Schisandra chinensis Stem Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity via Activation of the Nrf2 Pathway and Inhibition of the MAPKs and NF-κB Pathways

The beneficial value of the stems of Schisandra chinensis (SSC) in neurological diseases is unclear. We examined whether SSC aqueous extract (SSCE) alleviates striatal toxicity in a 3-nitropropionic acid (3-NPA)-induced mouse model of Huntington's disease (HD). SSCE (75, 150, or 300 mg/kg/day, p.o.) was given daily before or after 3-NPA treatment. Pre- and onset-treatment with SSCE displayed a significant protective effect and pretreatment was more effective as assessed by neurological scores and survival rate. These effects were related to reductions in mean lesion area, cell death, succinate dehydrogenase activity, microglial activation, and protein expression of inflammatory factors including interleukin (IL)−1β, IL-6, tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclooxygenase-2 in the striatum after 3-NPA treatment. Pretreatment with SSCE stimulated the nuclear factor erythroid 2-related factor 2 pathway and inhibited phosphorylation of the mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways in the striatum after 3-NPA treatment. The gomisin A and schizandrin components of SSCE significantly reduced the neurological impairment and lethality induced by 3-NPA treatment. These results indicate for the first time that SSCE may effectively prevent 3-NPA-induced striatal toxicity during a wide therapeutic time window through anti-oxidative and anti-inflammatory activities. SSCE has potential value in preventive and therapeutic strategies for HD-like symptoms.


INTRODUCTION
Huntington's disease (HD) is an inherited neurological disorder caused by abnormal expansion of a CAG (cytosineadenine-guanine) repeat within exon 1 of the huntingtin (Htt) gene, located on chromosome 4 (Damiano et al., 2010;Ross and Tabrizi, 2011). The aggregation of mutant Htt protein leads to multiple issues, including toxic neuronal aggregates, transcriptional dysregulation, excitotoxicity, mitochondrial dysfunction with altered energy metabolism, and changes in axonal transport and synaptic dysfunction within the striatum and the cortex (Damiano et al., 2010;Ross and Tabrizi, 2011). HD is characterized clinically by subtle cognitive, motor, and psychiatric changes, which are collectively termed prodromal disease (Damiano et al., 2010;Ross and Tabrizi, 2011). Symptomatic treatments for abnormal motor functioning (mainly chorea), such as tetrabenazine (Xenazine R , the only US Food and Drug Administration-approved medication for HD) (Huntington Study Group, 2006) or neuroleptics (Barr et al., 1988) have limited benefits and are associated with disabling adverse effects, such as sedation/somnolence, insomnia, and depression. Development of efficient and safe drugs that forestall the outbreak or delay the onset of HD is critical.
Oxidative stress is considered a potential contributing factor to HD pathogenesis (Damiano et al., 2010;Ross and Tabrizi, 2011). Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of the antioxidative cellular response, interacts with antioxidant response element (ARE), and has a variety of cytoprotective roles against oxidative stress, apoptosis, and inflammation in the nervous system (Copple, 2012;Joshi and Johnson, 2012;Suzuki et al., 2013). Mice with Nrf2 deletions are inherently more susceptible to drug-induced toxicity and oxidative stress-induced diseases, including neurological diseases, while the overexpression of Nrf2 ameliorates the destructive effects of oxidative stress in various in vivo and in vitro disease models, including models of Parkinson's disease and HD (Copple, 2012;Joshi and Johnson, 2012;Suzuki et al., 2013). The Nrf2 signaling pathway is involved in the suppression of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB), associated with inflammatory effects (Juge et al., 2007). The pathways are activated by pro-inflammatory cytokines, neurotrophic factors, neurotransmitters, neural injury, seizure activity, and proteins implicated in neurodegenerative disorders, including HD (Harper and Wilkie, 2003;Memet, 2006), and are upregulated by treatment with 3-nitropropionic acid (3-NPA) or kainate in the striatum, which mimics the pathology caused by mutant Htt (Sugino et al., 2000;Khoshnan et al., 2004). Although more studies are needed to fully identify the role of the Nrf2 and MAPKs/NF-κB pathways, it is reasonable to suggest that pharmacological modulation of these pathways may provide a new therapeutic target in HD.
Schisandra (S.) chinensis (Turcz.) Baill (Omija in Korean; wǔ wèi zi in Chinese; literally "five-flavor berry, " the common name) belongs to the genus Schisandra of the family Schisandraceae and is distributed and cultivated in northeastern China, far-eastern Russia, Japan, and Korea (Panossian and Wikman, 2008). The fruits of S. chinensis (FSC) have long been used in Oriental medicine to treat various diseases, such as gonorrhea, asthma, dysentery, enuresis, and dermatitis, and to relive excessive thirst (Panossian and Wikman, 2008). FSC extract and its constituents have gained attention for their potential role in the treatment of cardiovascular diseases like hypertension and myocardial infarction (Young Park et al., 2012;Chen et al., 2013), respiratory diseases including that caused by Chlamydia pneumonia and acute respiratory distress syndrome , metabolic diseases like osteoporosis and diabetes (Kim et al., 2014), digestive diseases including hepatotoxicity (Wang, K. P. et al., 2014), and neurological diseases like ischemia (Jiang et al., 2014), which corroborates the observed effects of S. chinensis in traditional settings. These beneficial effects of S. chinensis are the result of anti-oxidant, anti-inflammatory, and antiapoptotic activities related to the regulatory role of the Nrf2 and MAPKs/NF-κB pathways of chemical constituents including the lignans schizandrin, deoxyschisandrin (schizandrin A), gomisins, and pregomisin (Young Park et al., 2012;Chen et al., 2013;Jiang et al., 2014;Kim et al., 2014;Wang, K. P. et al., 2014;Zhou et al., 2014). The main constituents of FSC, including Schizandrin A/B/C and gomisin A/N/J, have been isolated from the stems of S. chinensis (SSC) (Lu and Chen, 2009;Zheng et al., 2014;Zhu et al., 2015). Schizandrins and gomisins have strong antioxidant and anti-inflammatory effects (Young Park et al., 2012;Chen et al., 2013;Jiang et al., 2014;Kim et al., 2014;Wang, K. P. et al., 2014;Zhou et al., 2014).
These findings strongly suggest the possibility of an essential role of SSC in physiological and pathological functions. However, to the best of our knowledge, there have been no reports on the beneficial effects of SSC. Herbal medicines including S. chinensis have traditionally been used as aqueous extract (Huh, 1613). Therefore, we investigated whether SSC aqueous extracts (SSCE) have a medicinal effect in a 3-NPA-induced striatal toxicity model. 3-NPA is an irreversible inhibitor of mitochondrial succinate dehydrogenase (SDH). It causes degeneration of striatal and cortical neurons in animals and results in gait abnormalities in an animal model of HD (Tunez et al., 2010;Mehrotra and Sandhir, 2014). We demonstrate that SSCE can be used as a beneficial tool to treat HD-like symptoms through the activation of Nrf2 signaling and inhibition of MAPKs and NF-κB signaling pathways.

Animals and Ethical Approval
Adult male C57BL/6N mice (Narabiotec Co., Ltd., Seoul, Republic of Korea, 8-10 weeks of age, 22-25 g body weight were purchased from Taconic Biosciences Inc. (Hudson, NY, USA). The mice were housed at a constant temperature of 23 ± 2 • C with a 12-h light-dark cycle (lights on from 08:00 to 20:00) and provided with food and water ad libitum. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Kyung Hee University. Proper randomization of laboratory animals and handling of data were performed in a blinded manner in accordance with recent recommendations from an NIH workshop on preclinical models of neurological diseases (Landis et al., 2012).

Preparation of SSCE
SSC was obtained from Mungyeong-si Distribution Corporation (Mungyeong, Republic of Korea). The material was air-dried in a shaded lot and cut into small pieces (3-5 mm-thickness; Figure 1A). A voucher specimen (KHKM0030) was deposited at the Laboratory of Autoimmune and Neurodegenerative Diseases, Department of Convergence Science, College of Korean Medicine, Kyung Hee University. The SSC (200 g) was extracted with distilled water (2 L) for 1.5 h at room temperature, further extracted by continuous heating for 1.5 h under reflux conditions with hot water, and subsequently filtered by vacuum filtration. The filtrate was concentrated using a rotary vacuum evaporator (EYELAN-1200A, EYELA; Rikakikai Co. Ltd, Tokyo, Japan), lyophilized using a freeze drying system (OPR-FDA-8612; Operon Co. Ltd, Gimpo, Republic of Korea), and stored in a freezer (-80 • C). The final yield of the SSCE was 10.55%.
Quantitation of SSCE Using Ultraviolet High Performance Liquid Chromatography (HPLC-UV) Gomisin A, gomisin N, and Schizandrin were obtained from Wuhan ChemFaces Biochemical Co., Ltd. (Wuhan, China) and Sigma-Aldrich Co. (St. Louis, MO, USA). Quantitation of SSCE using HPLC-UV was accomplished by using a modification of a previously described protocol . Briefly, standard solutions containing each lignin were prepared in concentrations ranging from 5 to 250 ng/mL. One hundred milligrams SSCE was dissolved in 1 ml of distilled water. All standard and SSCE solutions were filtered through a 0.20 µm syringe filter (Merck Millipore Co., Schwalbach, Germany). The HPLC apparatus consisted of a model 1260 pump (Agilent Technologies, Santa Clara, CA, USA), a model G1367D autosampler, and a model G1315C DAD. Masshunter software (Agilent Technologies) was used. A YMC TRIART C18 column (50 × 2.0 ID-1.9 µm) was used for the HPLC analysis. The detection wavelength was set to 254 nm. The mobile phase contained acidified water with formic acid (0.1%, solvent A; Wako Pure Chemical Industries, Ltd., Osaka, Japan) and acidified acetonitrile (J.T. Baker Chemicals Co., Center Valley, NJ, USA) with formic acid (0.1%, solvent B). The gradient program consisted of 10% solvent B for 2 min, 50% solvent B for 20 min, and then was linearly increased to 80% solvent B for another 30 min. This linear gradient was followed by isocratic elution for 35 min and reconditioning steps to return to the initial mobile phase. The flow rate was 0.2 ml/min, and the injection volume of the standards and SSCE was 5 µL. A calibration curve was established using a methanol stock solution containing each dibenzocyclooctane lignan diluted to a specified concentration. The coefficient values (r 2 ) were 0.999 and 1, demonstrating that the linearity in this range was sufficient to provide a highly accurate estimate of the content of gomisin A, gomisin N, and schizandrin in SSCE. Precision was determined using triplicate measurements of each standard, and the relative standard deviations (RSDs) were <2.2% (Figure 1).

Treatment with 3-NPA and SSCE
3-NPA was administered as previously described (Huang et al., 2006;Jang et al., 2013Jang et al., , 2014Jang and Cho, 2016). Briefly, 3-NPA was dissolved in physiological saline to a concentration of 100 mg/ml (pH 7.4), passed through a 0.2 µm filter to remove any bacteria, and kept at -80 • C until use. The 3-NPA solution was given intraperitoneally (i.p.) twice daily for 2 days at 12h intervals (8:00 a.m. and 8:00 p.m.) at a dose of 60 mg/kg on the first day and 80 mg/kg on the second day (60-60-80-80 dose regimen). SSCE was prepared in physiological saline and administered at doses of 75, 150, or 300 mg/kg to determine the most effective dose. Subsequently, 300 mg/kg SSCE was given at the time of onset, progressive, and peak stages to investigate its therapeutic time window. Each experiment was repeated at least three times using the same protocol. Total daily dose of SSCE for mice was determined by formula for dose translation based on body surface area (Reagan-Shaw et al., 2008) after considering body weight of animals, final extract yield, and traditional dose in humans. Gomisin A (20 mg/kg), gomisin N (20 mg/kg), and schizandrin (45 mg/kg) were treated peritoneally once daily from 30 min before 3-NPA treatment.

Neurological Semi-quantitative Assessment
To assess the severity of 3-NPA-induced neurological disorders, the previously described motor behavioral scale (Fernagut et al., 2002) and our standard for each subcriteria (Jang et al., 2014) were utilized. Briefly, the three-level (0, 1, and 2) scale was used to measure the severity of the following five items (maximal score = 10), which constituted the main motor symptoms observed: hindlimb clasping, global activity in a free-moving environment, hindlimb dystonia, truncal dystonia (kyphotic posture), and balance adjustment to a postural challenge. The neurological test was performed by an experimenter who was unaware of the experimental conditions and was done under constant temperature (23 ± 2 • C) and humidity (55 ± 5%) in a quiet room, 24 h or 5 days after the final treatment.

Histological Assessment of Striatal Damage
We used our previous protocol to evaluate the histopathological changes in the striatum (Jang et al., 2013(Jang et al., , 2014Jang and Cho, 2016). Briefly, 24 h after the last 3-NPA treatment, the mice were anesthetized with ethyl ether and then perfused intracardially with saline and cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains (n = 5 in the sham group, n = 5 in the 3-NPA group, n = 7 in the 3-NPA + SSCE 75 mg/kg group, n = 7 in the 3-NPA + SSCE 150 mg/kg group, n = 7 in the 3-NPA + SSCE 300 mg/kg group, and n = 5 in the SSCE group) were immediately removed, post-fixed overnight in the same fixative at 4 • C, and serially cryoprotected in 10, 20, and 30% sucrose in PBS for 48 h at 4 • C. Sequential coronal sections (30 µm thickness) were acquired on a model CM3050S freezing microtome (Leica Biosystems, Wetzlar, Germany), starting from the anterior aspect of the corpus callosum and proceeding through the entire striatum (bregma 1.40-1.30 mm), according to the mouse brain atlas (Franklin and Paxinos, 2008). For histological assessment, every tenth section (at intervals of 300 µm) was processed for cresyl violet staining, dehydrated, and coverslipped with Permount (Fisher Scientific, Waltham, MA, USA). The stained sections (n = 3 per brain) from the level of the mid-striatum were captured using a DP70 image analysis system (Olympus, Tokyo, Japan) and the level of 3-NPA-induced striatal damage was analyzed using the NIH Image J program (http://rsb. info.nih.gov/ij/).

Determination of SDH Activity
A previously developed protocol (Jang et al., 2013(Jang et al., , 2014Jang and Cho, 2016) was used to investigate SDH activity in the striatum and the calf muscle. Briefly, fresh brains (n = 3 per group) were rapidly frozen in isopentane, sectioned at a thickness of 10 µm, and mounted on glass slides. The sections were incubated for 15 min in PBS at 37 • C to activate the SDH. The three brain sections were washed in PBS and incubated in 0.05 M sodium succinate (Sigma-Aldrich), 0.3 mM nitroblue tetrazolium (Sigma-Aldrich), and 0.05 M PB (pH 7.6) for 20 min at 37 • C. The sections were then rinsed in cold PBS for 5 min and dried at room temperature. Each section was digitally imaged using a DP70 image analysis system (Olympus) for quantitative microscopy. Quantification was performed as described previously using the Image J program. SDH activity was quantified in the cerebral cortex, and the striatum as regions of interest (ROIs) delineated on a digitized image. Extraction of color in each section was taken as the saturation value, and this value was used to represent SDH activity (% of sham group).

Detection of Apoptosis with the Tunel Assay
The fragmentation of DNA was examined using an ApopTag R Peroxidase in situ Apoptosis Detection Kit (S7100) (Millipore) according to the manufacturer's instructions. Briefly, brain sections from each mouse (n = 5 per group) were subjected to enzymatic digestion with 20 µg/ml proteinase K for 5 min, treated with 5% hydrogen peroxide for 20 min to exhaust endogenous peroxidase activity, and washed with PBS. They were then immersed in ApopTag R equilibration buffer for 10 min to label the 3′-OH ends of fragmented DNA and incubated with terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) at 37 • C for 1 h. After being washed with PBS, the sections were incubated with DAB to detect signs of apoptotic cell death.

Toxicological Evaluation of SSCE
To examine whether SSCE has a toxic effect when used over the long term in mice, SSCE (16, 80, 400, and 2,000 mg/kg/day) was orally administrated to normal, 9-week-old, male mice for 15 days. Body weight, food intake, and water intake were measured daily and serum was obtained at 24 h after the last administration of SSCE. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured using enzymatic or ultraviolet assays with an ALT, AST, or LDH detection kit (Roche, Basel, Switzerland) according to the manufacturer's instructions with a Cobas 8,000 modular analyzer (Roche) using a previously described protocol (Jang et al., 2014;Choi et al., 2015). General histological structure was evaluated by hematoxylin & eosin (H&E) staining as previously described.

Statistical Analyses
Statistical analyses were performed using the SPSS 21.0 package (SPSS Inc, Chicago, IL, USA) for Windows. Two-sample comparisons were carried out using the Student's t-test and multiple comparisons were made using two-way ANOVA with Tukey's post hoc test. All data are presented as means ± S.E.M. and statistical difference was identified at the 5% level unless otherwise indicated.

Therapeutic Time Window of SSCE
To determine the therapeutic time window of SSCE treatment against 3-NPA-induced neurotoxicity, mice were treated with SSCE (300 mg/kg/day; the most effective dose in Figures 2A-H) at various times during or after 3-NPA treatment. Groups were designated as pre-, onset-, progress-, and peak-treated groups. Based on the level of neurological impairment seen in Figures 2A-F and as we previously reported (Jang et al., 2014;Jang and Cho, 2016), we defined onset (initial stage of neurological score, 0-3), progression (middle stage of neurological score, 3-6), and peak stages (peak stage of neurological score, 6-10) of neurological symptoms for this experiment. The severity of neurological signs in the SSCE pre-(5.5 ± 0.2) and onset-treated groups (6.9 ± 0.8) were significantly lower than those in the 3-NPA group (8.3 ± 0.5).
The survival rate in the 3-NPA group was 44.4% (n = 4/9) at the end of the experiment, but was increased in a time-dependent manner by SSCE pre-(66.6%, n = 6/9) and onset-treatment (77.7%, n = 7/9). However, progression-and peak-treatment with SSCE did not significantly improve neurological impairment or survival rate (Figures 2I-L). The results suggest that SSCE can reduce 3-NPA-induced neurological impairment.

Protective Effect of SSCE on Striatal Cell Death Induced by 3-NPA Treatment
3-NPA-induced neurological dysfunction results from neuronal death in the striatum (Fernagut et al., 2002;Jang et al., 2013Jang et al., , 2014Jang and Cho, 2016). Appropriately, we investigated whether SSCE reduced striatal cell death. Twenty-four hours after the last (4th) 3-NPA treatment, brain slices including the striatum were stained with cresyl violet dye. Figure 3 shows representative striatal images from the sham, 3-NPA, and 3-NPA + SSCE (75, 150, and 300 mg/kg/day) groups. Fifty-five percent (n = 5/11) of the surviving 3-NPA-treated mice had visible bilateral striatal lesions (pale areas surrounded by dotted line), while the percentage of mice with striatal lesions was decreased to 36.3% (n = 4/11) and 20% (n = 2/10) in the groups pretreated with 75 and 150 mg/kg/day of SSCE, respectively (Figures 3A-G). Interestingly, visible striatal lesions were not detected in the group pretreated with 300 mg/kg/day of SSCE (0%; 0/10; Figures 3E,G). The ratio of the mean lesion area to the entire striatum was 28.7%, while the ratio was significantly reduced to 9.2% in the 150 mg/kg/day SSCE group (Figure 3H). Additionally, β-III-tubulin, a microtubule element of the tubulin family that is found almost exclusively in neurons, showed decreased expression in the striatum in the 3-NPA group (0.2 ± 0.01) compared with the striatum in the sham group (0.3 ± 0.01), whereas the decrease in expression was significantly rescued by pretreatment with SSCE (0.3 ± 0.01) ( Figure 3I). The findings suggest that SSCE can reduce 3-NPA-induced striatal toxicity.
At 24 h after the last 3-NPA treatment, the level of neurological impairment (I,K) and survival rate (J,L) were measured from each group (n = 4 or 9). N.D. in the graphs means not detected. The data are expressed as mean ± SEM. ANOVA testing was performed; ## P < 0.01 and # P < 0.05 vs. sham group; **P < 0.01 and *P < 0.05 vs. 3-NPA group.
Frontiers in Pharmacology | www.frontiersin.org Cor, St, and dotted outline indicate the cortex, striatum, and striatal lesion, respectively. Scale bar = 100 µm. N.D. in the graphs means not detected. The data are expressed as mean ± SEM. ANOVA testing was performed; # P < 0.05 and ## P < 0.01 vs. the sham group; * P < 0.05 and ** P < 0.01 vs. the 3-NPA group.
The number of TUNEL-positive cells was significantly increased in the striatum in the 3-NPA group (Figures 5B,I) compared to the sham group ( Figure 5A). But, the number of TUNELpositive cells was significantly decreased in the 3-NPA + SSCE (300 mg/kg/day) group (Figures 5C,I). Also, the number of cleaved caspase-3-immunoreactive cells was increased in the striatum of the 3-NPA group (Figure 5F), as compared with the sham group (Figure 5E), while that number was significantly decreased in the 3-NPA + SSCE group ( Figure 5G) as compared to the 3-NPA group, which corresponded to the expression pattern of cleaved caspase-3 protein as assessed by Western blot analysis ( Figure 5J). SSCE itself did not induce apoptosis in the striatum as compared to the sham group. The results suggest that SSCE may reduce neurological dysfunction and striatal toxicity via inhibiting apoptosis in the striatum after 3-NPA treatment.

Effect of SSCE on Microglial Activation in the Striatum after 3-NPA Treatment
Microglia are activated in neurological diseases including HD, and activated microglia release pro-and anti-inflammatory cytokines (Pavese et al., 2006;Lobsiger and Cleveland, 2007;Ferger et al., 2010). Therefore, we examined whether SSCE could inhibit microglial activation in striatal lesions after 3-NPA treatment (Figure 6). In striatal tissue from the 3-NPA group, Iba-1 (a marker for microglia/macrophage lineage cells)immunoreactive cells; (Ferger et al., 2010;Jang et al., 2013Jang et al., , 2014Jang and Cho, 2016) displayed activated morphology with enlarged cell bodies and short and thick processes ( Figure 6B) compared to the sham group (Figure 6A), which generally showed the typical forms of resting cells including small cell bodies and thin processes. However, the level of activation of Iba-1-immunoreactive cells was reduced in the striatum in the 3-NPA + SSCE group (Figure 6C) compared to the 3-NPA group (Figure 6B), corresponding to a change in protein expression of Iba-1 as measured by immunoblot analysis (Figure 6I). Microglia were not activated by treatment only with SSCE (300 mg/kg) (Figures 6D,I). Astrocytes may be activated by the debris from dead cells or by activated microglia within or around neurodegenerative lesions (Lobsiger and Cleveland, 2007). However, in the current study, GFAP-immunoreactive astrocytes were not significantly affected within or around the striatal lesions 24 h after the last (4th) 3-NPA treatment compared with those in the sham and SSCE groups (Figures 6E-H), which reflects a lack of change in GFAP protein expression ( Figure 6J). The findings suggest that SSCE inhibits microglial activation, but not astroglial activation, and is associated with a reduction in neurological impairment and striatal cell death.

Effect of SSCE on Inflammatory Mediators in the Striatum after 3-NPA Treatment
Since activated microglia within or around lesions secrete factors that can be either beneficial or detrimental to neuronal survival (Pavese et al., 2006;Lobsiger and Cleveland, 2007;Ferger et al., 2010), we examined whether the inhibition of microglial activation induced by SSCE (Figures 6A-D,I) is closely connected with the down-regulation of inflammatory factors in the striatum. Immunoblot analysis determined that protein expression levels of IL-1β, IL-6, TNF-α, iNOS, and COX-2 were markedly increased by 0.49-, 1.01-, 1.10-, 1.23-, and 0.98-fold, respectively, in the striatum 24 h after the last (4th) 3-NPA treatment, while their expression levels were significantly decreased by 36, 32, 34, 41, and 31%, respectively, in striatal tissue from the 3-NPA + SSCE group (300 mg/kg/day) compared with the 3-NPA group (Figures 6K-O). These results suggest that SSCE can mitigate the neurological dysfunction and
Collectively, these results indicate that SSCE exerts its antioxidant activity through stimulation of the Nrf2 pathway in the striatum.

Effect of SSCE on MAPKs and the NF-κB Pathway in the Striatum after 3-NPA Treatment
Since MAPKs and the NF-κB pathway are activated in the striatum by 3-NPA treatment (Sugino et al., 2000;Khoshnan et al., 2004;Zheng et al., 2014), we tested the modulatory effect of SSCE on these pathways in the striatum after 3-NPA treatment. As expected, phosphorylation of ERK, JNK, and p38 proteins was significantly increased by 66.4-, 223.2-, and 42.9fold, respectively, in the striatum 24 h after the last (4th) 3-NPA treatment, and by 59, 47, and 69%, respectively, compared   with phosphorylation in the sham and SSCE alone groups, whereas the enhancement in activation was significantly reduced in the striatum in the 3-NPA + SSCE group (Figures 7D-F).
We examined whether SSCE modulates the expression of NF-κB in the striatum after 3-NPA treatment. Protein expressions of p-NF-κB and p-IκBα were significantly increased by 68.0and 49.4-fold, respectively, in the striatum 24 h after the final 3-NPA treatment, by 49 and 54%, respectively, compared to expression in the sham group, while the enhancement in activity was significantly decreased by SSCE treatment (300 mg/kg/day; Figures 7G,H). Activation of MAPKs or the NF-κB pathway was not increased or decreased by SSCE treatment alone (Figure 7). These results indicate that SSCE may reduce striatal toxicity and neurological dysfunction after 3-NPA treatment by suppressing MAPKs and the NF-κB pathways.
These results indicate that gomisin A, gomisin N, and schizandrin may be the active components responsible for the beneficial effect of SSCE on 3-NPA-induced striatal toxicity.

Administration of SSCE Does Not induce Toxicity
Finally, to examine if SSCE has toxicity, we orally treated to normal male mice (9-weeks-old) with SSCE (16-2,000 mg/kg/day) for 15 days and assessed toxicity.

DISCUSSION
SSCE may alleviate 3-NPA-induced striatal toxicity by activating the Nrf2 pathway and inhibiting MAPKs and the NF-κB pathways. SSCE pretreatment alleviated neurological impairment, increased the survival rate, and reduced striatal cell death after 3-NPA treatment. Moreover, SSCE repressed SDH activity, apoptosis, microglial activation, and protein expression of IL-1β, IL-6, TNF-α, iNOS, and COX-2 in the striatum after 3-NPA treatment. SSCE stimulated the Nrf2 pathway and inhibited MAPK and NF-κB pathways. To the best of our knowledge, this is the first study to show that SSCE may attenuate 3-NPA-induced striatal toxicity by regulating various inflammatory cascades (Nrf2, MAPKs, and NF-κB pathways) and may be a potential therapeutic agent for HD-like symptoms.
The main ingredients of SSC, which include gomisin A/J/N and schisandrin (schizandrin) A/B/C, were also separated from the FSC (Lu and Chen, 2009;Zheng et al., 2014;Zhu et al., 2015). FSC extract and its constituents have beneficial effects in the treatment of various diseases including neurological diseases, such as cognitive and motor dysfunction (Panossian and Wikman, 2008), through anti-apoptotic (total extract and schisandrin B), anti-oxidative (total extract, schisandrin A, and schisandrin B), synaptic transmission (deoxyschizandrin), and anti-neuroinflammatory activities (schisandrin B and gomisin A) (Chen et al., 2008;Fu et al., 2008;He et al., 2014;Wang, X. et al., 2014;Giridharan et al., 2015;Zhang et al., 2015), which confirms the effects of FSC that have been reported by practitioners of traditional medicine (Huh, 1613). SSC also includes anwuweizic acid, daucosterol, (+)-deoxyschizandrin, (-)-dihydroguaiaretic acid, gomisin N, henrischinin A-C, schicagenin A-C, schinchinenin A-H, schinchinenlactone A-C, γ-schizandrin, β-sitosterol, tetradecanoic acid, and wuweizisu C (Shi et al., 2011;Song et al., 2013;Zheng et al., 2014). Anwuweizic acid, schicagenin A-C, schinchinenin A-H, and schinchinenlactone A-C have not been identified from FSC and their functions have not been investigated. The collective results strongly suggest that SSC may have a similar effect to FSC or a more impressive effect on the pathophysiological mechanisms of CNS. However, to the best of our knowledge, the beneficial effects of SSC have not yet been reported. The present findings demonstrate that SSCE has a neuroprotective effect against 3-NPA-induced striatal toxicity through anti-apoptotic, anti-inflammatory, and anti-oxidant activities that are related to the activation Nrf2 pathway and the inhibition of the MAPKs and NF-κB pathways.
The primary mechanism of 3-NPA-induced striatal toxicity involves irreversible inhibition of SDH, a key enzyme located in the inner mitochondrial membrane that is responsible for the oxidation of succinate to fumarate. Thus, regulating SDH activity in the striatum may be a therapeutic treatment option for diseases related to mitochondrial dysfunction. The representative medicinal herbs that alleviate neurological symptoms and striatal cell death by significantly preventing 3-NPA-induced inhibition of SDH activity include Ginkgo biloba extract (Mahdy et al., 2011) and Korean red ginseng (Jang et al., 2013), the antidepressant compound sertraline (Kumar and Kumar, 2009), and ethyl pyruvate (Jang et al., 2014). In the present study, SSCE also significantly ameliorated impaired SDH activity in the striatum following 3-NPA treatment, which was associated with improvement in behavioral impairment and beneficial mechanisms that included reduced apoptosis, inflammation, and oxidantion. Since impaired SDH activity can role as trigger of striatal cell death, maintenance of SDH activity by SSCE is an important finding.
We characterized the level of neurological impairment at various times to determine the therapeutic time window of SSCE in the 3-NPA-induced HD model. SSCE (300 mg/kg) applied prior to and following exposure to 3-NPA significantly alleviated neurological scores and lethality. However, progression-and peak-treatment with SSCE did not significantly improve neurological impairment or survival rate. This suggests that SSCE has preventive and therapeutic effects in the early stages of striatal toxicity caused by 3-NPA treatment and that its positive efficacy is related to the diminution of apoptosis via SDH inhibition, delayed cell death (secondary apoptosis) induced by inhibition of microglial activation and upregulation of pro-inflammatory cytokines, and anti-oxidation involving stimulation of the Nrf2 pathway in the striatum after 3-NPA treatment. This scenario is supported by the findings that 3-NPA induces secondary apoptosis through the generation of superoxide radicals after SDH inactivity (Dedeoglu et al., 2002) and that 3-NPA induces delayed cell death by activated microglial products surrounding the cells that die first (Brouillet, 2014).
Microglia are recruited and activated around or within neurodegenerative lesions earlier than astrocytes. Activated microglia release compounds that can be either beneficial or harmful to neuronal survival (Lobsiger and Cleveland, 2007). Clinical and positron emission tomography studies have shown that the level of microglial activation increases in proportion to the severity of HD symptoms (Pavese et al., 2006). Therefore, controlling recruitment and activation of microglia is an attractive therapeutic strategy for neurological disorders including HD (Lobsiger and Cleveland, 2007). Results demonstrate that SSCE inhibits microglial activation and protein expressions of IL-1β, IL-6, TNF-α, iNOS, and COX-2 in the striatum after 3-NPA treatment. Although the anti-inflammatory effects of SSCE have not yet been fully investigated, gomisin A/J/N and schisandrin B/C exert anti-neuroinflammatory activity in lipopolysaccharide-induced microglia by inhibiting NF-κB or/and MAPKs pathways (Oh et al., 2010;Zeng et al., 2012;Wang, X. et al., 2014). The findings support the possibility that SSCE has potent anti-inflammatory activity against 3-NPAinduced neurotoxicity.
Antioxidants can be used in preventive and therapeutic strategies for neurological disorders because oxidative stress contributes to numerous neurodegenerative diseases, such as HD. The Nrf2 pathway is one of the major regulators of oxidative stress (Copple, 2012;Joshi and Johnson, 2012). Mice with Nrf2 deletions exhibit aggravated 3-NPA-and malonateinduced motor dysfunction, striatal SDH and LDH activity, and striatal lesions as compared with wild type mice. However, oral administration of the Nrf2 activator tert-butylhydroquinone attenuates 3-NPA-induced striatal toxicity in wild type mice but not in Nrf2 knockout mice (Tarozzi et al., 2013). Schizandrin C is a component of FSC and SSC. It significantly induces the expression of phase II detoxifying/antioxidant enzymes including HO-1 and NQO1, inhibits lipoteichoic acid-stimulated reactive oxygen species (ROS) production in microglia (Park et al., 2013), and ameliorates learning and memory deficits caused by Aβ-induced oxidative stress in mice (Mao et al., 2015). Schisandrin B also protects against solar irradiationinduced oxidative stress in rat skin tissue (Lam et al., 2011). In the present study, pretreatment with SSCE increased protein expression of Nrf2-dependent gene products (HO-1 and NQO1) by stimulating the Nrf2 pathway in the striatum after 3-NPA treatment (Figure 7). Our previous study demonstrated that preactivation of the Nrf2 pathway by pretreatment with other Nrf2 activators (dimethyl fumarate and antioxidant response element inducer-3) reduces 3-NPA-induced neurological impairment and lethality (Jang and Cho, 2016). The collective findings indicate that SSCE may include an essential inducible factor that protects against complex II inhibitor (3-NPA)-mediated neurotoxicity.
JNK, ERK, and p38 MAPKs are phosphorylated in polyglutamine-expanded Htt-mediated neuronal toxicity (Khoshnan et al., 2004;Wild and Tabrizi, 2014), in the striatal neurons of YAC128 and R6/2 transgenic mice (Wild and Tabrizi, 2014), and in a 3-NPA-induced neurotoxicity model (Sugino et al., 2000;Jang et al., 2013Jang et al., , 2014Jang and Cho, 2016). However, inhibition of the phosphorylation of ERK or p38 by intrathecal administration of an ERK inhibitor (PD98059) or p-p38 inhibitor (SB203580) alleviates 3-NPA-induced motor dysfunction and increases the survival rate in the 3-NPA-induced HD model (Jang et al., 2013). SSCE significantly decreased 3-NPA-induced phosphorylation of ERK, JNK, and p38 MAPKs in the striatum (Figure 7), which corresponded to improvements in neurological symptoms (Figure 2) and striatal cell death (Figure 3). Gomisin J, gomisin N, schisandrin A, schisandrin B, and schisandrin C reportedly exert anti-inflammatory effects by down-regulating the ERK, JNK, or p38 MAPKs signaling pathways in lipopolysaccharide-treated RAW 264.7 cells (Ci et al., 2010;Oh et al., 2010) and in a dextran sulfate sodiuminduced acute colitis model . In addition, schisandrin B prevents doxorubicin-induced cardiac dysfunction by inhibiting the p38 MAPK pathway . Taken together, despite the relative lack of information on the efficacy and critical mechanisms of action of SSCE, our findings indicate that SSCE may be used to regulate the phosphorylation of MAPK pathways in the therapeutic approach to HD-like symptoms.
The activation of the NF-κB pathway is increased in striatal cells following 3-NPA treatment and in cultured cells expressing mutant Htt. Preventing the degradation of NF-κB inhibitors with a dominant-negative ubiquitin ligase β-transducin repeatcontaining protein decreases the toxicity of mutant Htt in MSNs. Inhibition of IKK activity with an N-terminally truncated form of IKK gamma (γ) reduces mutant Htt-induced toxicity in MSNs (Khoshnan et al., 2004). Sulforaphane, ethyl pyruvate, and Korean red ginseng extract prevent mitochondrial dysfunction by inhibiting the NF-κB p65 pathway in the striatum following 3-NPA treatment (Jang et al., 2013(Jang et al., , 2014Jang and Cho, 2016). Furthermore, blocking the NF-κB pathway by intrathecal injection of IκB inhibitor (pyrrolidine dithiocarbamate) before 3-NPA intoxication decreases lethality in an animal model (Jang et al., 2014). Various strategies have been attempted to clarify the critical role of the NF-κB pathway in the pathogenesis of HD and to determine the therapeutic safety and efficacy of its modification in HD. In this study, we demonstrate for the first time that SSCE alleviates NF-κB activity (expression of p65 and p-IκBα), which agrees with the reduced microglial activation and decreased protein expression of IL-1β, IL-6, TNF-α, iNOS, and COX-2 in the striatum following 3-NPA treatment (Figure 6). Taken together, our findings suggest that SSCE might alleviate 3-NPA-induced striatal toxicity by inhibiting activation of the NF-κB pathway and that SSCE could be useful as a treatment for HD-like syndromes.
The main components of S. chinensis, including gomisin A, gomisin N, and schizandrin, have neuroprotective effects (Chen et al., 2008;Fu et al., 2008;Park et al., 2013;Jiang et al., 2014;Wang, X. et al., 2014;Giridharan et al., 2015;Mao et al., 2015;Zhang et al., 2015). In the present study, pretreatment with gomisin A (20 mg/kg, i.p.) and schizandrin (45 mg/kg, i.p.) significantly reduced neurological impairment and lethality compared to administration of 3-NPA alone. Although not statistically significant (p = 0.0542), gomisin N (20 mg/kg, i.p.) showed a tendency to reduce neurological impairment. And gomisin N significantly reduced lethality (Figure 8). These results suggest that certain components, including gomisin A, gomisin N, and schizandrin, may be active in the protective effects of SSCE on 3-NPA-induced neurotoxicity, and that the essential efficacy and cellular mechanisms of action of the active components of SSCE may be exploited as useful adjuvant therapies and in functional foods to benefit patients with neurological diseases, including HD.
The relationship between Nrf2 and NF-κB is not well characterized. However, the identification of NF-κB binding cites in the promoter region of the Nrf2 gene suggests cross-talk between these two regulators of inflammatory processes. NF-κB signaling inhibits the Nrf2 pathway through the interaction of p65 and KEAP1 (Sandberg et al., 2014). Furthermore, the protective up-regulation of the Nrf2 pathway is mediated by activation of several kinases including JNK, ERK, and p38 (Sandberg et al., 2014). In the present study, SSCE activated the Nrf2 pathway (Nrf2, HO-1, and NQO-1) and inhibited activation of MAPK pathways (JNK, ERK, and p38) and NF-kB pathways (IkBα and NF-κB p65) in the striatum after 3-NPA treatment (Figure 7). Although further studies are necessary to demonstrate the details of the mechanism, our findings suggest that SSCE may exert a neuroprotective effect by regulating crosstalk between Nrf2, MAPKs, and NF-κB pathways.

CONCLUSIONS
The most appropriate therapeutic strategy for the treatment of HD is currently unclear. Herein, we demonstrate for the first time the potential neuroprotective value of SSCE for the treatment of HD-like symptoms. Pre-and onset-treatment with SSCE improved neurological scores and lethality after 3-NPA treatment, but the same positive effects were not observed following progression-and peak-treatment. Pretreatment with SSCE significantly reduced SDH activity, apoptosis, microglial activation, and protein expression of IL-1β, IL-6, TNF-α, iNOS, and COX-2 in the striatum after 3-NPA treatment, corresponding to the activation of the Nrf2 pathway and attenuation of MAPKs and NF-κB pathways in the striatum following 3-NPA treatment. Additionally, gomisin A, and schizandrin treatment significantly improved neurological impairment and lethality compared to treatment with 3-NPA alone. These findings strongly indicate that SSCE has beneficial effects against 3-NPA-induced striatal toxicity that are due to anti-oxidative and anti-inflammatory activities. Further studies including those that address the molecular mechanism of SSCE and its constituents are required before it is used as a preventive and/or therapeutic strategy for neurological disorders, such as HD-like syndromes.

EK
performed the behavioral experiments, immunohistochemistry, and Western blots analysis, and prepared the figures. MJ carried out apoptotic analysis and contributed to data interpretation. ML and JC assisted with behavioral experiments and animal maintenance. SK and DJ analyzed and commented the results. IC conceived all experiments, analyzed the results, and wrote the manuscript. All authors have read and approved the final manuscript.