Scrophularia buergeriana Extract Improves Memory Impairment via Inhibition of the Apoptosis Pathway in the Mouse Hippocampus

Scrophularia buergeriana (SB) Miq. (Scrophulariaceae) has been used to help cure swelling and fever and has reported antioxidant and neuro-protective effects. However, few mechanism–based studies have evaluated the memory-improving effects in a beta-amyloid induced memory loss model. As a result of Scrophularia buergeriana extract (SBE) administration (30 and 100 mg/kg) for 28 days significantly recovered beta-amyloid-induced amnesia in the passive avoidance test and improved the impairment of spatial memory in the Morris Water Maze (MWM) task. Furthermore, SBE up-regulated superoxide dismutase-1 (SOD)-1, SOD-2, glutathione peroxidase-1, and B-cell lymphoma (Bcl)-2 protein expression levels. Additionally, SBE downregulated Bcl-2-associated X protein, cleaved caspase-9, cleaved poly (adenosine diphosphate-ribose) polymerase, and Aβ protein expression levels and inhibited the phosphorylation of the tau protein of Aβ-treated mice hippocampus. These results demonstrate that SBE improved memory impairment by reducing beta-amyloid induced neurotoxicity and regulated oxidative stress, anti-apoptotic pathways.


Introduction
Alzheimer's disease (AD) is one type of dementia and a progressive neuro-degenerative disease featured by deposits of extracellular amyloid β (Aβ) peptide and flame-shaped neurofibrillary tangles of hyper-phosphorylated tau protein, inducing neurotoxicity accompanied by cognitive impairment and memory loss [1][2][3][4].
The Aβ plaque is composed of Aβ 1-40 and 1-42, major forms of Aβ found in the brains of AD patients. The Aβ 1-42 protein is more neurotoxic and induces more oxidative damage than Aβ 1-40 [5]. An important factor in AD development is considered to be Aβ accumulation, since oxidative stress is followed by Aβ cytotoxicity [1,6]. The overproduction of reactive oxygen species (ROS) induced Aβ accumulation and oxidative stress damages cellular components resulting in structural damage, functional disorder, and cell apoptosis [7].
Cells have an antioxidant defense system protecting them from ROS attacks using various enzymes, such as superoxide dismutase (SOD), glutathione peroxidases (GPx), glutathione reductases (GR) [8]. SOD is known as the first detoxification enzyme and the most powerful endogenous antioxidant in the cell. It acts as a catalyst and converse of the superoxide (O 2− ) radical into ordinary oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ), leaving the harmful superoxide anion less dangerous.

Passive Avoidance Test
The passive avoidance test equipment (Twin County Med Associates, Hudson, NY, USA) was used to perform passive avoidance experiments. This apparatus is divided into light and dark compartments with a guillotine door in the middle. The bottom is gridded to administer an electric shock. The tests were carried out for 3 days at the same time every day at 24 h intervals. For adaptation training, the animals were placed in the shaded area for 2 min, and then placed back in the illuminated area. When the animal moved into the shaded area, it was immediately placed in the illuminated compartment (day 15). Twenty-four hours later (day 16), two training sessions were performed every 2 min. After 60 s of adaptation, the animals were allowed to move between the two compartments freely for 120 s. However, on moving to the shaded area, the guillotine door was closed and a 0.20 mA scrambled shock was applied for 2 s. The animals that failed to move were excluded from and 8 mice per group participated in the experiments. On the last day of testing, (day 17), the animal was located on the illuminated area and the guillotine door was opened. The time taken to move to the shaded area was measured.

Morris Water Maze (MWM) Test
The MWM test was performed 22 days after the Aβ 1-42 peptide injection. The platform was placed in one of the four designated release points in the water pool, allowing the animal a search time of 60 s. The mice that found the platform were left on it for 30 s, but the mice that failed to find the platform within 60 s were placed on the platform and allowed to rest for about 30 s. All mice tested twice a day, and the position of the platform was randomly changed in the water pool. Using this method, we measured the time taken to locate the platform by repeating the test for 7 consecutive days (training: 2 days, behavioral test: 4 days, probe trial: 1 day). On the last 7 days (Day 28), a probe trial was conducted. Here, 1 h after vehicle or drug administration, the platform was taken out of the pool, and the number of times the mice passed the platform was measured for 60 s on Day 28. The entire experiment plan is exhibited in Figure 1.

Passive Avoidance Test
The passive avoidance test equipment (Twin County Med Associates, Hudson, NY, USA) was used to perform passive avoidance experiments. This apparatus is divided into light and dark compartments with a guillotine door in the middle. The bottom is gridded to administer an electric shock. The tests were carried out for 3 days at the same time every day at 24 h intervals. For adaptation training, the animals were placed in the shaded area for 2 min, and then placed back in the illuminated area. When the animal moved into the shaded area, it was immediately placed in the illuminated compartment (day 15). Twenty-four hours later (day 16), two training sessions were performed every 2 min. After 60 s of adaptation, the animals were allowed to move between the two compartments freely for 120 s. However, on moving to the shaded area, the guillotine door was closed and a 0.20 mA scrambled shock was applied for 2 s. The animals that failed to move were excluded from and 8 mice per group participated in the experiments. On the last day of testing, (day 17), the animal was located on the illuminated area and the guillotine door was opened. The time taken to move to the shaded area was measured.

Morris Water Maze (MWM) Test
The MWM test was performed 22 days after the Aβ 1-42 peptide injection. The platform was placed in one of the four designated release points in the water pool, allowing the animal a search time of 60 s. The mice that found the platform were left on it for 30 s, but the mice that failed to find the platform within 60 s were placed on the platform and allowed to rest for about 30 s. All mice tested twice a day, and the position of the platform was randomly changed in the water pool. Using this method, we measured the time taken to locate the platform by repeating the test for 7 consecutive days (training: 2 days, behavioral test: 4 days, probe trial: 1 day). On the last 7 days (Day 28), a probe trial was conducted. Here, 1 h after vehicle or drug administration, the platform was taken out of the pool, and the number of times the mice passed the platform was measured for 60 s on Day 28. The entire experiment plan is exhibited in Figure 1.

Preparation of Tissue Samples
The mice were anesthetized and sacrificed after behavioral tasks for biochemical studies. The hippocampus was separated from the brain tissue and then immediately stored at −80 • C until further assessment.

Glutathione Reductase (GR) Activity
GR enzyme activity was determined using the Glutathione Reductase Assay Kit (Abcam, Cambridge, UK). Hippocampal tissues were homogenized in cold assay buffer, and centrifuged at 10,000× g for 15 min at 4 • C. The separated supernatant from hippocampus was taken and kept at −80 • C until further analysis. This assay is based on the reduction of glutathione by nicotinamide adenine dinucleotide phosphate (NADP)H in the presence of GR. GR activity can be detected by measuring the change in absorbance at 405 nm.

Western Blotting
Hippocampal tissues were homogenized with RIPA buffer and 1% protease inhibitor cocktail (Roche, Mannheim, Germany) and the lysate was centrifuged at 10,000× g for 15 min at 4 • C. The separated supernatant from hippocampus was taken, and protein concentrations were measured using a BCA protein assay kit (Thermo, Waltham, MA, USA). The proteins were separated using 8% or 12% SDS-PAGE and were then moved to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were initially incubated to block with 5% non-fat skimmed milk in Tris-buffered saline containing 0.1% Tween-20 for 30 min. Next, they were incubated with specific primary antibodies against SOD-1, SOD-2, GPx-1, Aβ (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax, Bcl-2, cleaved Caspase-9, cleaved PARP, phospho-tau, tau, and β-actin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at 23 • C. The membranes were then incubated in the corresponding horseradish peroxidase-conjugated anti-rabbit, anti-mouse immunoglobulin G (1:10000; GenDEPOT, Barker, TX, USA) for 1 h at 23 • C. The membrane was detected using the ECL system (Atto, Tokyo, Japan). The intensity of the bands on the membrane was detected by Image-Pro Plus software (6.0 Version; Media Cybernetics, Silver Spring, MD, USA)

Statistical Analysis
The experimental results are expressed as standard error of the mean (SEM) and were assessed using the SPSS program (version 22.0, SPSS Inc., Chicago, IL, USA). Difference value of between treatment groups were analyzed by Student's t-test and one-way analysis of variance (ANOVA), and performed following multiple comparisons correction using Dunnett's post-hoc test using Origin 7.0 software (OriginLab, Northampton, MA, USA). p < 0.05 indicates that there is a statistical difference, and p < 0.01 was considered statistically highly significant between mean values.

Effects of the Scrophularia Buergeriana Extract (SBE) on the Passive Avoidance Test in Aβ 1-42 Treated Mice
To determine the effect of SBE on memory deficits, we conducted the passive avoidance test in the Aβ-induced mouse model. Aβ-injected mice demonstrated a remarkable reduction in the time to move from the illuminated compartment to the shaded area compared to normal mice (p < 0.01), implying that the learning capacity was lowered. However, the step-through latency was dose-dependently increased in mice administered with SBE 30 mg/kg (p < 0.05) and 100 mg/kg (p < 0.01) ( Figure 2) compared to Aβ-injected mice.

of the SBE on the MWM Test in Aβ 1-42 Induced Memory Deficit Mice
estigate the memory-enhancing effect of SBE, we next performed the MW ice. Normal mice quickly located the platform during four consecutive behavi ected group exhibited a significantly delayed escape latency compared to th days 25-27. However, mice treated with SBE 30 mg/kg and 100 mg/kg demo escape latency from days 25-27 ( Figure 3A). Furthermore, we observed that A nstrated significantly increased swim distances to find the platform compar trol mice ( Figure 3B). We confirmed that SBE 30 mg/kg (p < 0.05) and 100 mg/kg ecreased the distance traveled to locate the platform. In the probe trials (Figur mice reported a decreased number of crossings over the previous platform he control mice. The number of crossings was recovered by treatment with SBE d 100 mg/kg (p < 0.01) compared to the Aβ-injected mice and the recovery was s we confirmed that SBE enhanced spatial recognition in the MWM test.

Effects of the SBE on the MWM Test in Aβ 1-42 Induced Memory Deficit Mice
To investigate the memory-enhancing effect of SBE, we next performed the MWM test in C57BL/6 mice. Normal mice quickly located the platform during four consecutive behavioral tests. The Aβ-injected group exhibited a significantly delayed escape latency compared to the normal group from days 25-27. However, mice treated with SBE 30 mg/kg and 100 mg/kg demonstrated a shortened escape latency from days 25-27 ( Figure 3A). Furthermore, we observed that Aβ-injected mice demonstrated significantly increased swim distances to find the platform compared to the normal control mice ( Figure 3B). We confirmed that SBE 30 mg/kg (p < 0.05) and 100 mg/kg (p < 0.01) on day 27 decreased the distance traveled to locate the platform. In the probe trials ( Figure 3C), the Aβ-treated mice reported a decreased number of crossings over the previous platform position against as the control mice. The number of crossings was recovered by treatment with SBE 30 mg/kg (p < 0.05) and 100 mg/kg (p < 0.01) compared to the Aβ-injected mice and the recovery was significant. Therefore, we confirmed that SBE enhanced spatial recognition in the MWM test.

Effects of the SBE on Glutathione Reductase (GR) Activity in the Hippocampus
To evaluate the effects of SBE on GR activity of Aβ treatment mice, GR activity was measured in the hippocampal tissue. GR activity was significantly reduced in mice treated with Aβ compared with the normal group. However, mice treated with SBE 30 mg/kg (p < 0.05) and 100 mg/kg (p < 0.05) significantly increased hippocampal GR activity compared to the Aβ-treated group. Furthermore, the efficacy of SBE was comparable to the normal group mice (Figure 4). β-treated mice reported a decreased number of crossings over the previous platform positio ainst as the control mice. The number of crossings was recovered by treatment with SBE 30 mg/k < 0.05) and 100 mg/kg (p < 0.01) compared to the Aβ-injected mice and the recovery was significan erefore, we confirmed that SBE enhanced spatial recognition in the MWM test.

Effects of the SBE on Glutathione Reductase (GR) Activity in the Hippocampus
To evaluate the effects of SBE on GR activity of Aβ treatment mice, GR activity was measured in the hippocampal tissue. GR activity was significantly reduced in mice treated with Aβ compared with the normal group. However, mice treated with SBE 30 mg/kg (p < 0.05) and 100 mg/kg (p < 0.05) significantly increased hippocampal GR activity compared to the Aβ-treated group. Furthermore, the efficacy of SBE was comparable to the normal group mice (Figure 4).

Effects of the SBE on the Antioxidant Enzymes in Hippocampus of Aβ 1-42 Treated Mice
To investigate the effects of SBE on antioxidant enzymes in the hippocampus of Aβ treated mice, the protein levels of SOD1, SOD2, and GPx-1 were evaluated. The protein levels of SOD1, SOD2, and GPx-1 were markedly decreased in mice treated with Aβ against the control group. Furthermore, treatment with SBE 30 mg/kg increased GPx-1 protein expression by 1.3-fold. In addition, SOD1,

Effects of the SBE on the Antioxidant Enzymes in Hippocampus of Aβ 1-42 Treated Mice
To investigate the effects of SBE on antioxidant enzymes in the hippocampus of Aβ treated mice, the protein levels of SOD1, SOD2, and GPx-1 were evaluated. The protein levels of SOD1, SOD2, and GPx-1 were markedly decreased in mice treated with Aβ against the control group. Furthermore, treatment with SBE 30 mg/kg increased GPx-1 protein expression by 1.3-fold. In addition, SOD1, SOD2, and GPx-1 protein levels were further increased in mice administered SBE 100 mg/kg by 4.2-, 1.7-and 2.9-fold, respectively, compared to the Aβ-treated group ( Figure 5).

Figure 4.
Effects of SBE on glutathione reductase (GR) activity in the mouse hippocampus. The hippocampus was lysed, and the supernatant was used to measurement GR activity. The results were calculated as a unit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized per protein and expressed as means ± SEM of independent experiments (n = 3). ## p < 0.01 vs. Control group; $$ < 0.01 vs. Control group; * p < 0.05 vs. Aβ (1-42) group.

Effects of the SBE on the Antioxidant Enzymes in Hippocampus of Aβ 1-42 Treated Mice
To investigate the effects of SBE on antioxidant enzymes in the hippocampus of Aβ treated mice, the protein levels of SOD1, SOD2, and GPx-1 were evaluated. The protein levels of SOD1, SOD2, and GPx-1 were markedly decreased in mice treated with Aβ against the control group. Furthermore, treatment with SBE 30 mg/kg increased GPx-1 protein expression by 1.3-fold. In addition, SOD1, SOD2, and GPx-1 protein levels were further increased in mice administered SBE 100 mg/kg by 4.2-, 1.7-and 2.9-fold, respectively, compared to the Aβ-treated group ( Figure 5).

Effects of the SBE on Apoptosis in the Hippocampus of Aβ 1-42 Treated Mice
To demonstrate the protective effect of SBE on Aβ-induced apoptosis, the protein expression levels of Bax, Bcl-2, caspase-9, and cleaved PARP were analyzed in the hippocampal tissue. Aβ treatment significantly increased the protein levels of Bax, cleaved Caspase-9, and cleaved PARP. Conversely, the Bcl-2 protein levels decreased in the Aβ-treated group. Bcl-2/Bax are known as important proteins related to apoptosis in the mitochondria. The Bax protein level was dose-dependently reduced with SBE treatment by 31% and 51%, and SBE 100 mg/kg (p < 0.01) significantly increased the levels of Bcl-2 and Bcl-2/Bax by 1.41-and 2.87-fold, respectively, compared to the Aβ-treated group in Figure 6. SBE 30 mg/kg and 100 mg/kg significantly reduced the levels of cleaved caspase-9 by 61%, and 76%, respectively, and reduced cleaved PARP by 33% and, 74%, respectively, compared to the Aβ treatment group. Therefore, we confirmed that SBE exhibited a prominent neuro-protective effect.
dependently reduced with SBE treatment by 31% and 51%, and SBE 100 mg/kg (p < 0.01) significantly increased the levels of Bcl-2 and Bcl-2/Bax by 1.41-and 2.87-fold, respectively, compared to the Aβtreated group in Figure 6. SBE 30 mg/kg and 100 mg/kg significantly reduced the levels of cleaved caspase-9 by 61%, and 76%, respectively, and reduced cleaved PARP by 33% and, 74%, respectively, compared to the Aβ treatment group. Therefore, we confirmed that SBE exhibited a prominent neuroprotective effect.

Effects of the SBE on Aβ Accumulation and Hyper-Phosphorylation of Tau Proteins in Hippocampus of Aβ 1-42 Treated Mice
The pathological hallmarks of AD include Aβ accumulation and tau hyper-phosphorylation. The hyper-phosphorylation of the tau protein, can contribute to neuronal degeneration. Using western blot analysis, we confirmed that the Aβ injection promoted Aβ accumulation in the mouse hippocampus ( Figure 7). The administration of SBE 30 mg/kg (p < 0.01) and 100 mg/kg (p < 0.01) significantly reduced Aβ accumulation in the hippocampus against as the Aβ injection group by 22% and 53%, respectively ( Figure 7B). Based on the protective effect of SBE on Aβ accumulation, we confirmed the effect of SBE on tau phosphorylation in Aβ treated mice. Aβ treatment increased the tau protein phosphorylation compared to the normal group, and treatment with SBE 30 mg/kg and 100 mg/kg significantly attenuated Aβ induced hyper-phosphorylation of tau by 69% and 71%, respectively, compared to the Aβ treatment group.
significantly reduced Aβ accumulation in the hippocampus against as the Aβ injection group by 22% and 53%, respectively ( Figure 7B). Based on the protective effect of SBE on Aβ accumulation, we confirmed the effect of SBE on tau phosphorylation in Aβ treated mice. Aβ treatment increased the tau protein phosphorylation compared to the normal group, and treatment with SBE 30 mg/kg and 100 mg/kg significantly attenuated Aβ induced hyper-phosphorylation of tau by 69% and 71%, respectively, compared to the Aβ treatment group.

Discussion
Previously, we reported that SBE extracts demonstrate potent anti-amnesic activity and antiapoptotic effects in a scopolamine-induced memory impairment mouse model and in the SH-SY5Y cell line [11,12]. In vitro studies have shown that SBE treatment inhibited cell death induced by glutamate. Furthermore, the in vivo study indicates that acute and prolonged treatment with SBE demonstrates the highest anti-oxidant activity in the scopolamine-induced memory impairment model. However, research on amyloid-beta, which affects in the development of AD, is insufficient. The first report aimed to identify the neuro-protective activities, including amyloid-beta accumulation prevention effects and related mechanisms of chronic SBE administration in the Aβ 1-42-induced cognitive deficit mice model.
AD is a progressive, neurodegenerative disorder that causes damage to the brain and is characterized by cognitive decline, and irreversible memory loss. Amyloid-beta peptides, and hyper-

Discussion
Previously, we reported that SBE extracts demonstrate potent anti-amnesic activity and anti-apoptotic effects in a scopolamine-induced memory impairment mouse model and in the SH-SY5Y cell line [11,12]. In vitro studies have shown that SBE treatment inhibited cell death induced by glutamate. Furthermore, the in vivo study indicates that acute and prolonged treatment with SBE demonstrates the highest anti-oxidant activity in the scopolamine-induced memory impairment model. However, research on amyloid-beta, which affects in the development of AD, is insufficient. The first report aimed to identify the neuro-protective activities, including amyloid-beta accumulation prevention effects and related mechanisms of chronic SBE administration in the Aβ 1-42-induced cognitive deficit mice model.
AD is a progressive, neurodegenerative disorder that causes damage to the brain and is characterized by cognitive decline, and irreversible memory loss. Amyloid-beta peptides, and hyper-phosphorylated tau protein comprising neurofibrillary tangles have been considered critical factors of pathological hallmarks of AD [10]. Indeed, Aβ levels were higher in the brain of AD patients than normal aged brain samples, indicating clinical signs such as spatial memory loss [20,21].
To assess cognitive deficits, passive avoidance and MWM tasks were performed to investigate learning and memory skills. The passive avoidance task is used to confirm the memory function by measuring the escape time from the space. The MWM task is the most widely used laboratory behavioral test to assess hippocampal-dependent cognitive deficits and learning functions in mice and rats [20,22,23]. Injection of Aβ 1-42 resulted in severe cognitive impairments and memory loss in the passive avoidance test and MWM, as well as neurodegeneration. However, SBE treatment dose-dependently increased the step-through latency in the passive avoidance task and decreased escape latency and swim distance significantly for 4 days of MWM trials. This implied that SBE administration improves cognitive performance and ameliorates memory deficits, although not as completely as the normal model that did not cause memory loss.
In the previous study, we have demonstrated that neuronal cell death and cognitive deficiency due to oxidative stress are related to Aβ accumulation in the brain. [24]. Aβ is a known neuro-toxic peptide that promotes oxidative stress and lipid peroxidation in the intermembrane and also causes ROS generation by serving as a source of ROS [5,6,25]. The accumulation of Aβ may result in an increased production of ROS, subsequently leading to neuronal death through apoptotic pathways [25,26]. To prevent ROS generation and regulate the steady-state O 2 concentration, cells have anti-oxidant defense systems such as SODs, GR and GPx-1 enzymes [11,12,27]. Aβ 1-42 injection attenuated hippocampal antioxidant enzyme activities and protein expression levels, the same as in previous studies [28,29]. Nevertheless, we observed that the administration of SBE (30, 100 mg/kg) could significantly enhance GR activity and SODs, GPx-1 protein levels in the Aβ 1-42-treated mouse hippocampus.
The mitochondria are much more sensitive to oxidative stress and Aβ-mediated oxidative stress increased mitochondrial dysfunction leading to apoptotic cell death. The accumulated Aβ triggers neuronal death in the hippocampus by releasing caspase activators [24,29,30]. Aβ-induced apoptosis is regulated by mediators such as caspases, Bcl-2, and Bax [10]. The proportion of Bax and Bcl-2 affects whether a cell undergoes or escapes apoptosis. The caspase pathway promotes apoptosis and releases apoptosis-promoting factors when the Bax/Bcl-2 ratio is increased [20]. In our study, SBE treatment significantly lowered Bax, cleaved caspase-9, and cleaved PARP protein expression levels and upregulated Bcl-2 protein expression levels at the same time in mice hippocampal tissue.
Aβ injections increased the accumulation of Aβ and induced hyper-phosphorylation of the tau protein in the brain. The brains of AD patients demonstrate abnormally phosphorylated tau, 4-8-fold higher than normal brains [31]. In this study, the Aβ-injection increased the phosphorylated tau protein expression levels about 4-5 times as against the normal control. We observed that the Aβ and phosphorylated tau protein expression levels dose-dependently decreased in response to treatment with SBE compared to the normal group.
However, this pre-clinical study has some limitations and we will confirm the anti-inflammatory effects of SBE in a mouse model induced neuroinflammation and memory loss in a further study. Further studies are also needed for immunohistochemical analysis and the effect of SBE in clinical trials.
Collectively, our results indicate that SBE treatment improved memory impairment through reduction of Aβ accumulation and the regulation of oxidative stress, anti-apoptotic pathways, and tau protein hyper-phosphorylation in the Aβ 1-42 memory impairment-induced hippocampal tissue.

Conclusions
This study demonstrated that SBE possesses antioxidant and neuroprotective effects against a β-amyloid induced memory loss model. Amyloid beta generates oxidative stress as an early event in AD and causes tau phosphorylation, mitochondria dysfunction and ROS generation. Consecutively, it leads to apoptosis and neurodegeneration. SBE treatment ameliorates memory deficits and improves learning and memory function. SBE also exhibited remarkable neuroprotective effects against Aβ 1-42 induced neurotoxicity via the apoptosis pathway ( Figure 8). This study offers SBE at a dose of 100 mg/kg which suggests the possibility of development as a health functional food for the improvement of learning and memory impairment.
against Aβ 1-42 induced neurotoxicity via the apoptosis pathway ( Figure 8). This study offers SBE at a dose of 100 mg/kg which suggests the possibility of development as a health functional food for the improvement of learning and memory impairment.

Conflicts of Interest:
The authors declare no conflict of interest in this study.