Neurotrophic And Neurorescue Effects Of Echinacoside in The Subacute MPTP Mouse Model Of Parkinson's Disease

Contact: ali.ma@wecistanche.com

Qing Zhao, et al

Abstract

Many experiments support the notion that augmentation of neurotrophic factors' (NTFS) activity, especially glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) could prevent or halt the progress of neurodegeneration in Parkinson's disease (PD). However, the application of NTFS as therapeutic agents for PD (Parkinson's disease) is hampered by the difficulty in delivering them to specific brain regions safely and effectively. Another potential strategy is to stimulate the endogenous expression of NTFS. In this study, we investigated the effects of Echinacoside (ECH), a monomer extracted from herbs, on rescuing dopaminergic function in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)- lesioned mice. We found that oral administration of ECH (Echinacoside) (30 mg/kg/day for 14 days) to MPTP-treated mice, commencing after impairment of the nigrostriatal system, suppressed the reduction of nigral dopaminergic neurons, striatal fibers, dopamine, and dopamine transporter to 134.24%, 203.17%, 147.25% and 154.72 of MPTP-lesioned animals respectively (p< 0.05). There was a relative elevation in the expression of GDNF and BDNF mRNA (2.94 and 3.75-fold) and protein (184.34% and 185.93%) in ECH treated mice compared with vehicle-treated MPTP-lesioned mice (p< 0.05). In addition, the apoptosis cells and Bax/Bcl-2 ratio of mRNA and protein in MPTP-lesioned mice significantly increased, and these effects could be prevented by ECH. At the 7th and 14th days of ECH treatment, the gait disorder displayed obvious improvement (p< 0.05). These findings demonstrate that ECH is probably a novel, orally active, a non-peptide inducer of NTFS and inhibitor of apoptosis, and they provide preclinical support for the therapeutic potential of this compound in the treatment of PD.

Keywords: Parkinson's disease, MPTP, Echinacoside, GDNF, BDNF, Neurorescue

Echinacoside in Cistanche has Neurotrophic and neurorescue effects on Parkinson's disease

Click to cistanche extract benefits for Parkinson's disease

1. Introduction

Parkinson's disease (PD) is characterized by progressive dopaminergic neuronal cell death in the substantia nigra, resulting in severe motor deficits. In contrast to neuroprotective strategies, which aim to slow degeneration and thus must be initiated before significant damage has occurred, neurorescue or restorative therapies aim to obliterate neuronal deficits and degeneration after the initiation of impairments. Neurotrophic factors (NTFs) are secreted proteins that regulate the survival, functional maintenance, and phenotypic development of neuronal cells, in particular, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are widely recognized as potent survival factors for the dopaminergic neurons and the nigrostriatal pathway that degenerate in PD (Parkinson's disease). While the neuroprotective or neurorescue activities of these NTFs have been demonstrated in a number of in vitro and in vivo experiments (Peterson and Nutt, 2008), their application as therapeutic agents for PD has been hampered by the difficulties in delivering them into the brain because the macromolecule peptides cannot pass the brain-blood barrier (BBB). Thus clinical trials testing treatment of PD (Parkinson's disease) using either surgery or gene therapy must solve the problem of efficient delivery and expression of NTFs (Dietz et al., 2006; Torp et al., 2006). As an alternative to such invasive approaches, a potential strategy is to stimulate the endogenous expression of NTFs. Recently, several non-peptide NTF inducers, such as XIB4035 and PYM50028, have been shown to attenuate the loss of dopaminergic neurons and might have beneficial effects in the treatment of PD (Tokugawa et al., 2003; Visanji et al., 2008). These low molecular weight agents can be administered systemically, and they would thus overcome most of the problems associated with the efficacy and safety of delivering NTFS into the brain by such methods as induction of NTF expression by viral vectors or with the use of encapsulated NTF producing cells (Bespalov and Saarma, 2007). Thus, molecules that either induce the endogenous expression of NTFs or enhance their signaling have been given increasing attention as alternative therapeutic options for PD (Parkinson's disease).

Echinacoside (ECH, Fig. 1) is one of the principal constituents of phenylethanoid glycosides (PhGs) extracted from a famous traditional Chinese medicine, Herba Cistanches (the stems of Cistanche deserticola, Cistanche salsa, or Cistanche tubulosa). It is also the main phenolic component in the roots of Echinacea Angustifolia and E. pallida (Pellati et al., 2004), which are widely used in Europe and North America for their immunoregulation properties (Hostettmann, 2003). As a polyphenol natural product, ECH (Echinacoside) has various bioactivities, such as powerful antioxidant (Sloley et al., 2001; Dalby-Brown et al., 2005), free radical scavenging, and reduce the amount of NO radical generated by activated macrophages (Xiong et al., 2000) so that it can protect against hepatotoxicity (Wu et al., 2007) and acute lung injury in rats (Zhang et al., 2007, Xiong et al., 1998). It also has anti-inflammatory, antitumor, anti-aging, and improving learning memory activities (reviewed by He et al., 2009). Several studies have reported ECH (Echinacoside) has anti-apoptotic and neuroprotective properties in neurodegenerative disease, for example, ECH (Echinacoside) shows significant neuroprotective effects on H2O2 injured PC-12 cells and rescues SH-SY5Y neuronal cells from TNF-α induce apoptosis through the mitochondrial pathway (Kuang et al., 2009; Deng et al., 2004). In vivo, the compound has protected dopaminergic neurons in 6-hydroxydopamine (6-OHDA)-lesioned rats by attenuating the diminution of dopamine (DA) and its intermediates (Chen et al., 2007a,b). Another study reported that ECH had neuroprotective effects and behavioral improvement in the mouse model of MPTP-induced dopaminergic neuronal damage, while further investigation revealed that ECH induced down-regulation of caspase-3 and caspase-8 activation in MPP+ -induced apoptosis of cerebellar granule neurons (Geng et al., 2007). But these studies only investigated the anti-apoptotic or neuroprotective roles of ECH by the method of pre-treatment. The questions of whether ECH affects other dopaminergic neuronal prosurvival and death-related factors (such as NTFs and the Bcl-2 family) and whether it has neurorescue effects have not yet been addressed. Through reviewing the sparse literature on NTF induction by herbs or herbal compounds, we found that Rehmannia glutinosa and catalpol, two herbs with similar functions to Cistanches according to the traditional Chinese medical theory, could induce GDNF gene and protein expressions both in vitro and in vivo then attenuate MPTP-induced neuronal degeneration of nigral-striatal dopaminergic pathway (Yu et al., 2006; Xu et al., 2010). We, therefore, hypothesized that ECH may also have analogous efficacy. On the other hand, the subacute MPTP mouse model is a quite popular regimen that involves one injection of 30 mg/kg daily for five consecutive days. This regimen causes apoptosis and depletes striatal dopamine by 40–50% in young adult C57BL/6 mice, and the dopaminergic lesion stabilizes by 21 days after MPTP administration (Tatton and Kish, 1997; Jackson-Lewis and Przedborski, 2007). So our current study chooses this model and investigates the effects of ECH on expressions of GDNF and BDNF, two of the most potent NTFs associated with dopaminergic neurons survival, and it further demonstrates the neurorescue and anti-apoptotic capabilities of this compound after subacute MPTP lesion induction in a mouse model of PD (Parkinson's disease).

2. Results

2.1. ECH (Echinacoside) improves gait dysfunctions in a subacute MPTP mouse model of PD (Parkinson's disease)

Shortened stride length is one of the chief characteristics of abnormal gait in PD (Parkinson's disease). The results of our study demonstrate a significant decrease in the forelimb and hindlimb stride lengths in the MPTP-lesioned mice treated with vehicle (saline) on the 9th day after the last administration of MPTP, i.e., the 7th day of vehicle treatment, compared with the normal control group (p= 0.023, 0.014, Figs. 2A, B). Meanwhile, the stride lengths of mice in the high-dose ECH (Echinacoside) (30 mg/kg/day) treatment group is longer than those of MPTP-vehicle animals, especially for the hindlimbs (p= 0.024, 0.009). Moreover, the improvement associated with the high-dose ECH (Echinacoside) was also observed on the 14th day of treatment (p= 0.015, 0.012), although there was also a minor spontaneous recovery in the MPTP-lesioned mice. The moderate (20 mg/kg/day) and low (10 mg/kg/day) dose ECH (Echinacoside) treated groups showed a tendency towards longer stride lengths, however, the differences between them and the MPTP-vehicle treated mice were not significant.

Fig. 2 – ECH (Echinacoside) improved the gait impairment induced by subacute MPTP injections. A: Stride length of the forelimb. The forelimb lengths in the gait analysis test displayed a significant reduction in the MPTP+NS (saline) group compared with the NS+NS group on the 7th day of MPTP-vehicle treatment, but these deficits were prevented by the high dose of ECH (Echinacoside) (30 mg/ kg/day) when measured at the 7th and 14th day of treatment. B: Stride length of the hindlimb. The hindlimb stride length of MPTP-lesion mice decreased obviously compared to the saline controls on the 7th day of the MPTP-vehicle treatment, and it was also significantly improved by the high dose of ECH (Echinacoside). Data are expressed as means±SEM (n=10 each). * p< 0.05 vs. NS+NS group; # p< 0.05 vs. MPTP+NS group.

Echinacoside in Cistanche has Neurotrophic and neurorescue effects on Parkinson's disease

2.2. ECH (Echinacoside) attenuates the loss of nigral TH-positive neurons and striatum TH-positive axons in subacute MPTP-lesioned mice

After finishing 14 days of treatment with ECH (Echinacoside) or the vehicle and the completion of behavioral assessment, half of the mice in each group were sacrificed for Nissl and TH-immunoreactive detections. Representative coronal mesencephalon sections containing TH-positive neurons and fibers in the substantia nigra compacta (SNpc) and striatum of mice are shown in Figs. 3A, B, C, D. There were significant reductions in the number of Nissl-positive neurons (only cresyl violetpositive) and TH-positive neurons (both TH and cresyl violet positive neurons) in SNpc for the vehicle-treated MPTP lesioned group compared with non-lesioned control mice (71.89% and 67.97% of saline control, p= 0.000, 0.000, Figs. 3A, B, E). In mice receiving daily oral administration of high-dose ECH (Echinacoside) the number of Nissl-positive and TH-positive neurons was significantly higher than those in MPTP-vehicle treated animals (135.39% and 134.24% of MPTP control, p= 0.000), and it was even close to those of the non-lesioned control mice (97.34% and 91.25%). A similar but weaker effect was also seen in the moderate-dose ECH treatment group (125.39% and 124.59% of MPTP control, p= 0.002, 0.004). But the low-dose ECH treatment did not provide a significant rescuing effect. On the other hand, the similar tendency of changes for Nissl-positive and TH-positive neurons in SNpc excluded the interference on results due to a regulation of the TH enzyme.

Accompanying the loss of DA neurons in the MPTP model of PD (Parkinson's disease) is a reduction in TH-positive fibers through the striatum. We observed an apparent fragmentation of TH-positive axons in the striatum of MPTP-lesioned animals but the damage was improved in the ECH (Echinacoside) treatment group, especially in the case of high-dose ECH (Echinacoside) (Fig. 3D). Furthermore, the optical density (OD) analysis of TH-positive fibers revealed a significant loss of DA terminals in the MPTP-vehicle treated group (47.37% of saline control, p= 0.001, Fig. 3F). In contrast, an obvious relative elevation in the density of TH-positive fibers was observed in mice that received the high-dose ECH (Echinacoside) treatment compared with the MPTP-lesioned control animals (203.17% of MPTP control, p= 0.003, Figs. 3C, F), bringing it to 96.24% of the normal control levels. Meanwhile, the moderate-dose ECH group also displayed effects of significant but weaker neurorescuing effect (177.78% of MPTP control, p= 0.022), while the low-dose showed no benefits.

2.3. ECH (Echinacoside) inhibits the decrease of striatal DA in the subacute MPTP mouse model of PD (Parkinson's disease)

At the end of the experiment, the content of DA in the striatum in the vehicle-treated MPTP model was only 31.25% of normal controls (p= 0.000, Table 1). After administration of high-dose ECH (Echinacoside) for 2 weeks, DA content increased to 47.97% of normal and was significantly higher than MPTP-lesioned mice (p= 0.015). On the other hand, the content of the metabolites DOPAC decreased in the MPTP model was also elevated by ECH (Echinacoside) so that the ratio of DOPAC/DA was not significantly changed (p> 0.05). It seemed that ECH (Echinacoside) has no obvious regulations on the catabolism of DA.

2.4. ECH (Echinacoside) elevates the striatal DAT protein in subacute MPTP-lesioned mice

Western blot analysis showed that MPTP-induced a marked dopamine transporter (DAT) protein decrease in striatum tissue compared to the saline control 14 days after its last injection (p=0.000, Figs. 4A, B). However, the MPTP-lesioned animals treated with high and moderate doses of ECH (Echinacoside) inhibited the reduction of DAT protein (p=0.001, 0.046). A low dose of ECH showed no activity on the elevation of DAT expression.

2.5. ECH (Echinacoside) reduces the apoptosis cells in the nigral of subacute MPTP-lesioned mice

With the aid of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique, more apoptotic cells incorporating intense dark brown staining nuclei and noticeable shrinking soma were detected in the SNpc of subacute MPTP-lesioned mice compared with the normal control (p= 0.000, Figs. 5A, B, C). However, the MPTP-injected animal treated with high and moderate doses of ECH (Echinacoside) demonstrated a significant decrease of TUNELpositive cells compared with the saline-treated MPTP model (p= 0.000, 0.003).

2.6. ECH (Echinacoside) increases the relative mRNA and protein levels of GDNF and BDNF in the substantia nigra of subacute MPTP-lesioned mice

To define the changes in GDNF and BDNF expression at both transcriptional and protein levels after the different treatments real-time PCR and Western blot analysis were used. There were no significant differences in mRNA and protein levels of GDNF and BDNF in the ventral midbrain between the MPTP-vehicle treated animals and the saline control group at the end of the experiment (p> 0.05, Figs. 6A, B, C). However, after two weeks of treatment with the high-dose ECH (Echinacoside), the relative mRNA and protein levels of GDNF were significantly increased compared with vehicle-treated MPTP-lesioned mice (2.94-fold in mRNA and 184.34% in protein of MPTP control, p=0.005, 0.000). These elevations were also significant when compared with the saline control animals (1.97-fold and 142.63% of saline control, p = 0.003, 0.001, Figs. 6A, C). Meanwhile, the moderate-dose ECH (Echinacoside) displayed weaker but still obvious elevations in the mRNA and protein levels of GDNF (2.13-fold and 160.55% of MPTP control, p= 0.012, 0.001). There were similar changes in the expression of BDNF, which had significantly higher relative mRNA and protein levels in the high-dose ECH (Echinacoside) treated group compared with MPTP-lesioned or normal control mice (3.75 folds and 185.93% of MPTP control, p= 0.001, 0.000; 2.33 folds and 154.39% of saline control, p= 0.002, 0.001, Figs. 6A, C). Treatment with moderate-dose ECH (Echinacoside) elevated both BDNF mRNA and protein expression (2.51 folds and 162.69% of MPTP control, p= 0.044, 0.002). Thus ECH (Echinacoside) was able to increase both mRNA and protein expressions of GDNF and BDNF in some dose-dependent manner.

2.7. ECH (Echinacoside) attenuates the relative mRNA and protein ratio of Bax/Bcl-2 in the substantia nigra of subacute MPTP-lesioned mice

The effects of ECH (Echinacoside) on apoptosis, known to be another important factor related to the loss of nigral dopaminergic neurons in PD (Parkinson's disease), were assessed by analyzing the Bax and Bcl-2 expression by Real-time PCR and Western Blot. The relative mRNA levels of the pro-apoptotic factor Bax increased in SN of MPTP-lesioned mice (2.14-fold of saline control, p=0.036), while those of the anti-apoptotic factor Bcl-2 were not significantly different with normal control mice (p>0.05, Fig. 7A). However, the mRNA ratio of Bax/Bcl-2 was increased significantly in the lesioned mice (1.99-fold of saline control, p=0.002), and this elevation could be reversed by the high-dose ECH (Echinacoside) after 14 days of treatment (37.12% of MPTP control, p=0.000, Fig. 7A) due to a slight down-regulation of Bax (82.71% of MPTP control, p>0.05) and an obvious up-regulation of Bcl-2 gene expression (2.39-fold of MPTP control, p=0.000). There were similar but weaker effects on the Bax/Bcl-2 mRNA ratio for the moderate-dose ECH (Echinacoside) compared with the vehicle-treated MPTP-lesioned mice (63.88% of MPTP control, p=0.016, Fig. 7A). With one accord, the protein ratio of Bax/Bcl-2 was significantly increased in MPTP-lesioned mice (265.97% of saline control, p=0.000) and reversed by the high and moderate doses of ECH (Echinacoside) (52.33%, 67.25% of MPTP control, p=0.000, 0.001 Figs. 7B, C), but not by the 10 mg/kg dose of ECH, mirroring the effects seen on Bax/ Bcl-2 ratio of mRNA.

3. Discussion

In the present study, we demonstrate for the first time that ECH (Echinacoside), a monomer extracted from a traditional Chinese herb, has neurorescue activities in the subacute MPTP mouse model of PD (Parkinson's disease) as well as the potential to induce the expression of NTFs at both transcription and protein levels. After 14 days of oral administration, ECH (Echinacoside) demonstrated multifunctional effects on rescuing dopaminergic neurons function, relieving the reduction of DAT, inhibiting the growth in a number of apoptosis cells in SNpc, and improving the gait disorder compared with the saline-treated MPTP-lesioned mice. We presume that the neurorescue activities of ECH are at least partially related to the relative elevation of mRNA and protein expression of GDNF and BDNF as well as the regulation on Bax and Bcl-2, two key molecules of the Bcl-2 family involved in apoptosis of dopaminergic neurons in the SN.

Although the findings regarding different therapeutic avenues from a variety of in vitro and in vivo PD (Parkinson's disease) models are promising, neuroprotection in PD (Parkinson's disease) remains an elusive goal. Compared with the identified strategies of neuroprotection, which almost always required pre-treatment (i.e., administration before injury), neurorescue or restorative strategies are more in line with the mode of clinical pharmacologic intervention at the early stage of PD. In our study, ECH (Echinacoside) administration was begun after the MPTP lesion, differing from previous research, and it exhibited clear benefits. Another purpose of the delayed-start treatment was to ensure that any observed effects were neither due to the compound interfering with the conversion of MPTP to its active metabolite MPP+, a reaction mediated by MAO-B nor potential rapid down-regulated DAT function and then block the uptake of MPP+ (Sagi et al., 2007; Lai et al., 2009).

It is well known that neuronal survival requires trophic support. GDNF and BDNF are the most promising trophic factors in the neurorescue or restorative treatment of neurodegenerative diseases, particularly in PD (Parkinson's disease). To date, a number of clinical trials have been undertaken to study the delivery of trophic factors, especially GDNF, directly to the PD brain, but they yielded mixed results. The problem may be due to the site and method of delivery, namely the distribution of GDNF after different injection or infusion techniques(Lang et al., 2006; Morrison et al., 2007; Nutt et al., 2003; Slevin et al., 2007). In treatments utilizing cell-based delivery systems or gene therapy applied with viral vectors, overexpression of the GDNF gene and protein could promote cell viability in the dopaminergic midbrain neurons and induce symptomatic improvement of rats with 6-OHDA lesions and of monkeys treated with MPTP (Andereggen et al., 2009; Kong et al., 2008). However, these therapies still have critical issues regarding their clinical application due to questions about the efficacy and stability of gene transfer and the possibility of immune responses (Mochizuki, 2009).
While there have been efforts to overcome the problems in cell or gene therapy associated with delivery through neurosurgery, increasing attention is now being paid to strategies that induce the expression of endogenous trophic factors or enhance their signaling as alternative therapeutic options for PD (Parkinson's disease) (Chen et al., 2007a,b; Saavedra et al., 2008). In this study, we report for the first time that ECH (Echinacoside) has neurorescue and neurotrophic effects in a PD (Parkinson's disease) mouse model, and these effects are associated with up-regulation of GDNF and BDNF at mRNA and protein levels. The significant difference in the number of axons or fibers in ECH (Echinacoside) treated mice compared with vehicle-treated MPTP-lesioned mice demonstrated that this compound promoted neurite outgrowth and regeneration. Notably, the density of fibers in treated mice approached normal uninjured levels (∼96.24% for high-dose ECH). A potential explanation for the apparently higher number of TH-positive neurons in ECH treated lesioned mice is that ECH mimics or enhances the roles of GDNF and BDNF, GDNF, in particular, may strongly modulate the viability and phenotypic expression of dopamine ventral mesencephalic neurons and may compensate for cell loss (Palfi et al., 2002).In other words, it is possible that ECH up-regulates the transcription of the TH gene, similar to GDNF (Xiao et al., 2002). Therefore our study not only confirmed ECH is beneficial as a natural low molecular weight medicine in the MPTP mouse model but also demonstrated potential mechanisms for these effects consistent with mediation by trophic factors.

Echinacoside in Cistanche has Neurotrophic and neurorescue effects on Parkinson's disease

Another interesting finding in our study is there are no obvious signs of activation for MAPK/ERK1/2 or Ras-PI3K-Akt survival pathway which has been reported as contributing to neurorescue effects of NTFs (Yu et al., 2006; Sagi et al., 2007). Because p-ERK1/2 (the activated phosphorylated form of MAPK) and p-Akt (the activated mark of Ras-PI3K-Akt pathway) were not detected through Western blot analysis in the midbrain of MPTP-lesioned animals treated with highdose ECH (data not showed). One of the possible explanations is the delayed time of detection because ERK activity is usually increased rapidly after medicine stimulation (Hisaoka et al., 2007) and another probability is the noninvolvement of these signaling pathways for ECH's neurorescue or neurotrophic effects in subacute MPTP mouse model of PD (Parkinson's disease) which needs to be investigated further.

In our study, the subacute MPTP regimen in the PD (Parkinson's disease) mouse model causes apoptosis and the dopaminergic lesions that stabilize by 21 days after MPTP administration, which is consistent with the record of previous literature (Jackson-Lewis and Przedborski, 2007). Suppression of apoptosis might protect neurons against injuries ranging from trophic factor deprivation to pathological stimuli (Yuan and Yankner, 2000). Additionally, NTFs can protect neurons against the mitochondria-induced apoptosis that occurs due to the activation of the phosphatidylinositol-3-kinase pathway (Toth et al., 2002). It is known that apoptosis is regulated by several pathways. Previous research regarding the Bcl-2 proteins supports the notion that such pro-apoptotic family members participate in neuronal death in a variety of PD (Parkinson's disease) models (Levy et al., 2009). The apoptosis-promoting proteins, including Bax, act on cytochrome in the mitochondrial membrane while the apoptosis-preventing proteins, such as Bcl-2, act by heterodimerizing with Bax proteins and preventing their activity. Therefore, the ratio of Bax/Bcl-2 is known to determine whether some cells will live or die (Tanaka et al., 2004). Previous research from our lab has demonstrated the potential neuroprotective and anti-apoptotic roles of ECH (Echinacoside) on MPP+ -induced SH-SY5Y cells through flow cytometry and other apoptosis-related tests (Wang et al., 2007). Thus we proceeded to analyze the molecular basis of the anti-apoptotic effects of ECH (Echinacoside) in vivo in the present study. We find that high and moderate doses of ECH treatment inhibit the elevation both on the number of TUNEL-positive apoptosis cells in SNpc and the Bax/Bcl-2 ratio at gene and protein levels compared with the MPTP-lesioned group. These observations provide some clues about the potential anti-apoptotic mechanisms of ECH outside of effects on caspase families. Though our preliminary study has not determined the phenotype of apoptotic cells previous research hints that TH-positive neurons undergo apoptotic degeneration and lose their TH activity but not glial cells in the non-acute MPTP mouse model of PD (Novikova et al., 2006). In addition, our study further confirms the involvement of Bcl-2 family members in the pathogenesis of a subacute MPTP-induced mouse model of PD (Parkinson's disease). It remains unclear whether there are common pathways with respect to the relationship between Bcl-2 family members and NTFs. Nonetheless, our current results suggest that increasing the endogenous GDNF and/or BDNF levels and regulating expressions of Bcl-2 pro-apoptotic family members may be a useful strategy for neuronal regeneration therapy

Finally the effects of ECH (Echinacoside) on relieving gait impairment in the MPTP mouse model illustrate its potential as symptomatic medication focusing on nondopaminergic targets, which are currently the focus of promising investigation in clinical trials. In PD (Parkinson's disease), motor symptoms such as bradykinesia and rigidity respond well to dopamine replacement medications. Balance and gait problems may also be reversed by dopaminergic agents early in the course of the disease, but they usually become resistant with disease progression (Meredith and Kang, 2006). In our study, short-term (7 days) administration with a high dose of ECH (Echinacoside) had a positive effect on the stride length of mice, suggesting that it may be a promising supplement for dopamine replacement medications.

It should be noted that our study also investigated the glial cell expression by means of immunofluorescence, but no change of astroglial or activated microglial expression was observed in MPTP-lesioned mice treated with ECH (Echinacoside) when compared with saline-treated MPTP mice (data not shown). Notwithstanding we can suggest that the effects of ECH (Echinacoside) are not confined to the above poles of the dopaminergic system, and next studies will be required to define the full roles of ECH and mechanisms in rodent or primate models of PD (Parkinson's disease).

In conclusion, the present study demonstrates that ECH (Echinacoside) exhibited neurorescue effects on MPTP-induced loss of dopaminergic neurons and fibers in vivo as well as on impairment of behavior. These effects appeared to be correlated with the multifactorial activities of this compound, including relieving MPTP toxicity to DAT, enhancing expressions of neurotrophic factors GDNF/BDNF, and suppressing abnormal apoptosis in the ventral midbrain of subacute MPTPlesioned mice through the regulation of Bcl-2 family members. Combined with the results from our previous study regarding the neuroprotective effects of ECH (Echinacoside) in vitro, we presume that this compound has the potential not only to delay the progression of PD (Parkinson's disease) but also to reverse existing neurodegenerative damage and even to improve symptoms. In concert, these data suggest that ECH (Echinacoside) is a promising, clinically applicable approach for the treatment of PD (Parkinson's disease).

4. Experimental procedures

4.1. Animal protocols

All procedures were approved by the Animal Ethical Committee of Zhongshan Hospital Fudan University and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experiments were conducted using male C57BL/6 mice purchased from the Shanghai Slac Laboratory Animal Company (Shanghai, China) at 10 weeks of age and weighing 24–26 g. The animals were maintained in standard conditions (12 ⁄ 12 h light ⁄ dark cycle, 21± 2 °C, and relative humidity of 40%) and allowed access to food and water ad libitum.

4.2. Administration of MPTP

Mice received daily intraperitoneal (i.p.) injections of vehicle (saline) or MPTP (30 mg/kg/day; Sigma, USA) dissolved in physiological saline for five consecutive days to induce Parkinsonism. Each treatment group included ten mice. ECH was purchased from Chengdu Push Bio-Technology Co. Ltd (Sichuan, China). The extract complies with the Enterprise Standard and was assayed by HPLC≥98%. ECH (Echinacoside) was dissolved in physiological saline and adjusted the pH to 7.4 before it was given by means of intragastric (i.g.) administration. The effects of ECH (Echinacoside) on MPTP-induced parkinsonism were studied in the following experimental groups: Group I, NS (saline, i.p.)+NS (i.g.); Group II, MPTP (i.p.)+NS (i.g.); Group III, MPTP (i.p.)+ECH 10 mg/ kg/day (i.g.); Group IV, MPTP (i.p.)+ECH 20 mg/kg/day (i.g.); Group V, MPTP (i.p.) + ECH 30 mg/kg/day (i.g.); Group VI, NS (i.p.) + ECH 30 mg/kg/day (i.g.).

Animals in Groups I–VI received saline or MPTP (i.p.) daily for 5 days; vehicle (saline) or ECH (Echinacoside) treatment (i.g.) was started 72 h after the last MPTP administration and given daily for 14 days. Mice were killed by cervical dislocation or perfusion after the last vehicle or ECH administration and behavior observation.

4.3. Gait test

The gait test was performed according to previously published methods (Fernagut et al., 2002; Fleming et al., 2004; Tillerson et al., 2002) with a minor modification. The apparatus was composed of a runway, 4.5 cm (width )× 40 cm (length) ×12 cm (height) illuminated by a light (60 W) and a black wooden box, 20 cm (width)× 17 cm (length)× 10 cm (height) placed at one end of the runway. Animals' fore and hind paws were wet with pencil blue ink and they were allowed to trot on a strip of paper (4.5 cm width, 40 cm length) down the brightly lit runway towards the dark goal box. Stride lengths were measured manually as the distance between two paw prints. The three longest stride lengths (corresponding to maximal velocity) were measured from each run. Pawprints made at the beginning (7 cm) and at the end (7 cm) of the run were excluded because of velocity changes. Runs in which the experimenter observed mice making stops or obvious decelerations were excluded from the analysis. The behavioral assessment was performed 3 days before the first MPTP injection and on the 7th and 14th day of ECH (Echinacoside) or vehicle treatment.

4.4. Perfusion and tissue processing

At the end of the experiment, half of the animals (n= 5) in each group were sacrificed under 10% chloral hydrate overdose anesthesia (360 mg/kg) then perfused via intracardial infusion with saline (0.9%) followed by 4% paraformaldehyde (PFA), pH7.4. After intracardial perfusion, brains were collected and post-fixed in 4% PFA for 24 h at 4 °C, embedded in paraffin, and cut into 5 µm coronal sections encompassing the entire substantia nigra compact part (SNpc) and striatum (ST) (anteroposterior levels: Bregma −2.92 mm to−3.64 mm and +0.02 to+0.86 mm) with the aid of the mouse brain atlas (Franklin and Paxinos, 1996).

Another 5 animals in each group were killed by cervical dislocation and the tissue of their ventral midbrain and striatum were dissected rapidly on ice (Jackson-Lewis and Przedborski, 2007) frozen in liquid nitrogen and stored at −80 °C until used.

Echinacoside in Cistanche has Neurotrophic and neurorescue effects on Parkinson's disease

4.5. Tyrosine hydroxylase and Nissl immunohistochemistry

Immunohistochemistry of brain tissues was performed as previously described (He et al., 2008; Yokoyama et al., 2008) with minor modifications. Briefly, one set of sections through the SNpc (consisting of 14–15 sections) was prepared for Nissl staining (cresyl violet) using routine procedures while another set through the SNpc and one set through the ST (consisting 15–16 sections) was processed for tyrosine hydroxylase (TH) immunohistochemistry. The latter sections were incubated with 0.3% hydrogen peroxide (H2O2) for 10 min at room temperature to remove the endogenous peroxidase activity and then placed in a blocking buffer containing 10% goat serum with 0.2% Triton X-100 in 0.01 M PBS (pH 7.2) for 30 min at 37 °C. In each treatment, the slides were washed at least three times with 0.01 M PBS each for 5 min. They were then incubated at 4 °C overnight with mouse anti-TH monoclonal antibody (Sigma, USA) at 1:2000 dilution in 0.01 M PBS containing 1% goat serum and 0.2% Triton X-100. On the second day, sections were treated for 30 min with biotinylated anti-mouse IgG and then processed with streptavidin–peroxidase complex (ABC kit, Vector, USA). The peroxidase reaction was visualized by 0.05% diaminobenzidine (DAB) with 0.03% H2O2 in Tris–HCl buffer. Sections through SNpc were counterstained with cresyl violet (Sigma, USA) as well for anatomical reference. For controls, sections were processed as described above, except that there was no primary antibody used. Control sections were immunonegative

4.6. Quantification of TH-immunoreactive neurons and fibers

Every 10th section (total 14–15 sections) through SNpc corresponding to the Bregma −2.92 mm to −3.64 mm was analyzed using an Olympus BX51 microscope with a DP71 camera (Olympus, USA). After delineation of the SNpc at low magnification (4× objective) according to the mouse brain atlas, Nissl-stained and TH-immunoreactive cells in the right side were counted by two observers who were blind to treatment at higher magnification (20× objective). TH-positive (TH and cresyl violet positive) and Nissl-stained (cresyl violet positive) cells with nucleus were both counted to ensure that the treatment effects were related to TH cell increase rather than an up-regulation of the enzyme. Note that the analyses of the TH-immunoreactive profiles were restricted to the SNpc and thus excluded the ventral tegmental area.

The optical density (OD) of striatal dopaminergic fibers was analyzed on a computer using Image-Pro Plus software, following the previously described optical dissector method (Burke, et al., 1990; Wu et al., 2002). The average labeling for each area was calculated on five automatically matched brain sections of the same animal corresponding to the Bregma +0.38 mm–+0.50 mm in a mouse brain atlas (n= 5 each group). The right side of striatal images was acquired at the same magnification (10× objective) and the OD of staining was thus assessed for the entire region of the sections automatically. Background intensities taken from the corpus callosum devoid of TH staining were subtracted from every measurement.

4.7. TUNEL assay

Serial brain coronal sections through the SNpc corresponding to the Bregma −2.92 mm to −3.64 mm were collected with the aid of the mouse brain atlas (Franklin and Paxinos, 1996). At least one in every 10 sections (total 14–15 sections) from each animal were processed for in situ end-labeling of fragmented DNA staining (TUNEL) by using the In Situ Cell Death Detection Kit, AP (Roche, Germany) according to the manufacturer's instructions and literature with minor modification (Novikova et al., 2006; He et al., 2008). Negative controls were performed in parallel without the terminal deoxynucleotidyl transferase. Sections were finally counter-stained with hematoxylin. The total number of TUNELpositive cells on one slide (total 14–15 sections, right side) from each animal were counted blindly under a light microscope at 20× objective magnification within the anatomical boundaries of SNpc.

4.8. HPLC

Striatal tissue was assayed for levels of DA and its metabolites using high-pressure liquid chromatography (HPLC) Coulochem III systems with electrochemical detection (ESA Inc., Chelmsford, MA) as previously described with minor modification (Petroske et al., 2001; Schneider et al., 2006). Briefly, the striatum was dissected out and weighed (left side), placed in plastic tubes, homogenized by sonication for 10 s in 0.4 M perchloric acid, and centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatants were passed through a 0.2-μm millipore filter. The mobile phase, consisting of 100 mM NaH2PO4, 50 mM citric acid, 6% methanol, 1.7 mM 1-octane sulphonic acid, 50 μM EDTA, 10% acetonitrile, was delivered at a flow rate of 0.6 ml/min at 25 °C to the reversed-phase column. Aliquots of 20 μl of the supernatant solution were injected by HPLC autosampler with the cooling module set at 4 °C. Data were calibrated with an external standard. The levels of dopamine and its metabolites were calculated and expressed as ng/mg wet tissue weight.

4.9. Real-time PCR

Total RNA was isolated by homogenizing frozen tissue of the ventral midbrain (left side) in 1 ml of Trizol reagent (Invitrogen, USA) followed by isopropanol precipitation (n= 5). Pellets were washed with 70% ethanol and suspended in RNase-free water, and the concentration of RNA was determined using a GeneQuant RNA/DNA calculator (Amersham Biosciences, NJ). RNA (1 μg) was taken as a template and total cDNA synthesis was performed using a PrimeScriptTM RT reagent Kit (Takara, Japan). SYBR Green PCR amplification was performed in a Realplex4 S Real-time PCR instrument (Eppendorf, German; Premix Ex TaqTM, Takara). As an endogenous control, β-actin was labeled with a reporter dye. The thermocycling profile conditions were as follows: 95 °C 3 min initial denaturation, then 40 cycles of 95 °C 15 s denaturation, 61 °C 15 s annealing, with a 72 °C 45 s final extension. The relative fold changes were determined by the method of 2− ΔΔCt as described previously (Livak and Schmittgen, 2001). Primer sequences were as follows in Table 2. All primers were designed according to the relevant literature and synthesized by Genemed Company.

4.10. Western blot

For Western blot analysis, isolated tissues from striatum (for DAT and β-actin assay) or ventral midbrain (for GDNF,BDNF, Bax,Bcl-2 and β-actin assay) were homogenized in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1%NP–40, 0.5% sodium deoxycholate, 0.1% SDS and sodium orthovanadate, sodium fluoride, EDTA, leupeptin) and centrifuged at 13,000 rpm at 4 °C for 5 min. The total protein content in the supernatant was determined using a BCA Protein Assay Kit with a spectrophotometer, and it was diluted to an appropriate final concentration with the homogenization buffer and a protein solubilization solution. The sample was boiled for 3 min, and 20 μl (containing 30 μg of protein) of each sample was applied to an SDS polyacrylamide (10%) gel for electrophoresis. The gel was run at 80 V for 2 h then electrophoretically transferred to a nitrocellulose membrane in transfer buffer using Trans-Blot SD (Bio-Rad) at 20 V for 1.5 h, then blocked by immersion in TBST containing 1% BSA for 4 h at RT and incubated at 4 °C overnight with respective primary antibodies: rabbit anti-DAT (1:200, Chemicon International Inc, USA), mouse anti-GDNF, BDNF (1:200, Santa Cruz Biotechnology, USA), mouse anti-Bax, Bcl-2,β-actin (1:1000, Santa Cruz). After rinsing three times in TBST for 10 min, blots were incubated for 2 h at RT with anti-rabbit IgG-peroxidase conjugated (1:2000, Santa Cruz). Immunoreactivity was visualized by an ECL fluorescence detection system (GE, U.S.A.). The blots were scanned by KODAK In-Vivo Multispectral Imaging Systems FX (Carestream Health, U.S.A) for a 5 min exposure time, and pictures were acquired by CCD automatically. The intensity of protein bands was measured by densitometry and expressed as a ratio to β-actin intensity to ensure that changes in proteins levels were not simply due to differences in amounts of sample loading (Matsunaga et al., 2006)

4.11. Statistical analyses

All data were represented as mean± SEM. One-way ANOVA followed by a post-hoc analysis of Tukey's HSD and Student– Newman–Keuls multiple comparisons test was performed for statistical purposes. A value of p< 0.05 was considered statistically significant.

Acknowledgments

This work was supported by the Scientific and Technical Supporting Program, the Ministry of Science and Technology of China (No. 2006BAI04A11-3), and the Medical Leader Sponsorship by Shanghai Municipal Government (No. 2007- 057). We thank Professor Fang Huang and Professor Danian Zhu for their guidance on the experiments and manuscript.

Echinacoside in Cistanche has Neurotrophic and neurorescue effects on Parkinson's disease

From: ' Neurotrophic and neurorescue effects of Echinacoside in the subacute MPTP mouse model of Parkinson's disease ' by Qing Zhao, et al

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