its analog MADP. Our data showed that, with comparison to salidroside, MADP exhibits more potent ability for alleviating cell apoptosis induced by glutamate in the hippocampal neurons. One report also showed that MADP has stronger protective function against hypoglycemia and serum limitation induced cell apoptosis in PC12 cells (Meng et al., 2011). To explore the mechanisms responsible for the different anti-apoptotic ability between MADP and salidroside, we measured some pro-apoptotic and anti-apoptotic protein expressions. Cells that undergo survival or apoptosis are determined by the level of Bcl-2 family
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Fig. 6. Effects of MADP or salidroside on cell apoptotic and survival pathways. (A) The protein levels of Bcl-2 and Bax. (B) The protein levels of phosphorylated Akt (p-Akt) and total Akt. (C) The protein levels of phosphorylated JNK (p-JNK) and total JNK. (D) The protein levels of phosphorylated Erk1/2 (p-Erk1/2) and total Erk1/2. Tubulin was used as loading control. *p b 0.05, **p b 0.01 vs only glutamate treated neurons. #p b 0.05, ##p b 0.01 vs glutamate plus salidroside (120 μM) treated neurons. ΔΔp b 0.01 vs glutamate plus salidroside (240 μM) treated neurons. The data were expressed as mean ± SD.
proteins (Kroemer, 1997; Meng et al., 2011; Yang and Korsmeyer, 1996). Among them, Bcl-2 and Bcl-XL are anti-apoptotic proteins, while Bax, BclXs, Bad, Bak and Bik are pro-apoptotic proteins (Chen et al., 2008). Upon physiological and/or pathological insults, the pro-apoptotic proteins belonging to Bcl-2 family will translocate into mitochondria and trigger the release of cytochrome c and the activation of terminal caspases which cause apoptosis. On the contrary, the anti-apoptotic proteins Bcl2 and Bcl-XL inhibit caspase activation. In the present study, the protein levels of Bcl-2 and Bax were not changed by MADP or salidroside, indicating that MADP and salidroside that attenuate cell apoptosis might not through stimulation of anti-apoptotic protein expression. This result is consistent with the previous report in which salidroside was found to have no effects on the protein levels of Bcl-2 and Bax in the glutamatetreated hippocampal neurons (Chen et al., 2008). PI3K/Akt pathway activation is closely related to cell survival in various cellular systems (Chen et al., 2008; Hsu et al., 2013; Zhang et al., 2011a). Akt phosphorylation was reduced by glutamate exposure in the hippocampal neurons, indicating that cell apoptosis was initiated by glutamate. The reduction in Akt phosphorylation was also observed in cells challenged with other insults including serum limitation (Liang et al., 2013), glucose deprivation (Xu et al., 2012) and hydrogen peroxide (Lee et al., 2013). MADP or salidroside markedly improves Akt phosphorylation compared with the cells treated with glutamate
alone. The data indicate that cell death induced by glutamate was partially rescued by MADP or salidroside. It is worthy to note that the protein level of p-Akt in the MADP-treated cells was much higher than that in the salidroside-treated group. These results indicate that MADP promotes cell survival under glutamate insult possibly through PI3K/Akt activation. This speculation still needs further ascertainment and pharmacological inhibitors of PI3K/Akt will be applied to address the issue. Unlike the PI3K/Akt pathway, MAPK pathway was generally considered as a cell death sign (Karin, 1998). MAPK family contains JNK, Erk and p38 kinase, which was activated by lots of stimuli including ultra violet (UV), osmotic stress, heat shock and proinflammatory cytokines (Johnson and Lapadat, 2002). On the contrary, MAPK also links cell survival other than cell apoptosis (Liu and Lin, 2005). Accumulating evidence suggests that JNK is involved in cell survival or anti-apoptosis process. There is an enhanced apoptosis in hindbrain and forebrain regions of mice deficient with both jnk1 and jnk2 at E10.5 (Kuan et al., 1999; Liu and Lin, 2005; Sabapathy et al., 1999). These observations suggest that JNK may link cell survival in these brain regions during development. Herein, we found that JNK activity was remarkably declined in the glutamate-injured hippocampal neurons. The decline was partially restored by MADP, while no significant alteration was found by salidroside. Therefore, we preclude that JNK is probably involved in cell survival other than cell apoptosis in the current cell model. Several
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reports have shown that Erk activation plays beneficial roles against cell apoptosis induced by a variety of environmental stimuli (Kuan et al., 1999; Lee et al., 2011; Liu and Lin, 2005; Sabapathy et al., 1999; Yu et al., 2010). However, our results showed that Erk activity was not affected by glutamate, salidroside or MADP. Taken together, we investigated the neuron-protective effects of salidroside and its new analog MADP against glutamate-induced cell apoptosis in the cultured rat hippocampal neurons. The protective effect of MADP is stronger than that of salidroside, suggesting that MADP might be a better candidate for developing novel agents treating neuron loss related disorders such as Alzheimer's disease and Huntington's disease. Conflict of interest statement The authors declare that there are no conflicts of interest.
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