Neuroprotective effects of extract of Acanthopanax senticosus harms on SH-SY5Y cells overexpressing wild-type or A53T mutant α-synuclein

Phytomedicine 21 (2014) 704–711

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Neuroprotective effects of extract of Acanthopanax senticosus harms on SH-SY5Y cells overexpressing wild-type or A53T mutant ␣-synuclein Xu-zhao Li a , Shuai-nan Zhang a , Ke-xin Wang a , Hong-yu Liu b , Zhi-ming Yang a , Shu-min Liu a,c,∗ , Fang Lu a,∗∗ a Chinese Medicine Toxicological Laboratory, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China b School of Basic Medical Sciences, Nanjing University of Chinese Medicine, Nanjing 210046, PR China c Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, Harbin 150040, PR China

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Article history: Received 26 August 2013 Received in revised form 16 September 2013 Accepted 11 October 2013 Keywords: Acanthopanax senticosus harms Parkinson’s disease ␣-Synuclein

a b s t r a c t Extract of Acanthopanax senticosus harms (EAS) has been shown to have neuroprotective effects on dopaminergic neurons in Parkinson’s disease (PD) mice model. ␣-Synuclein is a key player in the pathogenesis of PD, the elevated level of which is deleterious to dopaminergic neurons, and enhancing its clearance might be a promising strategy for treating PD. To assess the potential of EAS in this regard, we investigated its effect on the SH-SY5Y cells overexpressing wild-type ␣-synuclein (WT-␣-Syn) or A53T mutant ␣-synuclein (A53T-␣-Syn), and the implicated pathway it might mediate. After treatment with EAS, the changes of ␣-synuclein, caspase-3, parkin, phospho-protein kinase B (Akt), phospho-glycogen synthase kinase 3 beta (GSK3␤), and phospho-microtubule-associated protein tau (Tau) in WT-␣-Syn or A53T-␣-Syn transgenic cells were reverted back to near normal levels, demonstrated by the western blotting and quantitative real-time PCR outcomes. The neuroprotective effects of EAS may be able to protect WT-␣-Syn or A53T-␣-Syn transgenic SH-SY5Y cells from ␣-synuclein overexpression and toxicity. Therefore, we speculate that EAS might be a promising candidate for prevention or treatment of ␣-synuclein-related neurodegenerative disorders such as PD. © 2013 Elsevier GmbH. All rights reserved.

Introduction Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s disease, is characterized primarily by a progressive degeneration of the dopaminergic neurons in

Abbreviations: A53T-␣-Syn, A53T mutant ␣-synuclein; Akt, protein kinase B; DMSO, dimethyl sulfoxide; EAS, extract of Acanthopanax senticosus harms; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GSK3␤, glycogen synthase kinase 3 beta; IOD, integral optical density; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PD, Parkinson’s disease; Tau, microtubule-associated protein tau; TCM, traditional Chinese medicine; WT␣-Syn, wild-type ␣-synuclein. ∗ Corresponding author at: Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China. Tel.: +86 451 82193278; fax: +86 451 82193278. ∗∗ Corresponding author at: Chinese Medicine Toxicological Laboratory, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, PR China. Tel.: +86 451 87266814; fax: +86 451 87266814. E-mail addresses: [email protected] (S.-m. Liu), lufang [email protected] (F. Lu). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.10.012

the substantia nigra leading to a dopamine deficit in the striatum (Winklhofer and Haass, 2010). ␣-Synuclein, the elevated level of which is deleterious to dopaminergic neurons, is a key player in the pathogenesis of PD based on genetic, neuropathologic, and cellular/molecular lines of evidence (Eriksen et al., 2003; Lim et al., 2003; Mouradian, 2002). ␣-Synuclein accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons (Junn et al., 2009). Insulin signaling pathway of the central nervous system is also involved in the pathogenesis of PD (Fernandes et al., 2001; Takahashi et al., 1996), which is generally thought to proceed through receptor-mediated tyrosine phosphorylation of insulin receptor substrate-1 and/or 2. This leads to activation of phosphoinositide 3-kinase, which phosphorylates and activates protein kinase B (Akt) (Li et al., 2010). The activation of Akt (phosphoAkt) can protect neuronal cell against glycogen synthase kinase 3 beta (GSK3␤) activity through phosphorylating it into phosphoGSK3␤ (Crespo-Biel et al., 2007; Gavalda et al., 2004; Hetman et al., 2000). In addition, microtubule-associated protein tau (Tau) is also linked with PD, the major biological function of which is to stabilize microtubules and facilitate axonal transport (Lei et al., 2010).

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The capacity of Tau to maintain its normal biological function is dependent upon its phosphorylation state (Lei et al., 2010). Tau hyperphosphorylation (phospho-Tau) may be associated with apoptosis and contribute to the brain damage, which is due to an increase in GSK3␤ activity in combination with an inactivation of Akt (Crespo-Biel et al., 2007; Duka et al., 2009; Wen et al., 2004; Yeung et al., 2010). ␣-Synuclein may also lead to hyperphosphorylation of Tau, and the presence of both proteins in Lewy bodies may imply a physiological or pathophysiological interaction in PD (Jellinger, 2011; Lei et al., 2010; Muntane et al., 2008). The earliest description of PD was traced in the Yellow Emperor’s Internal Classic, a book written 2000 years ago. In traditional Chinese medicine (TCM), PD is termed as “shaking palsy”, a syndrome characterized by tremors, numbness and limpness and weakness of the four limbs, and the pathologic features of PD are liver-kidney Yin deficiency and qi-blood deficiency (Li et al., 2013b). TCMs are gaining more attention for the treatment of PD, due to their specific theory and long historical clinical practice. They are composed of various kinds of components and using them would act on multiple targets and control a complex syndrome well, such as PD (Li et al., 2013b). Acanthopanax senticosus (Rupr. and Maxim.) Harms is a widely used traditional Chinese herb, the geographical distribution of which is mainly indigenous to south-east Asia, northern China, and south-eastern part of the Russian Federation, etc. (WHO, 2004). In the theory of TCM, it is described as following: Indications hypofunction of the spleen and the kidney marked by general weakness, lassitude, anorexia, aching of the loins and knees; insomnia and dream-disturbed sleep (ChPC, 2010). In modern pharmacological researches, we found that Acanthopanax senticosus Harms has neuroprotective features (Bocharov et al., 2010, 2008), stressprotective and adaptogenic activities, etc. (Panossian and Wikman, 2010). Eleutheroside B and eleutheroside E are the major chemical constituents of Acanthopanax senticosus harms. Eleutheroside B has neuroprotective effect on PC12 cells against apoptosis induced by 1-methyl-4-phenylpyridinium ion (Lu et al., 2011). Eleutheroside B and eleutheroside E showed obvious protective effects against A␤ (25–35)-induced atrophies of axons and dendrites (Bai et al., 2011). Our previous studies have indicated that EAS can protect C57BL/6 mice against 1-methyl-4-phenyl-1,2,3,6tetrahydro-pyridine-induced dopaminergic neuronal damage (Liu et al., 2012), the neuroprotection of which can be connected with the regulation of tyrosine metabolism, mitochondrial betaoxidation of long chain saturated fatty acids and fatty acid metabolism, etc. (Li et al., 2013a). Here, we investigate the repression of ␣-synuclein overexpression and toxicity by EAS in WT-␣-Syn or A53T-␣-Syn transgenic SH-SY5Y cells in order to find a possible therapeutic application of this natural extract to PD. Materials and methods Reagents 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma–Aldrich (U.S.A.). Fetal bovine serum (FBS) was purchased from ExCell biology, Inc. (Australia). DMEM/F-12 medium was purchased from HyClone (Thermo Scientific, Beijing, PR China). Primary antibodies for western blotting were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (PR China). The secondary antibodies and anti-GAPDH (loading control) antibody were purchased from Zhongshan Goldenbridge Biotechnology Co., Ltd. (PR China). Plant material and extraction The crude drug is the root and rhizome of Acanthopanax senticosus (Rupr. Et Maxim.) Harms, which was collected in Wuchang (N44

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39 , E127 35 ) of Heilongjiang province, PR China. The voucher specimen (hlj-201003) of the herb was authenticated by Professor Ke Fu, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine. EAS was prepared with the crude drug, which was extracted 3 times with 10 volume of 80% ethanol for 2, 2 and 2 h, respectively, separated and purified by AB-8 macroporous adsorption resin (polarity: weak-polar, particle diameter: 0.3–1.25 mm, surface area: 480–520 m2 /g, average pore diameter: 130–140 nm, moisture content: 67.3%). The process for purification was as follows: the concentration of sample solution was 500 g/l; adsorption flow rate was 2 bed volumes/h; eluent was 30% ethanol; the dosage was 9 bed volumes; elution flow rate was 1 bed volume/h. The ethanol phase was evaporated under vacuum and then oven dried at 60 ◦ C to give the extracts. The yield of the extract is 1.3% (w/w) (Liu et al., 2012). The contents of eleutheroside B and eleutheroside E in EAS were 7.63 ± 0.34% (w/w) and 10.90 ± 0.22% (w/w) respectively. The fingerprint analysis of EAS was shown in Fig. 1.

Cell culture SH-SY5Y cells, a human neuroblastoma cell line purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, PR China), were maintained in DMEM/F-12 medium supplemented with 10% FBS and 1% penicillin–streptomycin (Hyclone) in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦ C. The media was replaced every 2 days.

Stable transfection of SH-SY5Y cells For stable transfection of SH-SY5Y cells, the pReceiverLv122 expression vectors (GeneCopoeia, U.S.A.) containing a cytomegalovirus promoter were used. The destination vectors are designed to allow high-level expression of recombinant fusion proteins in dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. WT-␣-Syn or A53T-␣-Synenhanced green fluorescent protein (EGFP) fusion constructs were PCR amplified and expression clones were created in the pReceiverLv122 expression vectors at the downstream of a cytomegalovirus promoter. The orientation and sequence of each construct was confirmed by restriction analysis and DNA sequencing. Lentivirus encoding WT-␣-Syn or A53T-␣-Syn-EGFP fusion constructs were generated by co-transfecting the pReceiver-Lv122 expression construct together with the Lenti-PacTM HIV Expression Packaging Kit (GeneCopoeia, U.S.A.) into the 293T lentiviral packaging cells according to the manufacturer’s protocols. Then WT-␣-Syn or A53T-␣-Syn constructed in lentivirus was transfected into SH-SY5Y cell line. EGFP fluorescence was visible in transfected cells after 3–4 days (shown in Fig. 2); the transfection efficiency of the primary cells was over 70%. The individual stably transfected colony was subsequently selected in the presence of puromycin (2 ␮g/ml). Protective effects of EAS on cell viability The cells were seeded in a 96-well plate (5000 cells/well), and maintained in DMEM/F-12 medium with 10% FBS at 37 ◦ C in an atmosphere of 5% CO2 for 48 h. Various concentrations of EAS, such as 0, 50, 100, 200, 300 and 400 ␮g/ml, were added in each well, and subsequently incubated at 37 ◦ C in 5% CO2 incubator for 48 h. Then the media were removed and 100 ␮l of DMEM/F-12 medium containing MTT (final concentration is 0.5 mg/ml) was added to each well and further incubated for 4 h. The MTT solution was aspirated off, and the cell crystals were dissolved using 150 ␮l dimethyl sulfoxide (DMSO). Finally, color intensity was measured using a

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Fig. 1. The UPLC-TOF/MS BPI profile of EAS in (A) positive and (B) negative ESI mode. Waters acquityTM UPLC was equipped with a reversed-phase ACQUITY UPLCTM HSS T3 column (2.1 mm × 100 mm, 1.8 ␮m, Waters Corp, Milford, USA). The analytical column was maintained at a temperature of 30 ◦ C and the mobile phases was composed of phase A (water with 0.1% formic acid) and phase B (acetonitrile containing 0.1% formic acid). A solvent gradient system was used: 0–1.5 min, 18% A; 1.5–2 min, 18–35% A; 2–3 min, 35–100% A; 3–4.5 min, 100% A. The flow rate was 0.3 ml/min. Injection volume was 5 ␮l. The UPLC system was connected to a time-of-flight mass spectrometer Waters-Micromass LCT Premier TOF equipped with an electrospray interface operating in the positive and negative ion mode, using the following parameters: capillary voltage, 1000 V; sample cone voltage, 40 V; source temperature, 100 ◦ C; desolvation temperature, 350 ◦ C; desolvation gas flow, 700 l/h; cone gas flow, 20 l/h.

Tecan Infinite M200 microplate reader (Tecan Inc., Maennedorf, Switzerland) at 570 nm with the reference of 630 nm. Sample treatment for cellular ultramicrostructure, western blotting and quantitative real-time PCR analysis

A53T-␣-Syn transgenic SH-SY5Y cells for 48 h, respectively; WT-␣Syn+EAS and A53T-␣-Syn+EAS groups were incubated in 200 ␮g/ml EAS with WT-␣-Syn and A53T-␣-Syn transgenic SH-SY5Y cells for 48 h, respectively. Cellular ultramicrostructure analysis

The cells were divided into 5 groups: control, WT-␣-Syn and A53T-␣-Syn groups were incubated in DMEM/F-12 medium containing 10% FBS with normal SH-SY5Y cells, WT-␣-Syn and

For the electron microscopy study, cells were fixed in the 3% glutaraldehyde, and then processed by osmium tetroxide, dehydrated,

Fig. 2. EGFP fluorescence in (A) WT-␣-Syn or (B) A53T-␣-Syn transgenic SH-SY5Y cells were visualized by fluorescence microscopy under 100× magnification.

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and embedded in epoxy resin. 90 nm sections were examined and photographed using transmission electron microscope (TECNAI G2, Netherlands) at 100 kV.

Western blotting analysis The protein samples were extracted from SH-SY5Y cell line and the concentrations were determined with BCA Protein Assay Kit. Equal amounts (40 ␮g) of protein were separated in a polyacrylamide gel, transferred to nitrocellulose membranes at 50 mA for 40–60 min, and blocked for 2 h at room temperature with Tris-buffered saline containing 0.05% Tween 20 (TBST (pH 7.4)) and 5% nonfat dried milk. After washing 3 times for 10 min each in PBS, membranes were incubated with rabbit anti-human ␣synuclein, caspase-3, parkin, phospho-Akt, phospho-GSK3␤, and phospho-Tau antibodies, respectively, at 1:1000 concentration each overnight at 4 ◦ C. Membranes were then washed again with PBS, 3 times for 10 min each, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The protein bands were visualized with 4chloronaphthol (Sigma, U.S.A.). The integral optical density (IOD) of bands was determined by Gel-Pro 4.0 software (Media Cybernetics, Inc. U.S.A.). The levels of target protein were expressed as the ratio of target protein IOD to GAPDH IOD.

RNA isolation and quantitative real-time PCR Total RNA was extracted using RNApure rapid extraction kit (BioTeKe Corporation, PR China). Reverse transcription were performed with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, U.S.A.) for ␣-synuclein, caspase-3, parkin mRNA following standard protocols. Quantitative real-time PCR was performed using One Step SYBR® PrimeScript® RT-PCR Kit (Takara, PR China) with ABI-7500 PCR machine (Applied Biosystems, U.S.A.). Amplification procedure was 95 ◦ C for 30 s, followed by 40 cycles at 95 ◦ C for 5 s, 60 ◦ C for 34 s. Primers for ␣-synuclein mRNA were 5 -TGACGGGTGTGACAGCAGTAG-3 (forward) and 5 -CAGTGGCTGCTGCAATGC-3 (reverse), primers for caspase-3 mRNA were 5 -TGGTTCATCCAGTCGCTTTG-3 (forward) and 5 -TAGCCCTCTGCTCCATCCTG-3 (reverse), primers for parkin mRNA were 5 -AGCCTCCAAGCCTCTAAATG-3 (forward) and 5 -CACGGACTCTTTCTTCAT-3 (reverse). The relative levels of ␣-synuclein, caspase-3, parkin mRNA in SH-SY5Y cells were calculated against GAPDH mRNA (internal control) using the 2−CT method.

Statistical analysis Statistical analysis data were presented as mean ± standard deviation (¯x ± s). A one-way analysis of variance (ANOVA) and a multiple range least significant difference (LSD) were used for intergroup comparisons. Statistical software SPSS 18.0 was applied. Statistical significance was accepted if p < 0.05.

Results Protective effects of EAS on cell viability (shown in Fig. 3) Compared with that in control group (p < 0.05), the cell viability were increased by 13.0%, 38.7% and 24.9% in 200, 300 and 400 ␮g/ml EAS groups, respectively. The cell viability in 50 and 100 ␮g/ml EAS groups were also more than that in control group, but there was no significant difference.

Fig. 3. Protective effects of EAS on cell viability. Cell viability was measured by MTT method. Data are expressed as mean ± SE of the percentage of control of the three independent experiments. a p < 0.05 vs control group.

Ultrastructural changes in WT-˛-Syn or A53T-˛-Syn transgenic SH-SY5Y cells (shown in Fig. 4) The morphology of the WT-␣-Syn or A53T-␣-Syn transgenic SHSY5Y cells were confirmed by analysis at an ultrastructural level. As shown in Fig. 4A, the normal-appearing cells were easily detectable in control group. Cellular and nuclear membranes, and cell organ were complete. The nucleus contained a large, rounded, and distinctly bounded chromatin clump. In WT-␣-Syn and A53T-␣-Syn groups, morphological alterations were observed in the nucleus and cytoplasm (shown in Fig. 4B and C). Most of the cells were injured, including nuclear membrane shrinkage and lots of intracellular vacuoles formation. As shown in Fig. 4D and E, the impaired cells were significantly ameliorated after the treatment of EAS. Cellular and nuclear membranes were relatively complete. Within the cytoplasm, fewer of intracellular vacuoles were observed in the SH-SY5Y cells overexpressing WT-␣-Syn or A53T-␣-Syn. Western blotting analysis and the levels of target protein in SH-SY5Y cells (shown in Fig. 5) The levels of ␣-synuclein, caspase-3, and phospho-Tau in WT-␣Syn and A53T-␣-Syn groups were increased significantly, and the levels of parkin, phospho-Akt, and phospho-GSK3␤ in WT-␣-Syn and A53T-␣-Syn groups were decreased significantly, compared with those in control group (p < 0.05). Compared with those in WT-␣-Syn group (p < 0.05), the levels of ␣-synuclein, caspase-3, and phospho-Tau in WT-␣-Syn+EAS group were decreased significantly, and the levels of parkin, phospho-Akt, and phospho-GSK3␤ in WT-␣-Syn+EAS group were increased significantly. Compared with those in A53T-␣-Syn group (p < 0.05), the levels of ␣synuclein, caspase-3, and phospho-Tau in A53T-␣-Syn+EAS group were decreased significantly, and the levels of parkin, phosphoAkt, and phospho-GSK3␤ in A53T-␣-Syn+EAS group were increased significantly. Effects of EAS on the expressions of ˛-synuclein, caspase-3, parkin mRNA in SH-SY5Y cells (shown in Fig. 6) The expressions of ␣-synuclein and caspase-3 mRNA in WT-␣Syn and A53T-␣-Syn groups were up-regulated significantly, and the expressions of parkin mRNA in WT-␣-Syn and A53T-␣-Syn groups were down-regulated significantly, compared with those in control group (p < 0.05). Compared with those in WT-␣-Syn group (p < 0.05), the expressions of ␣-synuclein and caspase-3 mRNA in WT-␣-Syn+EAS group were down-regulated significantly, and the

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Fig. 4. Effects of EAS on the ultrastructural changes in WT-␣-Syn or A53T-␣-Syn transgenic SH-SY5Y cells. Panels A-E demonstrate representative SH-SY5Y cells from each group at 4200× magnification. (A) Control group: cellular and nuclear membranes, and cell organ were complete, and chromatin distributed uniformly; (B) WT-␣-Syn and (C) A53T-␣-Syn groups: most of the cells were injured, including nuclear membrane shrinkage (black arrow) and lots of intracellular vacuoles formation (open arrow); (D) WT-␣-Syn+EAS and (E) A53T-␣-Syn+EAS groups: the impaired cells were significantly ameliorated, and relatively complete nuclear membrane (black arrow) and fewer of intracellular vacuoles (open arrow) were observed in the SH-SY5Y cells overexpressing WT-␣-Syn or A53T-␣-Syn. Scale bar: 2 ␮m (A–E).

expressions of parkin mRNA in WT-␣-Syn+EAS group were upregulated significantly. Compared with those in A53T-␣-Syn group (p < 0.05), the expressions of ␣-synuclein and caspase-3 mRNA in A53T-␣-Syn+EAS group were down-regulated significantly, and the expressions of parkin mRNA in A53T-␣-Syn+EAS group were upregulated significantly. Discussion Based on MTT assay (shown in Fig. 3), various concentrations of EAS, and 200, 300 and 400 ␮g/mL of EAS in particular, can protect the cells and play a role in promoting normal SH-SY5Y cells growth. The protective abilities of EAS may be good for the inhibition of cell death induced by ␣-synuclein. Abnormal deposit of certain proteins is a common pathology for many neurodegenerative disorders, therefore, increasing their clearance might be a strategy applicable to all conditions involving protein aggregation (Lan et al., 2012). ␣-Synuclein, the elevated level of which is

deleterious to dopaminergic neurons, accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons (Junn et al., 2009). In the present experiment, the expressions of ␣-synuclein were significantly enhanced in WT-␣-Syn and A53T-␣-Syn groups. Meanwhile, we observed that the levels of ␣-synuclein were decreased in WT-␣-Syn+EAS and A53T-␣-Syn+EAS groups. The results may indicate that EAS could enhance the clearance of ␣synuclein and repress its cytotoxicity. As shown in Fig. 4, EAS could significantly ameliorate the impaired cells induced by WT-␣-Syn or A53T-␣-Syn, and reverse cell death. In addition, ␣-synuclein, the toxicity induced by which can be suppressed by parkin (Oluwatosin-Chigbu et al., 2003; Petrucelli et al., 2002), induces apoptotic cell death in the neuronal cells via a caspase-3 signaling cascade (Adamczyk et al., 2010). Parkin purges damaged organelles from the vital mitochondrial network (Tanaka, 2010) and rescues mitochondrial dysfunction induced by knockdown of mortalin, mutation in whose gene is the most common

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Fig. 5. Western blotting analysis and the levels of target protein in the SH-SY5Y cells. Membranes were incubated with rabbit anti-human (A) ␣-synuclein, (B) caspase-3, (C) parkin, (D) phospho-Akt, (E) phospho-GSK3␤ and (F) phospho-Tau antibodies, respectively, at 1:1000 concentration each. GAPDH was used as a loading control. The IOD of bands was determined by Gel-Pro 4.0 software. The levels of target protein were expressed as the ratio of target protein IOD to GAPDH IOD. Data represent the mean ± SE of each group (n = 6). Values are from three independent experiments. a p < 0.05 vs control group, b p < 0.05 vs WT-␣-Syn group, c p < 0.05 vs A53T-␣-Syn group.

cause of autosomal recessive PD (Yang et al., 2011). Pre-study has reported caspase-3 is expressed in postmitotic dopaminergic neurons and caspase-3 activation could result in dopaminergic neuronal apoptosis in PD model, which may represent a common integration point and constitute an attractive target for antiapoptotic therapy in PD (Hartmann et al., 2000). The results from our study were consistent with the previous report. In this experiment, parkin deficiency was unable to suppress the toxicity induced by ␣-synuclein, which could activate the expression of caspase3 to induce apoptotic cell death in WT-␣-Syn and A53T-␣-Syn

groups. In SH-SY5Y cells overexpressing WT-␣-Syn or A53T-␣Syn, EAS could revert back the abnormal expressions of parkin and caspase-3 to near normal levels. These results shown that EAS could repress ␣-synuclein overexpression and toxicity via up-regulating the expression of parkin, and then inactivate caspase-3 to inhibit apoptotic cell death in the neuronal cells. Intriguingly, insulin signaling pathway of the central nervous system is also involved in the pathogenesis of PD (Fernandes et al., 2001; Takahashi et al., 1996). In the present study, EAS could increase the levels of phospho-Akt and phospho-GSK3␤ and protect

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anti-PD mechanism of EAS for further clarification and studies in the pre-clinical and clinical area. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This article is supported by the National Natural Science Foundation of China (81073019), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20102327110004), and the outstanding innovative talent support programs of Heilongjiang University of Chinese Medicine. References

Fig. 6. The relative levels of (A) ␣-synuclein, (B) caspase-3, and (C) parkin mRNA in SH-SY5Y cells. These gene expressions were analyzed by quantitative real-time PCR and calculated against GAPDH mRNA (internal control) using the 2−CT method. Data represent the mean ± SE of each group (n = 6). Values are from three independent experiments. a p < 0.05 vs control group, b p < 0.05 vs WT-␣-Syn group, c p < 0.05 vs A53T-␣-Syn group.

neuronal cell against phospho-Tau activity, which is associated with apoptosis and contribute to the brain damage (Crespo-Biel et al., 2007; Duka et al., 2009; Wen et al., 2004; Yeung et al., 2010). The repression of ␣-synuclein expression by EAS could also lead to a decrease in the level of phospho-Tau, inhibit the presence of both proteins in Lewy bodies, and attenuate their physiological or pathophysiological interaction in PD (Jellinger, 2011; Lei et al., 2010; Muntane et al., 2008). In conclusion, the findings of this study illustrate the repression of ␣-synuclein overexpression and toxicity by EAS in WT-␣-Syn or A53T-␣-Syn transgenic SH-SY5Y cells, and demonstrate the neuroprotective effects of EAS could suppress ␣-synuclein overexpression and toxicity via activating the expression of parkin, and then inactivate caspase-3 activity to inhibit apoptotic cell death in the neuronal cells. EAS could also activate the insulin signaling pathway to inhibit phospho-Tau activity and its physiological or pathophysiological interaction with ␣-synuclein. Therefore, we speculate that EAS might be a promising candidate for prevention or treatment of ␣-synuclein-related neurodegenerative disorders such as PD. This study will provide better understanding of the

Adamczyk, A., Kazmierczak, A., Czapski, G.A., Strosznajder, J.B., 2010. Alphasynuclein induced cell death in mouse hippocampal (HT22) cells is mediated by nitric oxide-dependent activation of caspase-3. FEBS Letters 584, 3504–3508. Bai, Y., Tohda, C., Zhu, S., Hattori, M., Komatsu, K., 2011. Active components from Siberian ginseng (Eleutherococcus senticosus) for protection of amyloid beta(25–35)-induced neuritic atrophy in cultured rat cortical neurons. Journal of Natural Medicines 65, 417–423. Bocharov, E.V., Ivanova-Smolenskaya, I.A., Poleshchuk, V.V., Kucheryanu, V.G., Il’enko, V.A., Bocharova, O.A., 2010. Therapeutic efficacy of the neuroprotective plant adaptogen in neurodegenerative disease (Parkinson’s disease as an example). Bulletin of Experimental Biology and Medicine 149, 682–684. Bocharov, E.V., Kucherianu, V.G., Bocharova, O.A., Karpova, R.V., 2008. Neuroprotective features of phytoadaptogens. Vestnik Rossiiskoi Akademii Meditsinskikh Nauk, 47–50. ChPC, 2010. Radix et, Rhizoma seu Caulis Acanthopanacis Senticosi (Ciwaujia). Pharmacopoeia of the People’s Republic of China, vol. 1. China Medical Science Press, Beijing, China, pp. 192. Crespo-Biel, N., Canudas, A.M., Camins, A., Pallas, M., 2007. Kainate induces AKT, ERK and cdk5/GSK3beta pathway deregulation, phosphorylates tau protein in mouse hippocampus. Neurochemistry International 50, 435–442. Duka, T., Duka, V., Joyce, J.N., Sidhu, A., 2009. Alpha-synuclein contributes to GSK3beta-catalyzed Tau phosphorylation in Parkinson’s disease models. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 23, 2820–2830. Eriksen, J.L., Dawson, T.M., Dickson, D.W., Petrucelli, L., 2003. Caught in the act: alpha-synuclein is the culprit in Parkinson’s disease. Neuron 40, 453–456. Fernandes, M.L., Saad, M.J., Velloso, L.A., 2001. Effects of age on elements of insulinsignaling pathway in central nervous system of rats. Endocrine 16, 227–234. Gavalda, N., Perez-Navarro, E., Gratacos, E., Comella, J.X., Alberch, J., 2004. Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons. Molecular and Cellular Neurosciences 25, 460–468. Hartmann, A., Hunot, S., Michel, P.P., Muriel, M.P., Vyas, S., Faucheux, B.A., MouattPrigent, A., Turmel, H., Srinivasan, A., Ruberg, M., Evan, G.I., Agid, Y., Hirsch, E.C., 2000. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proceedings of the National Academy of Sciences of the United States of America 97, 2875–2880. Hetman, M., Cavanaugh, J.E., Kimelman, D., Xia, Z., 2000. Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 20, 2567–2574. Jellinger, K.A., 2011. Interaction between alpha-synuclein and tau in Parkinson’s disease comment on Wills et al.: elevated tauopathy and alpha-synuclein pathology in postmortem Parkinson’s disease brains with and without dementia. Exp Neurol 2010; 225: 210–218. Experimental Neurology 227, 13–18. Junn, E., Lee, K.W., Jeong, B.S., Chan, T.W., Im, J.Y., Mouradian, M.M., 2009. Repression of alpha-synuclein expression and toxicity by microRNA-7. Proceedings of the National Academy of Sciences of the United States of America 106, 13052–13057. Lan, D.M., Liu, F.T., Zhao, J., Chen, Y., Wu, J.J., Ding, Z.T., Yue, Z.Y., Ren, H.M., Jiang, Y.P., Wang, J., 2012. Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant alpha-synuclein. Neurochemical Research 37, 2025–2032. Lei, P., Ayton, S., Finkelstein, D.I., Adlard, P.A., Masters, C.L., Bush, A.I., 2010. Tau protein: relevance to Parkinson’s disease. International Journal of Biochemistry & Cell Biology 42, 1775–1778. Li, S., Brown, M.S., Goldstein, J.L., 2010. Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proceedings of the National Academy of Sciences of the United States of America 107, 3441–3446. Li, X.Z., Zhang, S.N., Lu, F., Liu, C.F., Wang, Y., Bai, Y., Wang, N., Liu, S.M., 2013a. Cerebral metabonomics study on Parkinson’s disease mice treated with extract of Acanthopanax senticosus harms. Phytomedicine 20, 1219–1229. Li, X.Z., Zhang, S.N., Liu, S.M., Lu, F., 2013b. Recent advances in herbal medicines treating Parkinson’s disease. Fitoterapia 84, 273–285.

X.-z. Li et al. / Phytomedicine 21 (2014) 704–711 Lim, K.L., Dawson, V.L., Dawson, T.M., 2003. The cast of molecular characters in Parkinson’s disease: felons, conspirators, and suspects. Annals of the New York Academy of Sciences 991, 80–92. Liu, S.M., Li, X.Z., Huo, Y., Lu, F., 2012. Protective effect of extract of Acanthopanax senticosus harms on dopaminergic neurons in Parkinson’s disease mice. Phytomedicine 19, 631–638. Lu, F., Dong, Y., Deng, L., Liu, S., Zhou, S., An, L., Tang, B., 2011. Neuroprotective effect of eleutheroside B on 1-methyl-4-phenylpyridinium ion-induced apoptosis in PC12 cells. Neural Regeneration Research 6 (18), 1375–1379. Mouradian, M.M., 2002. Recent advances in the genetics and pathogenesis of Parkinson disease. Neurology 58, 179–185. Muntane, G., Dalfo, E., Martinez, A., Ferrer, I., 2008. Phosphorylation of tau and alphasynuclein in synaptic-enriched fractions of the frontal cortex in Alzheimer’s disease, and in Parkinson’s disease and related alpha-synucleinopathies. Neuroscience 152, 913–923. Oluwatosin-Chigbu, Y., Robbins, A., Scott, C.W., Arriza, J.L., Reid, J.D., Zysk, J.R., 2003. Parkin suppresses wild-type alpha-synuclein-induced toxicity in SHSY-5Y cells. Biochemical and Biophysical Research Communications 309, 679–684. Panossian, A., Wikman, G., 2010. Effects of adaptogens on the central nervous system and the molecular mechanisms associated with their stress-protective activity. Pharmaceuticals 3, 188–224.

711

Petrucelli, L., O’Farrell, C., Lockhart, P.J., Baptista, M., Kehoe, K., Vink, L., Choi, P., Wolozin, B., Farrer, M., Hardy, J., Cookson, M.R., 2002. Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons. Neuron 36, 1007–1019. Takahashi, M., Yamada, T., Tooyama, I., Moroo, I., Kimura, H., Yamamoto, T., Okada, H., 1996. Insulin receptor mRNA in the substantia nigra in Parkinson’s disease. Neuroscience Letters 204, 201–204. Tanaka, A., 2010. Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network. FEBS Letters 584, 1386–1392. Wen, Y., Yang, S., Liu, R., Simpkins, J.W., 2004. Transient cerebral ischemia induces site-specific hyperphosphorylation of tau protein. Brain Research 1022, 30–38. WHO, 2004. Radix Eleutherococci. WHO Monographs on Selected Medicinal Plants, vol. 2. WHO, Geneva, pp. 83–96. Winklhofer, K.F., Haass, C., 2010. Mitochondrial dysfunction in Parkinson’s disease. Biochimica et Biophysica Acta 1802, 29–44. Yang, H., Zhou, X., Liu, X., Yang, L., Chen, Q., Zhao, D., Zuo, J., Liu, W., 2011. Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin. Biochemical and Biophysical Research Communications 410, 114–120. Yeung, L.Y., Wai, M.S., Fan, M., Mak, Y.T., Lam, W.P., Li, Z., Lu, G., Yew, D.T., 2010. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicology Letters 193, 189–193.