Transient exposure to echinacoside is sufficient to activate Trk signaling and protect neuronal cells from rotenone

Authors

  • Min Zhu,

    1. Laboratory of Neurology of Integrative Medical Institute, Zhongshan Hospital, Fudan University, Shanghai, P. R. China
    2. Institute of Neurology and Department of Neurology, Huashan Hospital, Fudan University, Shanghai, P. R. China
    3. The State Key Laboratory of Neurobiology, Shanghai Medical College, Fudan University, Shanghai, P. R. China
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  • Chuanzhen Lu,

    1. Institute of Neurology and Department of Neurology, Huashan Hospital, Fudan University, Shanghai, P. R. China
    2. The State Key Laboratory of Neurobiology, Shanghai Medical College, Fudan University, Shanghai, P. R. China
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  • Wenwei Li

    Corresponding author
    1. Institute of Neurology and Department of Neurology, Huashan Hospital, Fudan University, Shanghai, P. R. China
    2. Institutes of Brain Science, Fudan University, Shanghai, P. R. China
    • Laboratory of Neurology of Integrative Medical Institute, Zhongshan Hospital, Fudan University, Shanghai, P. R. China
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Addresss correspondence and reprint requests to Wenwei Li, Integrative medicine Department, Zhongshan Hospital, No.136, Yixueyuan Road, Shanghai, 200032, People's Republic of China.

E-mails: wenweili2000@yahoo.com.cn; 041104162@fudan.edu.cn

Abstract

Neurotrophins exert their physiological functions mainly through Trk receptors, and the neurotrophic signaling network is critical to the survival of neurons. However, therapeutic use of neurotrophins in treating neurodegenerative diseases is hampered by a number of pharmacological challenges, and the most significant challenge is their delivery into the central nervous system. Here, we reported that echinacoside, a small natural compound, elicits neuroprotection by activating Trk receptors and their downstream signal pathways. Echinacoside is the major active component of Cistanches Herba, a widely used Chinese herb with neuroprotective effects. We showed in this study that transient exposure to echinacoside is sufficient to protect neuronal cells and non-neuronal cells over-expressed with TrkA or TrkB against rotenone injury. Additional investigations on the mechanisms underlying suggested that transient treatment with echinacoside inhibits cytochrome c release and caspase-3 activation caused by ensuing rotenone exposure via activating Trk-extracellular signal-regulated kinase (ERK) pathway in neuronal cells. As echinacoside is able to cross the blood–brain barrier freely, it may have a promising potential in neurodegenerative diseases treatment.

Abbreviations used
BDNF

brain-derived neurotrophic factor

HEK

human embryonic kidney

COX IV

cytochrome c oxidase subunit IV

cyto c

cytochrome c

DMEM

Dulbecco's modified eagle's medium

FBS

fetal bovine serum

H2DCFDA

2,7-dichlorofluorescin diacetate

LDH

lactate dehydrogenase

MAPKs

mitogen-activated protein kinases

MFI

mean relative fluorescent density

NGF

nerve growth factor

NT-3

neurotrophin 3

NT-4/5

neurotrophin 4/5

pAkt

phosphorylated Akt

pERK

phosphorylated extracellular signal-regulated kinase

pJNK

phosphorylated c-Jun N-terminal protein kinase

pp38

phosphorylated p38 MAPK

ROS

reactive oxygen species

SDS

sodium dodecyl sulfate

The neurotrophin family is composed of four proteins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5). They play a key role in neuronal development, differentiation, and survival, through binding and activating Trk tyrosine kinase receptors (TrkA, TrkB, and TrkC) and p75NTR. NGF selectively binds to TrkA, BDNF and NT-4/5 primarily attach to TrkB, and NT-3 mainly interacts with TrkC. Neurotrophins interaction with Trk receptors provokes receptor homodimerization, autophosphorylation at cytoplasmic tyrosine residues, and downstream signal pathways, such as Ras/Raf/MAPK and PI3K/Akt. Those signals result in a variety of biological events, including neuronal survival and differentiation, cytoskeletal changes, and axon elongation (Kaplan and Stephens 1994; Chao 2003).

Among all neurotrophins, NGF and BDNF have been studied the most for the neuroprotection function, and showed great therapeutic potential in treating neurodegenerative diseases. NGF has been reported to improve the survival of cholinergic neurons and reduce cognitive decline in humans with mild Alzheimer's disease (Mufson et al. 2008). BDNF has been proposed to protect mesencephalic dopaminergic neurons in rats and monkeys of PD models (Frim et al. 1994; Pearce et al. 1999) and rescue the majority of striatal projection neurons in HD models (Perez-Navarro et al. 2000). However, the most significant challenge in the potential clinical use of neurotrophins for neurodegenerative diseases treatment is their delivery into the central nervous system. They have low oral bioavailability and poor blood–brain barrier permeability, thus could not enter the central nervous system following systemic administration (Saragovi and Gehring 2000). Therefore, tremendous efforts have been made to discover or synthesize small, proteolytically stable compounds with neurotrophic function.

Echinacoside (Fig. 1a) is a small natural compound of the highest concentration in the phenylethanoid glycosides isolated from Cistanches Herba, which is used as a traditional Chinese herbal medicine with neuroprotective effects. Increasing studies reported that echinacoside also elicits neuroprotection in vivo and in vitro, and it has protection on other kinds of cells, as well. Echinacoside shows significant neuroprotective effects on the mouse model of Parkinson's disease (Geng et al. 2007; Zhao et al. 2010) and rescues PC12 cells from H2O2-induced apoptosis (Kuang et al. 2009); it also protects against hepatotoxicity (Wu et al. 2007) and lung injury in rats (Zhang et al. 2007). However, the mechanisms underlying echinacoside neuroprotection are still unclear. As echinacoside contains caffeoyl and hydroxyphenylethyl moieties, both well known as antioxidants, its antioxidative activity spurred enormous interest. In this study, we showed that transient exposure to echinacoside selectively protects neuronal cells against rotenone injury by activating TrkA/TrkB receptors and their downstream signaling events. Hence, echinacoside may also initiate neuroprotection by mimicking neurotrophic functions.

Figure 1.

The chemical structure of echinacoside (a) and a schematic representation of echinacoside sustained treatment during rotenone exposure (b) and echinacoside transient exposure before rotenone treatment (c).

Materials and methods

Cell line culture

SH-SY5Y, Hela and human embryonic kidney 293 T (HEK293T) cells were obtained from American Type Culture Collection and used within 20 passages of the original vial. SH-SY5Y cells were grown in Dulbecco's modified eagle's medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 0.1 mg/mL streptomycin, and 3.7 g/L NaHCO3. Hela and HEK293T cells were grown in DMEM medium supplemented with 10% FBS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, and 3.7 g/L NaHCO3. Cell cultures were all kept at 37°C in a saturated humidity air atmosphere containing 5% CO2. At 80% confluence, growth medium was changed to 1% FBS containing medium for 2 h before addition of test agents.

Primary culture of rat cortical neurons

Primary rat cortical neuron cultures were prepared from embryonic days 17 Sprague-Dawley rat fetuses. Animal experiment protocols conformed to the Animal Care and Use Committee of Fudan University and all efforts were made to minimize the number of animals used and their suffering. Briefly, after pregnant rats (embryonic days 17; Shanghai Experimental Animal Center, Chinese Academy of Science, Shanghai, China) were killed, the embryonic brain cortex was carefully removed. A cell suspension was prepared by repeated passage through a pipette and filtration through an 80-μm nylon mesh. The cells were resuspended in Neurobasal medium (Gibco, Rockville, MD, USA), supplemented with B-27, glutamine (0.5 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL), and plated onto poly-l-lysine (100 μg/mL, Sigma, St. Louis, MO, USA)-pre-coated dishes at a density of 106 cells/mL. The culture dishes were kept at 37°C in 5% CO2 and 95% air in a humidified incubator for 8 days in vitro before drug treatments.

Chemicals

Rotenone was applied to inhibit mitochondrial respiratory chain and induce cell injury. K252a (1 μM) is a specific Trk inhibitor (Ohama et al. 2003). U0126 (5 μM) is a selective phosphorylated ERK blocker (Gao et al. 2003). All the drugs above were purchased from Sigma. Echinacoside was purchased from Chinese National Institutes for Food and Drug Control.

Hydrogen peroxide detection

H2O2 production was determined with the Amplex Red hydrogen peroxide assay kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, after drug treatments, 20 000 cells from each group suspended in 20 μL Kreds-Ringer phosphate buffer were added to 100 μL of the reaction mixture [containing 50 μM Amplex Red and 0.1 U/mL of horseradish peroxidase (HRP)], and then incubated for 30 min at 25°C. Fluorescence was detected at 590 nm using excitation at 530 nm.

Lactate dehydrogenase (LDH) assay

Cell culture medium was collected during drug treatments. LDH activity in the culture medium was measured with an LDH kit (Sigma) and a DU-640 spectrophotometer system (Beckman Instruments, Fullerton, CA, USA). All experiments were performed in triplicate. Data were then normalized to control levels.

Transfection

Full length human TrkA/B cDNA was subcloned into pcDNA3.0 expression vector. Plasmid DNA was first diluted in OPTI-MEM I (Gibco) and then mixed with the FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). The DNA-FuGENE HD Transfection Reagent mixture was added to Hela and HEK293T cells in the antibiotic-free transfection medium (DMEM containing glutamine) after a 20-min incubation period at 25°C. The medium was replaced with normal culture medium 6 h later. Cells were used for drug treatments 48 h after transfection.

Cell fractionation and isolation of cytosol

Cytosolic fraction was isolated from cells as described by Yang and colleagues (Yang et al. 1997). Briefly, cells were harvested after drug treatments, and resuspended with 5 volumes of isolation buffer (0.25 M sucrose, 20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreithol, 0.1 mM phenylmethanesulfonyl fluoride). The cellular suspension was homogenized with 15–20 strokes of a Teflon-glass homogenizer, and centrifuged at 750 g for 10 min. The pellet was resuspended in isolation buffer and recentrifuged. The supernatants obtained from the two low-speed spins were pooled and centrifuged at 15 000 g for 20 min at 4°C, with the resultant supernatant being used as the cytosolic fraction.

Protein preparation

The cells were collected after drug treatments, and treated with radio-immunoprecipitation assay lysis buffer [150 mM sodium chloride, 1% NP-40, 50 mM Tris, pH 8.0, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing 1 mM EDTA, 1 mM dithiothreithol, 0.1 mM phenylmethanesulfonyl fluoride, and protease inhibitors (Roche, EDTA free). The samples were centrifuged at 12 000 g for 20 min at 4°C to remove unsolvable substances. The protein concentration of total cells and cytosolic fraction was determined by a Bio-Rad protein assay (Hercules, CA, USA).

SDS-PAGE and immunoblotting

Protein samples (10–30 μg) were electrophoresed on 8–12% sodium dodecyl sulfate-polyacrylamide gel, and transferred to the polyvinyldifluoridine membrane (Bio-Rad). Membranes were blocked with 5% skim milk in TBS-0.1% Tween 20 and subsequently incubated overnight at 4°C with sheep antibody against cytochrome c (1 : 1000; Chemicon, Temecula, CA, USA), mouse antibody against BDNF (1 : 1000; Calbiochem, La Jolla, CA, USA), β-actin (1 : 2000; Sigma), rabbit antibodies against pTrkB817 (1 : 1000; Novus, Littleton, CO, USA), rabbit antibodies against pERK, pp38, pJNK, pTrkA490, ERK, p38 MAPK, JNK, TrkA, TrkB, NGF, cleaved caspase-3 or COX IV (1 : 1000; all from Cell Signaling, Beverly, USA). After washing, the membranes were treated with horseradish peroxidase-conjugated donkey anti-sheep IgG (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, USA), goat anti-mouse IgM (1 : 2000; Santa Cruz Biotechnology), or goat anti-rabbit Ig G (1 : 2000; Chemicon) for 1 h at 37°C. Peroxidase activity was visualized with the ECL substrate kit (Santa Cruz Biotechnology). Images were taken and the densities of the bands were measured using the Gel Documentation Systems (Bio-Rad).

Statistical analyses

The data are expressed as means ± SE and were subjected to statistical analysis via one-way anova. The level of statistical significance was set at < 0.05.

Results

Sustained treatment with echinacoside protects various kinds of cells against rotenone injury

Rotenone is a specific mitochondrial complex I inhibitor, and it results in cell damage through promoting peroxides generation and triggering mitochondria-dependent death pathways (Ramsay et al. 1991). Three kinds of human cell lines, including SH-SY5Y, Hela, and HEK293T cells, were exposed to 200 nM rotenone for 36 h to induce cell injury, which was monitored by detecting LDH release into the culture medium at 6, 12, 24, and 36 h. Meanwhile, H2O2 production was measured using the Amplex Red hydrogen peroxide assay kit. H2O2 levels (Fig. 2b) and LDH release (Fig. 2a) were both significantly increased by rotenone, suggesting that rotenone remarkably promotes H2O2 generation and causes severe damage to all three cell lines.

Figure 2.

Sustained treatment with echinacoside protects various kinds of cells against rotenone injury. Hela, HEK293T, SH-SY5Y, and primary rat cortical neuronal cells were all treated with rotenone (200 nM) and three doses of echinacoside (5, 10, 20 mg/L), which were added 2 h before rotenone. Lactate dehydrogenase (LDH) release (a) and H2O2 production (b) were both measured at 6, 12, 24 and 36 h of rotenone exposure. Data were normalized to control levels and are presented as means ± SE (= 4). R, rotenone; E, echinacoside. #< 0.05, ##< 0.01 compared to control; *< 0.05, **< 0.01 E+R groups versus R group at each time point.

To explore the effects of echinacoside on rotenone-induced H2O2 production and cell injury, three doses (5, 10 and 20 mg/L) of echinacoside were administrated to those cell lines 2 h ahead of rotenone (Fig. 1b). Cell damage and H2O2 levels were determined as described above. Rotenone-induced elevation of LDH release (Fig. 2a) and H2O2 generation (Fig. 2b) was modestly reduced in all three cell lines at 6 and 12 h by even the lowest dose of echinacoside, and higher doses provided much stronger protection. It indicates that echinacoside exerts strong antioxidative and protective effects against rotenone injury in different types of cells. However, the antioxidation function of echinacoside turned weak at 24 h and completely disappeared at 36 h of rotenone exposure (Fig. 2b), along with its protection in Hela and HEK293T cells, though SH-SY5Y cell injury was still significantly attenuated by echinacoside at 24 and 36 h of rotenone exposure (Fig. 2a). Those indicate that echinacoside selectively initiates prolonged protection in SH-SY5Y cells (Fig. 2a).

As SH-SY5Y cells are human neuroblastoma cells, we further questioned whether echinacoside has long-lasting protection in neuronal cells. We repeated the experiments above with primary rat cortical neuronal cell cultures, and found similar results as in SH-SY5Y cells. Echinacoside considerably suppressed neuronal injury through rotenone exposure (Fig. 2a), whereas its antioxidation function would not last over 24 h (Fig. 2b). The data above imply that sustained treatment with echinacoside selectively has prolonged protection on neuronal cells, where other mechanisms might be involved besides its antioxidative activity. Nonetheless, exposure to echinacoside alone barely affected the H2O2 production or cell survival in any of those four types of cells (Figure S1).

Transient exposure to echinacoside selectively attenuates neuronal cell injury caused by rotenone

Next, we treated those four kinds of cells above with different doses (5, 10 and 20 mg/L) of echinacoside for 2 h, and then exposed cells to rotenone alone for 36 h (Fig. 1c). Cell damage and H2O2 levels were also detected at 6, 12, 24, and 36 h of rotenone exposure. Echinacoside had little influence on H2O2 generation induced by ensuing rotenone exposure in any kind of cells (Fig. 3b). Nevertheless, echinacoside transient exposure remarkably suppressed rotenone-caused cell injury in SH-SY5Y and rat cortical neuronal cells, but not in Hela or HEK293T cells (Fig. 3a). This suggests that although echinacoside is a potent antioxidant, its transient exposure may selectively protect neuronal cells against rotenone injury via some other mechanism.

Figure 3.

Transient exposed to echinacoside selectively attenuates neuronal cell injury caused by rotenone. Hela, HEK293T, SH-SY5Y, and primary rat cortical neuronal cells were all pre-treated with three doses of echinacoside (5, 10, 20 mg/L) for 2 h, and then exposed to rotenone alone for 36 h. Lactate dehydrogenase (LDH) release (a) and H2O2 production (b) were detected at 6, 12, 24, and 36 h of rotenone exposure as described above (= 4). T-, transient exposure to. #< 0.05, ##< 0.01 compared to control; *< 0.05, **< 0.01 T-E+R groups versus R group at each time point.

To further investigate the mechanisms underlying, we chose 10 mg/L echinacoside in our following experiments, since it already provided the strongest protection in neurons or SH-SY5Y cells at this dose (Fig. 3a).

Echinacoside transient exposure protects neuronal cells against rotenone through activating TrkA/TrkB receptors

We then detected the effects of echinacoside transient exposure on the activity of TrkA/TrkB receptors in primary rat cortical neurons, because of the low levels of endogenous TrkA/TrkB in SH-SY5Y cells. The total protein levels and the phosphorylation levels of TrkA/TrkB were determined by immunoblotting at 6, 12, 24, and 36 h of rotenone exposure after brief treatment with echinacoside for 2 h. As seen from Fig. 4a and b, neither TrkA nor TrkB levels were altered during drug treatments. Meanwhile, pTrkA and pTrkB levels were both significantly reduced by rotenone, which were completely reversed and even further elevated by echinacoside. Furthermore, transient exposure to echinacoside also remarkably increased the levels of pTrkA and pTrkB in normal neurons, yet had no influence on TrkA or TrkB levels (Fig. 4e and f).

Figure 4.

Echinacoside transient exposure increases TrkA/TrkB activity and nerve growth factor/brain-derived neurotrophic factor (NGF/BDNF) levels in primary rat cortical neurons. NGF/BDNF levels and the total protein levels and the phosphoylation levels of TrkA/TrkB were detected by immuoblotting at 6, 12, 24, and 36 h of rotenone exposure after neurons were pre-treated with 10 mg/L echinacoside for 2 h. The phosphorylation levels of TrkA (a) and TrkB (b) levels are both reduced by rotenone, they are completely reversed by echinacoside, which are totally blocked when co-administrated with 1 μM K252a, a specific Trk inhibitor (c and d). The levels of pTrkA (e) and pTrkB (f) in normal neurons are remarkably increased by echinacoside treatment for 2 h, which is also reversed when co-administrated with K252a. NGF (g) and BDNF (h) levels were decreased by rotenone, which were both reversed by echinacoside. These effects of echinacoside were abolished by co-administration of K252a (i and j). NGF (k) and BDNF (l) levels in normal neurons were barely affected by echinacoside exposure for 2 h. The densities of the bands were normalized to β-actin. #< 0.05 compared to control; *< 0.05 T-E+R group versus R group at each time point; *< 0.05 T-(E+K) group versus T-E group at each time point; = 4.

To determine whether TrkA/TrkB activation is involved in the neuroprotection of echinacoside, neuronal cultures were treated with K252a (1 μM), a specific Trk inhibitor, and echinacoside for 2 h, and then exposed to rotenone alone for 36 h. Firstly, the total protein levels and the phosphorylation levels of TrkA/TrkB were detected at 6, 12, 24, and 36 h of rotenone exposure. As seen from Fig. 4c and d, K252a totally suppressed TrkA and TrkB phosphorylation stimulated by echinacoside without affecting their total protein levels, suggesting that Trk activity could be efficiently inhibited by 1 μM K252a. Next, cell damage was detected by LDH release measurement or cell apoptosis analysis at 6, 12, 24, and 36 h of rotenone exposure after treatment with K252a and echinacoside for 2 h. As shown in Fig. 6h and Figure S2a, the effects of echinacoside suppressing rotenone-induced LDH release elevation and cell apoptosis were both entirely reversed by K252a, indicating that transient exposure to echinacoside may protect neurons against rotenone injury via TrkA/TrkB stimulation. In addition, K252a also inhibited TrkA and TrkB activation triggered by echinacoside brief treatment in normal neurons (Fig. 4e and f).

Brief treatment with echinacoside reverses NGF and BDNF down-regulation in neurons exposed to rotenone

We further questioned whether transient exposure to echinacoside enhances TrkA/B activity by promoting the expression of their endogenous ligands in neurons. NGF and BDNF levels were determined at 6, 12, 24, and 36 h of rotenone exposure, and they were both slightly reduced at 6 h and remained down-regulation up to 36 h, which were totally reversed by echinacoside transient exposure (Fig. 4g and h). And these effects of echinacoside were abolished when co-administrated with K252a (Fig. 4i and j), indicating that echinacoside might reverse NGF and BDNF down-regulation in neurons exposed to rotenone via TrkA/TrkB activation. NGF and BDNF levels in normal neurons were slimly elevated by echinacoside transient exposure, though no statistical difference was seen (Fig. 4k and l).

Echinacoside transient exposure also protects non-neuronal cells over-expressed with TrkA/TrkB against rotenone injury

To test whether TrkA and TrkB are both required for the protective effects of echinacoside, Hela cells were over-expressed with TrkA or TrkB, and collected for immunoblot assay at 2 and 4 days after transfection. As seen from Fig. 5a, a robust band of TrkA or TrkB was detected at 2 and 4 days of over-expression.

Figure 5.

Transient exposure to echinacoside also protects Hela cells over-expressed with TrkA/TrkB against rotenone injury. TrkA and TrkB were detected by immuoblotting after Hela cells were transfected with an empty vector or over-expressed with TrkA or TrkB for 2 and 4 days (a). After transfection for 2 days, Hela cells were pre-treated with echinacoside (or co-administrated with K252a) for 2 h, and then exposed to rotenone alone for 36 h. The total protein levels and the phosphoylation levels of TrkA (b) and TrkB (c) were detected by immuoblotting at 6, 12, 24, and 36 h of rotenone exposure. Meanwhile, lactate dehydrogenase (LDH) release was detected as described above (d–f). #< 0.05, ##< 0.01 compared to control; *< 0.05 T-E+R groups versus R group at each time point; *< 0.05 T-(E+K) group versus T-E group at each time point; (= 3).

Next, Hela cells at 2 days of over-expression with TrkA/TrkB were treated with echinacoside for 2 h, and then exposed to rotenone alone for 36 h. Cell injury and Trk activity were measured as described above. Echinacoside showed no protection in Hela cells transfected with an empty vector (Fig. 5d and Figure S2b), while it modestly attenuated rotenone-induced LDH release elevation and cell apoptosis in cells over-expressed with TrkA (Fig. 5e and Figure S2c) or TrkB (Fig. 5f and Figure S2d), which were abolished when echinacoside was co-administrated with K252a. The data above suggest that TrkA and TrkB may be both involved in the protection of echinacoside.

As shown in Fig. 5b and c, neither TrkA nor TrkB protein levels were altered during drug exposure; however, pTrkA and pTrkB levels were both reduced by rotenone, yet they were totally reversed and even further increased by echinacoside. Those effects of echinacoside were abolished when it was co-administrated with K252a, which confirmed the central role of Trk activation in the protection of echinacoside. We also repeated the experiments in HEK293T cells, and found similar results (data not shown).

Echinacoside protects neuronal cells against rotenone injury through activating Trk-ERK pathway

Trk receptor autophosphorylation elicits its downstream mitogen-activated protein kinases (MAPKs) and Akt activation, which plays a key role in preventing apoptotic cell death (Kaplan and Stephens 1994; Chao 2003). We further tested the phosphorylation levels of Akt and MAPKs at 6, 12, 24, and 36 h of rotenone exposure after brief treatment with echinacoside for 2 h. As shown in Fig. 6, pAkt and pp38 levels were both increased during rotenone administration, while pERK levels were reduced and pJNK levels were hardly affected by rotenone. Echinacoside had no further influence on the levels of pAkt (Fig. 6e), pp38 (Fig. 6f), or pJNK (Fig. 6g), yet it tremendously elevated pERK levels at 6, 12, 24, and 36 h of rotenone exposure (Fig. 6a), which was entirely reversed when echinacoside was co-administrated with K252a (Fig. 6b).

Figure 6.

Echinacoside brief treatment protects neurons against rotenone injury via activating Trk-extracellular signal-regulated kinase (ERK) pathway. The changes in Akt and MAPKs phosphorylation in neurons were detected by immunoblotting at 6, 12, 24, and 36 h of rotenone exposure after pre-treated with echinacoside for 2 h. Rotenone treatment increases pAkt (e) and pp38 (f) levels, decreases pERK levels (a) but barely alters phosphorylated c-Jun N-terminal protein kinase (pJNK) levels (g). Echinacoside has no further influence on the levels of pAkt (e), pp38 (f), and pJNK (g), yet it tremendously elevates pERK levels at 6, 12, 24, and 36 h of rotenone exposure (a), which is completely abolished by co-administration of 1 μM K252a (b) or 5 μM U0126 (c). Echinacoside brief treatment for 2 h also increases pERK levels in normal neurons, which is reversed by co-administration of K252a or U0126 (d). Meanwhile, lactate dehydrogenase (LDH) release was detected as described above (h). #< 0.05 compared to control; *< 0.05 T-E+R group versus R group at each time point; *< 0.05 T-(E+K) group or T-(E+U) group versus T-E group at each time point; §p < 0.05 T-(E+U) group versus T-(E+K) group at each time point; = 4.

To investigate whether ERK activation is involved in the neuroprotection of echinacoside, neurons were treated with U0126 (5 μM), a specific pERK blocker, and echinacoside for 2 h, and then exposed to rotenone alone for 36 h. The phosphorylation levels of ERK and LDH release measurement were detected at 6, 12, 24, and 36 h of rotenone exposure. As shown in Fig. 6c, U126 completely reversed the elevation of pERK levels induced by echinacoside, implying that ERK activation could be efficiently inhibited by 5 μM U0126. As seen from Fig. 6h, the effects of echinacoside suppressing rotenone-induced LDH release elevation were abolished by co-administration of U0126. In addition, exposure to echinacoside for 2 h also robustly increased pERK levels in normal neurons, which was inhibited by co-administration of K252a or U0126 (Fig. 6d), although they caused no injury to normal cells (Figure S3).

The data above indicate that echinacoside transient exposure protects neuronal cells against rotenone injury via activating Trk-ERK pathway.

Echinacoside prevents cytochrome c release and caspase-3 activation in neuronal cells through activating Trk-ERK pathway

As a specific mitochondrial complex I inhibitor, rotenone results in severe mitochondrial dysfunction and causes subsequent cytochrome c (cyto c) release from mitochondria into cytosol, which further cleaves pro-caspase-3 to form active capase-3, and leads to cell death, eventually (Martinou et al. 2000). To explore whether echinacoside inhibits cyto c release and caspase-3 activation in rotenone-exposed neurons, cytosolic cyto c and cleaved caspase-3 levels were detected by immunoblotting at 6, 12, 24, and 36 h of rotenone exposure after brief treatment with echinacoside for 2 h. Cell fractions were first examined by immunoblotting for the presence of cytochrome oxidase subunit IV (COX IV) and β-actin. β-actin band was very strong while COX IV band was pretty weak in the cytosolic fraction (Fig. 7a and b), indicating that few mitochondria were detected in the cytosolic fraction. A robust band of cyto c was detected in the cytosolic fraction after treated with rotenone, suggesting that rotenone remarkably increased cyto c release in neurons (Fig. 7a). The elevation of cyto c release was suppressed by echinacoside, which was abolished by co-administration of K252a or U126 (Fig. 7a and b).

Figure 7.

Transient exposure to echinacoside prevents cytochrome c (cyto c) release and caspase-3 activation in neurons exposed to rotenone through stimulating Trk-extracellular signal-regulated kinase (ERK) pathway. The protein levels of cyto c in cytosolic fraction (a and b) and cleaved caspase-3 (c and d) were measured by immunoblotting at 6, 12, 24, and 36 h of rotenone exposure, and they are both remarkably increased. Echinacoside treatment for 2 h suppresses the elevation of both protein levels, which are both reversed by co-administration of K252a or U0126. #< 0.05 compared to control; *< 0.05 T-E+R group versus R group at each time point; = 4.

As shown in Fig. 7c and d, the levels of cleaved caspase-3 were far higher in neurons exposed to rotenone than in control cells, suggesting that caspase-3 is highly activated during rotenone exposure. Caspase-3 activation was prevented by echinacoside, and this effect was reversed by co-administration of K252a or U126. Echinacoside treatment alone or with K252a or U0126 for 2 h had no influence on cyto c release or caspase-3 activation in normal cells (data not shown).

Discussion

Here, we compared the protective effects of echinacoside sustained or transient treatment in four different types of cells, and found that the antioxidation function is implicated in echinacoside protection against rotenone injury in both neuronal and non-neuronal cells. More importantly, transient exposure to echinacoside has a specific protection in neuronal cells when no antioxidative effect is detectable. Further investigation revealed that transient exposure to echinacoside is sufficient to activate Trk-ERK pathway, and rescue cells by preventing rotenone-induced cyto c release and caspase-3 activation. Echinacoside transient exposure also exerts protection against rotenone injury in the non-neuronal cells over-expressed with TrkA/TrkB. In conclusion, aside from as an antioxidant, echinacoside may also elicit neuroprotection by mimicking neurotrophic function, where TrkA and TrkB are both involved.

NGF and BDNF are the endogenous ligands of TrkA and TrkB, respectively, and they are both critical to the survival of many neuronal populations. Nevertheless, their therapeutic use in treating neurodegenerative diseases is limited by the poor blood–brain barrier permeability (Saragovi and Gehring 2000; Melo et al. 2011). As such, tremendous efforts have been made to search for NGF and BDNF mimetics with better pharmacokinetic properties. Neurotrophin peptidomimetics can bind to Trk receptors and initiate neuroregenerative responses of neurotrophins, yet it is still difficult for them to cross blood–brain barrier (Saragovi and Gehring 2000; Dago et al. 2002). Some small non-peptide factors could increase the expression of neurotrophins, while others act by promoting neurotrophin action, although none of them is able to fully mimic NGF or BDNF function (Price et al. 2007; Webster and Pirrung 2008). We showed in this study that transient exposure to echinacoside not only enhances the activity of TrkA/TrkB, it also reverses NGF and BDNF down-regulation in neurons exposed to rotenone. Those indicate that echinacoside may be a potential non-peptide neurotrophic factor. However, further investigations are still needed on whether echinacoside activates TrkA/TrkB by directly binding to Trk receptors as neurotrophins or through other signaling pathways.

Most of previous studies indicate that echinacoside protects neuronal cells via free radical scavenging (Deng et al. 2004; Geng et al. 2007; Kuang et al. 2009). Lately, Zhao et al. noticed that some neurotrophic factors are increased in response to echinacoside in the mouse model of Parkinson's disease, which may be involved in the neuroprotection of echinacoside (Zhao et al. 2010), yet the details are so far unclear. We demonstrated here that echinacoside restores NGF and BDNF levels back to normal via TrkA/TrkB stimulation in cells exposed to rotenone, which in return, may contribute greatly to maintain TrkA/TrkB activity and the neuroprotective effects of echinacoside. This might shed a light into the mechanisms underlying echinacoside neuroprotection, although it needs to be confirmed in other kinds of neuronal injury.

Many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and Huntington's disease, result from progressive loss of neuron cells; and considerable evidence suggests that oxidative stress and mitochondrial dysfunction are the main causes of neuronal death in neurodegenerative diseases (Melo et al. 2011). We showed here that echinacoside not only has antioxidative activity, it also prevents mitochondria-dependent death pathway by mimicking neurotrophic function. As echinacoside can easily cross the blood–brain barrier and enter the central nervous system after systemic administration (Wei et al. 2011), it might have great potential in clinical treatment of neurodegenerative diseases.

Acknowledgements

There is no potential conflict of interest. This study was supported by China Postdoctoral Foundation (20080430076 & 200902205), Ph.D. Programs Foundation from China Ministry of Education (200802461038), and Shanghai Natural Science Foundation (10ZR1406100).

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