Dietary intake of branched-chain amino acids in a mouse model of Alzheimer's disease: Effects on survival, behavior, and neuropathology

Introduction High levels of plasmatic branched-chain amino acids (BCAA), commonly used as dietary supplements, are linked to metabolic risk factors for Alzheimer's disease (AD). BCAA directly influence amino acid transport to the brain and, therefore, neurotransmitter levels. We thus investigated the impact of BCAA on AD neuropathology in a mouse model. Methods 3xTg-AD mice were fed either a control diet or a high-fat diet from 6 to 18 months of age. For the last 2 months, dietary BCAA content was adjusted to high (+50%), normal (+0%), or low (−50%). Results Mice fed a BCAA-supplemented high-fat diet displayed higher tau neuropathology and only four out of 13 survived. Mice on the low-BCAA diet showed higher threonine and tryptophan cortical levels while performing better on the novel object recognition task. Discussion These preclinical data underscore a potential risk of combining high-fat and high BCAA consumption, and possible benefits from BCAA restriction in AD.

Introduction
Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, and the number of demented patients in the world is predicted to double within the next 15 years because of the aging population [1]. Given the lack of disease-modifying treatments, controlling environmental risk factors remains an attractive opportunity to reduce the incidence of AD [2]. Changing dietary habits has been proposed as a means of delaying the onset of the disease either through direct effects on brain function or indirectly by improving peripheral metabolic determinants [3]. Indeed, common nutrients such as lipids, amino acids, and polyphenols may have significant impact on cognitive performance [4][5][6]. However, the optimal diet to be used in prevention trials remains to be determined.
Branched-chain amino acids (BCAA; leucine, isoleucine, and valine) are essential amino acids, comprising approximately 20% of protein intake. BCAA are commonly used as dietary supplements, by doubling the daily intake of those amino acids, to increase muscular mass and recovery after exercise [7,8]. However, a BCAA-related signature is identifiable in a context of metabolic disorders: circulating BCAA are increased in people suffering from obesity and have been shown to predict insulin resistance and other complications related to metabolic diseases [9,10]. Evidence from metabolomics studies indicate that high-fat diet (HFD) acts in synergy with BCAA to induce metabolic disorders, at least in animal models [11].
BCAA enter the central nervous system via the large neutral amino acids transporter (LAT1) at the blood-brain barrier, where they compete with tryptophan and tyrosine, which are direct precursors to serotonin and dopamine, respectively [12]. Thus, circulating BCAA can modulate levels of these key neurotransmitters in the brain, thereby altering brain function and behavior [13][14][15][16]. Because cognitive impairments observed in AD have been associated with a neurotransmitter imbalance [17], we hypothesize that BCAA intake can modulate AD-like behavior. Indeed, the maple syrup urine disease is a neurometabolic disorder characterized by a toxic accumulation of BCAA and their metabolites, which leads to severe mental retardation and encephalopathy [18,19].
It is only recently that studies have provided clues to a potential link between BCAA and AD pathogenesis. A metabolomic analysis from the ADNI cohort recently reported inverse correlations between plasmatic valine concentrations and cognitive deficits, as well as ventricle volume in symptomatic stages [20]. Another metabolomic study in large prospective cohorts relates higher levels of plasma BCAA with a lower risk of dementia [21]. Conversely, a genome-wide association study suggests that singlenucleotide polymorphisms (SNPs) associated with higher plasma isoleucine levels are positively associated with the risk for developing AD [22]. However, no studies have focused yet on the effect of BCAA consumption on AD pathology. Thus, we investigated the impact of both BCAA supplementation and restriction in old 3xTg-AD mice, an animal model of age-related behavioral impairments and Ab/tau neuropathologies.

Animals and diets
The 3xTg-AD (APP swe , PS1 M146V , tau P301L ) mouse model of genetically induced AD-like neuropathology was used with an equal number of males and females in each group. Mice produced at our animal facility received either a control diet (CD; 12% kcal fat) or a high-fat diet (HFD; 60% kcal fat) for a 10-month period starting at the age of 6 months. At 16 months of age, groups of mice were exposed until 18 months to the same diets modified to include high (150%), normal (10%), or low (250%) BCAA content (Fig. 1A). Table 1 provides a detailed description of the amino acids measured in the diets using gas chromatography with flame ionization detector (see methods below), whereas Supplementary Table 1 contains the list of amino acids added to the formulations. Supplementary Table 2 shows the description of fatty acids measured in the diets using gas chromatography. Diets were formulated and custom-made by Research Diets, Inc. (New Brunswick, NJ) to ensure uniformity between groups and consistency with our previous studies with the 3xTg-AD mouse [23,24].
All mice were put under deep anesthesia with a ketamine/xylazine i.p. injection (100 mg/kg ketamine, 10 mg/ kg xylazine) and sacrificed by intracardiac perfusion with 50 mL of ice-cold 0.1 M phosphate buffer saline (PBS) with a cocktail of inhibitors of phosphatases (sodium pyrophosphate 1 mM and sodium fluoride 50 mM) and proteases (SigmaFAST protease inhibitor tablets; Sigma-Aldrich, St. Louis, USA) as described elsewhere [23]. Rapidly, one hemisphere of the brain was dissected and frozen at 280 C until processing for Western blot, ELISA, or measurements of amino acids and neurotransmitters. All experiments were performed in accordance with the Canadian Council on Animal Care and were approved by the Institutional Committee of the Centre Hospitalier de l'Universit e Laval (CHUL).

Biochemical analyses
Aspartate aminotransferase, alanine aminotransferase, creatine kinase, triglycerides, and cholesterol were analyzed in plasma from intracardiac blood (centrifuged 5 min, 3000 rpm) sampled just before intracardiac perfusion at sacrifice with a modular analyzer (Roche). Only samples with more than 100 mL of plasma could be analyzed with that method. When the hemolysis level reached more than 50 g/dL, samples were eliminated from the analysis. Creatinine was determined with an ELISA kit (B-Bridge International Inc, Santa Clara, CA, USA) according to the manufacturer's instructions.

Behavioral assessment
Recognition memory and locomotor activity were evaluated with the novel object recognition test and the open field test, respectively, as previously described (see the Supplementary Material) [25].

Amino acid quantification in diet, plasma, and brain
Amino acids in the diets, plasma, and cortex homogenates were analyzed by gas chromatography with flame ionization detector as described in Supplementary Material [26].

Protein extraction and Western immunoblotting
For Western immunoblotting and b-Amyloid ELISA, proteins were extracted from the parieto-temporal cortex. The protein extraction method results in a TBS-soluble fraction (intracellular and extracellular fraction), a detergent-soluble fraction (membrane fraction) and a detergent-insoluble fraction (insoluble proteins resuspended in formic acid) as previously described [25]. The detailed method for Western immunoblotting is described elsewhere [24]. The list of primary antibodies used in our experiments is available in Supplementary Table 5. Homogenates were all run on the same gel for each experiment.

Ab 40 and Ab 42 quantification
Ab 40 and Ab 42 were measured in TBS-soluble and detergent-insoluble fractions from the parieto-temporal cortex using sensitive human b-Amyloid ELISA kits (Wako, Osaka, Japan) according to the manufacturer's instructions, as shown [24]. Plates were read at 450 nm using a Synergy HT multidetection microplate reader (Biotek, Winooski, VT, USA).

Statistics
Data are presented as the mean 6 SEM. Statistical analysis used and the numbers of mice per group are specified in each figure or legend. Briefly, one-(one independent variable) or two-way (two independent variables) ANOVAs were used when more than two groups were compared and were followed by Tukey's post hoc (equal variance). In case of unequal variance, Dunnett's post hoc was used or a Kruskal-Wallis followed by Dunn's. The log-rank Mantel-Cox test was used for the analysis of the survival curves. Correlations between variables were investigated using linear regression analyses. All statistical analyses were performed with Prism 5 (GraphPad software, San Diego, CA, USA) or JMP (version 12.1.0; SAS Institute Inc., Cary, IL, USA) softwares, and statistical significance was set at P , .05.

Results
Experiments were performed in the 3xTg-AD mouse model of AD, which develops behavioral impairments as well as Ab and tau neuropathologies with age. Because age-related factors are essential to the development of AD [28,29], old 3xTg-AD mice were selected. To evaluate the impact of BCAA on AD pathogenesis, 3xTg-AD mice were fed a CD or HFD from 6 to 16 months of age and then both diets were either supplemented or restricted in BCAA by 50% for 2 months (Table 1; Fig. 1A). Animals were exposed to an HFD because it is more representative of prevalent dietary habits in Western societies [30], and also because HFD has been shown to accelerate AD-like pathology [23,24,31,32] and to synergize with BCAA to induce metabolic disorders [11] in animal models.
3.1. BCAA supplementation combined with HFD led to the premature death of two-thirds of 3xTg-AD mice At the end of the study, only four out of 13 mice (30.8%) in the group fed the HFD survived the 2-month supplementation with BCAA, which is significantly less than the group fed an HFD only, in which 10 mice out of 12 mice (83.3%) survived (Fig. 1B). Interestingly, no mice restricted in BCAA for the 2month period died prematurely, regardless of the base diet. Although a total of 15 animals from all groups died before the end of the planned study, we were able to sacrifice a few (n 5 7) in the high-BCAA HFD group while they were in critical conditions, allowing collection of intracardiac blood and brains as described in the Methods. These moribund mice fed the combination of HFD and BCAA (identified with red circles in Fig. 1) displayed higher aspartate aminotransferase, alanine aminotransferase, and creatine kinase plasmatic concentrations (Fig. 1C, D and E), suggesting major liver, muscular, or cardiac damage [33]. A two-way ANOVA revealed that mice fed the HFD, independently of BCAA intake, had lower plasmatic creatinine concentrations, possibly due to reduced muscle mass (Fig. 1F) [34]. No differences were found in plasma triglycerides and cholesterol levels between each group (Supplementary Table 4).

3.2.
Titrating BCAA supply modifies plasma and brain amino acids and monoamines levels in 18-month-old 3xTg-AD mice High BCAA consumption was reported to directly modify levels of plasmatic BCAA and aromatic amino acids (AAA) in humans [14]. Because tryptophan, threonine, tyrosine, valine, leucine, and isoleucine all compete for LAT1 for brain entry, specific changes in circulating amino acids may impact their balance in the brain, as well as downstream monoaminergic neurotransmitters [13,14]. We thus measured levels of amino acids in the plasma and parieto-temporal cortex (Fig. 2). We first confirmed that BCAA restriction decreased valine, leucine, and isoleucine concentrations in the plasma in both CD and HFD groups, but not in the cerebral cortex ( Fig. 2A, D). Lower BCAA intake also increased the ratio of circulating AAA to BCAA and levels of glycine and tyrosine under both types of diets (Fig. 2B, C). Although a 50% higher consumption of BCAA was not reflected in significantly higher plasmatic or cortical concentrations, increased asparagine levels were noted in the plasma of mice supplemented with BCAA in either the CD or HFD (Fig. 2C). Moreover, threonine levels were markedly modulated in the brain with higher and lower concentrations following low and high BCAA intake, respectively, whether in CD or HFD (Fig. 2F). Changes in cortical tyrosine levels were not significant, probably due to interindividual variations (Fig. 2F), whereas cortical concentrations of tryptophan, but not phenylalanine, were inversely associated with levels of BCAA in the plasma in both CD and HFD (Fig. 2F).
To confirm that modulations in plasmatic and brain amino acids impact neurotransmitter synthesis, we measured catecholamines (dopamine, DA) and indolamines (serotonin, 5-HT) and their metabolites in the prefrontal cortex and hippocampus of the 3xTg-AD mice (Fig. 3). Whereas BCAA intake did not affect DA concentrations in 3xTg-AD mice, a trend toward higher 5-HT content was observed in both the prefrontal cortex and hippocampus of mice fed an HFD reduced in BCAA compared with mice fed an HFD only (Fig. 3A, F). In addition, a low BCAA intake led to higher levels of metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in both the prefrontal cortex and hippocampus, compared to the HFD supplemented with BCAA (Fig. 3B, D, G).

Low BCAA dietary intake improves object recognition memory in old 3xTg-AD mice
To assess the effect of BCAA dietary intake on memory function, all groups of mice underwent the novel object recognition task at 18 months (Fig. 4A). This test requires minimal movement and induces no anxiety, which makes it a suitable choice for old 3xTg-AD mice [24]. The test first confirmed that 18-month-old 3xTg-AD mice, fed either a CD or a HFD, did not discriminate the novel from the old object (Fig. 4B, C), as previously shown [25]. Similarly, 3xTg-AD mice fed the high BCAA diet failed to recognize the novel object. In contrast, 3xTg-AD mice fed the low BCAA diets, either CD-low or HFD-low, spent significantly more time exploring the novel than the familiar object, suggesting memory improvement after BCAA restriction. The open field test confirmed that no group of mice demonstrated impaired voluntary locomotor activity, which could have affected exploratory behavior (Fig. 4D). The time the mice spent on a wire confirms that muscular strength was not affected by dietary treatments (Supplementary Fig.1). Interestingly, cortical threonine levels were correlated with the recognition index (Fig. 4E).

BCAA supplementation in an HFD context worsens tau hyperphosphorylation but not amyloid pathology in old 3xTg-AD mice
Tau hyperphosphorylation is one of the main neuropathological hallmarks of AD, correlating with cognitive deficits in patients with AD [28,35]. Here, we found that a 2-month BCAA supplementation combined with HFD was sufficient to increase soluble tau pSer202 (194%), pSer202/Thr205 (1271%), pThr231 (1680%), and pThr181 (193%) compared with control mice, without changing the level of total tau (Fig. 5A-D). Strikingly, phosphorylation of detergent-insoluble tau at pSer202 was also increased in the HFD-high group (1151%), whereas tau pSer396/404 and both total tau and total human tau were unchanged ( Fig. 5E-H). In contrast, reducing the BCAA resulted in a tau phosphorylation status similar to mice fed the CD. However, we did not find any changes in protein levels of the main kinases involved in tau phosphorylation (Supplementary Table 5).
Accumulation of brain Ab peptides and extracellular plaques are key markers of AD replicated in the 3xTg-AD mouse [25,28]. However, we did not observe any effect of BCAA consumption on Ab 40 or Ab 42 peptides in soluble or detergent-insoluble fractions (Fig. 5I, J). Finally, we found that the cytochrome oxidase protein, a marker of cellular activity, was greatly increased in the cortex of mice fed the HFD supplemented with BCAA (Fig. 5K). No changes in drebrin (Fig. 5L) or other AD-relevant markers were noted (Supplementary Table 5).

Discussion
The consumption of high levels of BCAA is prevalent in our contemporary society, but no study to our knowledge has directly probed for the impact of high or low BCAA intake in a context of AD and old age [8,36]. Correlative data linking BCAA and AD exist [20][21][22], but conclusions on causality require controlled intervention difficult to achieve in a clinical setting. In this study, 3xTg-AD mice were either supplemented or restricted in BCAA over a CD or HFD from 16 to 18 months of age. We found that BCAA supplementation combined with a HFD resulted in a significant mortality rate and strongly increased tau neuropathology. In contrast, reducing BCAA in both the CD and HFD did not result in any premature death, improved cognitive performance, and led to increased levels of threonine, tryptophan, 5-HT, 5-HIAA, and DOPAC in the brain.

BCAA toxicity
A first striking consequence of BCAA supplementation when combined with an HFD was a shortening of the lifespan of 3xTg-AD mice. Rises in plasmatic alanine aminotransferase, aspartate aminotransferase, and creatine kinase were observed in moribund animals and represent a possible signature of hepatic and muscular toxicity, corroborating previous studies reporting BCAA-induced liver injuries in HFD-fed obese mice, inflammation and oxidative stress [33,37]. Although the cause of death for all the animals included in our study is not definitive, major brain damages (atrophy, edema, etc.) were not observed, suggesting that hepatic and muscular toxicity played a more direct role in their premature death. Although little is known about BCAA metabolism in the elderly, some studies have revealed that the response to BCAA supplementation is different in old age [38]. Thus, 18month-old 3xTg-AD mice could display a failure in catabolizing BCAA, thus disturbing amino acids homeostasis in the brain and peripheral tissues.

Modulation of amino acids by BCAA intake: Plasma and brain levels
Our results further confirm that BCAA dietary supply modulates plasma and brain amino acids and catecholamines. Perhaps surprisingly, BCAA supplementation was not accompanied by a significant increase in plasmatic and cortical concentrations in the 3xTg-AD mouse, but rather by higher levels of asparagine in the plasma and lower cortical threonine. It should be kept in mind that the present measurement reflect an equilibrium reached following 2 months of altered BCAA intake, which is also affected by other factors such as distribution of amino acids in tissue where they integrate into proteins and elimination (metabolism and renal clearance). Still, BCAA restriction lowered  BCAA and increased tyrosine concentrations in the plasma, whereas in the cortex, levels of threonine and tryptophan were upregulated, suggesting complex interactions in amino acid homeostasis.
Previous studies have shown that BCAA administration decreases cortical tyrosine and tryptophan in the hippocampus of rats [39]. The likely mechanism is competition for amino acid transporters at the blood-brain barrier [12]. However, despite obvious trends in the means, no significant change in brain tyrosine content was detected here due to interindividual variation. More clearly, increases and decreases in BCAA intake induced a strong modulation in cortical concentrations of threonine, possibly due to the fact that brain uptake of threonine is highly sensitive to competition from BCAA [40]. Finally, although we did not observe a decrease in serotonin following BCAA supplementation as previously reported [15,39], a 2-month reduction in dietary BCAA combined to a HFD led to a trend toward increased serotonin and dopamine levels.
This tendency was supported by significant increases in serotonin metabolite 5-HIAA and dopamine metabolite DOPAC, changes typically interpreted as signs of increased secretion and metabolism of these neurotransmitters [41]. Overall, our results confirm that BCAA dietary supply modulates plasmatic and cerebral amino acids and neurotransmitters, thus providing a means by which they can modify brain homeostasis and function.

BCAA and memory
The observed failure of 18-month-old 3xTg-AD mice to recognize the novel object is consistent with previous work in the same model at similar ages [5,24,25]. As old transgenic mice did not recognize the novel object, it was predictable that further deterioration of memory could not be detected at this age even after BCAA supplementation. However, a 2-month reduction in BCAA intake improved recognition memory in mice fed either the CD or HFD. These results were intriguing because HFD is known to worsen cognitive defects in 3xTg-AD mice [24]. The observed increase in cortical amino acids and monoamines may underlie these memory improvements, as suggested by the fact that the animals with the higher levels of brain threonine performed better at the novel object recognition task. In particular, threonine, tryptophan, and serotonin are known to be involved in behavioral changes [15,42].

BCAA and AD neuropathology
BCAA have been only recently implicated in AD. Plasmatic valine has been linked to cognitive score and brain atrophy in an AD cohort [20], while a recent Mendelian randomization analysis indicates that genetic susceptibility to high plasmatic isoleucine is associated with a higher risk of developing the disease [22]. APP/PS1 mice, a mouse model   Fig. 5. BCAA-supplemented HFD worsens tau pathology but not amyloid pathology in 18-month-old 3xTg-AD mice. TBS-soluble tau phosphorylation at pSer202, pSer202/Thr205, pThr231, and pThr181 (A-D), detergent-insoluble tau phosphorylation at pSer202 and pSer396/404 (E, F), detergent-insoluble total tau and total human tau (G, H), TBS-soluble and detergent-insoluble Ab 42 /Ab 40 (I, J), detergent-soluble cytochrome oxidase 1 (K), and ratio of detergent-soluble on TBS-soluble drebrin (L) measured by Western blot in cortex homogenates. Data for tau phosphorylation were calculated as the ratio of the phosphoepitope to total tau concentration. Data are presented as mean 6 SEM. One-way ANOVA, Dunnett's (vs CD) post hoc analysis: *P ,.05, **P ,.01. Representative bands were selected from nonconsecutive samples run on the same gel. Abbreviations: BCAA, branched-chain amino acid; CD, control diet; HFD, high-fat diet; ctl, normal levels of BCAA; high, BCAA-supplemented diet by 50%; low, BCAA-reduced diet by 50%; OD, optical density; Ab, amyloid b. of amyloid pathology, spontaneously display increased plasmatic BCAA, without obesity, suggesting that AD pathology potentiates defects in BCAA metabolism, putting patients with AD at a higher risk of BCAA-induced brain damages [43,44]. Conversely, tau pathology is one of the major neuropathological markers of AD, strongly associated with antemortem cognitive deficits [35,45]. We found that a 2-month BCAA supplementation combined with a HFD strongly increased both soluble and detergentinsoluble tau hyperphosphorylation in the cortex of old 3xTg-AD mice. These results suggest that the combination of BCAA supplementation and HFD has synergistic deleterious effects on AD pathology and neuronal function. To our knowledge, it is the first time that BCAA supplementation has been implicated in tau phosphorylation. Although synaptic and amyloid pathologies were unaffected by BCAA modulation, the rise in cytochrome oxidase 1 protein in cortex homogenates of mice under BCAA-supplemented HFD suggests an increase in mitochondrial oxidative activity in those mice, possibly reflecting a state of neuronal oxidative stress [46].

Conclusion
There is scientific evidence that dietary intervention can reduce the risk of developing age-related cognitive impairment or AD [3,47]. Thus, nutritional strategies aimed at reducing AD pathology are of interest in the field. This study provides controlled preclinical data pinpointing the specific role of BCAA in an animal model of AD. For the first time, our results identify the potential risks of combining high BCAA consumption with a HFD, not only regarding peripheral toxicity but also with respect to the aggravation of AD-like tau neuropathology. On a more positive note, our results suggest that reducing the BCAA content in the diet supply, while maintaining protein intake, could have a beneficial effect on memory and could be considered in nutritional interventions aimed at improving cognitive performance and/or reducing the incidence of AD. Thus, it would be interesting to test whether protein supplementation with low BCAA concentration could prevent mood and memory impairment. Another easier intervention for the elderly would be to promote foods rich in protein but low in BCAA [36].


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Characterization of Apoptosis in a Breast Cancer Cell Line after IL-10 Silencing

Background Breast cancer is affected by the immune system in that different cytokines play roles in its initiation and progression. Interleukin-10 (IL-10), an anti-inflammatory cytokine, is an immunosuppressive factor involved in tumorigenesis. The present study was conducted to investigate the gene silencing effect of a small interference RNA (siRNA) targeting IL-10 on the apoptotic pathway in breast cancer cell line. Methods The siRNA targeting IL-10 and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) clone were introduced into MDA-MB-231 cells. Real-time PCR assays were used to determine IL-10 and GAPDH gene expression levels, in addition to those for protein kinase B (AKT), phosphoinositide 3-kinase (PI3K), B-cell lymphoma 2 (Bcl2), caspase-3 and caspase-9 genes related to apoptosis. Results Inhibition of IL-10 by the siRNA accelerated apoptosis and was accompanied by significant increase in caspase-3 and caspase-9 and a significant decrease in PI3K, AKT and Bcl2 expression levels compared to the non-transfected case. Conclusions In conclusion, the production of IL-10 may represent a new escape mechanism by breast cancer cells to evade destruction by the immune system. IL-10 gene silencing causes down regulation of both PI3K/AKT and Bcl2 gene expression and also increases the Bbc3, BAX caspase3, and caspase 3 cleavage expression levels. IL–10 might represent a promising new target for therapeutic strategies.

The IL-10 is immunosuppressive and anti-inflammatory cytokine. It inhibits antigen presentation to T cells via other cytokines (Ralph et al., 1992) and its up-regulation is associated with tumorigenesis, autoimmunity, and transplantation resistance (Katsikis et al., 1994;Yang et al., 2003). The IL-10 is produced mostly by monocytes, to some degree by lymphocytes (Fiorentino et al., 1989;Reddy et al., 2001) and by human tumor cells (Chen et al., 1994;Heckel et al., 2011) . However, other cancer cells such as ovarian cancer did not express IL-10 transcripts (Merogi et al., 1997). High serum levels of IL-10, IL-6 and TNF-α in the cancer patients are correlated with their survival outcome, resistant to the cytotoxicity of the chemotherapeutic drugs.
The apoptotic/anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members have been involved in the drug-resistant phenotype of many malignancies (Reed, 1995). The serine/threonine protein kinase AKT, a member of the phosphoinositide 3-kinase (PI3K) pathway, is involved in different cellular processes including cell proliferation and apoptosis (Franke et al., 2003). The activation of PI3K/Akt stimulates the cell survival via the phosphorylation and inactivation of several pro-apoptotic proteins, including Glycogen synthase kinase 3 (GSK-3), Bcl-2-associated death promoter (BAD) and caspase-9 (Cardone et al., 1998;Szanto et al., 2009;Lee et al., 2013). The hyper activation of PI3K/AKT is occurred in cancer (Sliva et al., 2002), therefore the inhibition of PI3K/Akt pathway may provide a new therapeutic approach for cancer treatment.
Small interference RNA (siRNA) is a genetic tool to facilitate the post-transcriptional sequence specific gene silencing (Okamoto and Murawaki, 2012). Therefore, this technique might be used for the inhibition of a specific gene in vitro and in vivo in order to understand its function. In siRNA process, a double stranded RNA combines with its homologous mRNA leading to mRNA degradation or silence of the translation process (Elbashir et al., 2001). This study aimed to evaluate the apoptotic gene regulation in human TNBC cell lines via knockdown IL-10 using siRNA.

IL-10 and GAPDH SiRNA Transfection
Transient transfection of siRNA was performed using lipofectamine transfection 3000 reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instruction. In brief, triple negative human breast cancer (MDA-MB -231) and human breast epithelial cell lines (MCF10A) were plated in triplicates with cells density of 2 × 104 per well in 12-well plates for 16 hours until cells reached approximately 70% confluence. One hour before transfection the cells were cultured in antibiotic-free medium. The cells were incubated with transfection mixtures containing 20 nM of GAPDH-or IL-10-siRNA for 8 hours, and then the medium was replaced with full culture medium and the cells incubated for 48 hours. Control cells (un-transfected cells) were transfected with lipofectamine 3000 reagent only. The RNA was extracted, reverse transcriptase polymerase chain reaction (RT-PCR) assay and the real time RT-PCR assay were done.

Detection of IL-10 by RT-PCR
To confirm the detection of IL-10 in both MDA-MB-231 and MCF-10 A, total RNA was extracted from non-transfected cell lines. The cDNA was generated from 1μg total RNA using High capacity cDNA reverse transcriptase kit (Applied Biosystem, CA, USA). The PCR reaction was done in 50 μl, contained 20-50ng cDNA, 1X PCR Master Mix (Promega, Madison, USA), 3 mM MgCl2, 300nM of forward primer GTGATGCCCCAAGCTGAGA and reverse primer CCCCCAGGGAGTTCACATG. The PCR was performed with the following cycling profile: incubation at 94°C for 5 min followed by 40 cycles of 1 min denaturation at 95°C, 1 min annealing at 55°C, and 1 min elongation at 72°C. The last cycle was followed by a final extension of 10 min at 72°C. Polymerase chain reaction master mix without cDNA was used as negative control. The PCR products were analyzed on a 2% agarose gel stained with ethidium bromide and visualized by UV-transillumination.

Measurement of caspase-3 activity
T h e c a s p a s e -3 a c t i v i t y w a s m e a s u r e d i n transfected-and nontranfected-MDA-MB-231 cells by using colorimetric assay ab39401 (Abcam, Cambridge, MA 02139-1517, USA) kit according to the manufacturer instructions. In brief, transfected MDA-MB -231 cells were incompletely homogenized in 50 µl cell lysis buffer on ice for 10 minutes. After centrifugation, the protein concentrations were adjusted to 50-200 μg protein per reaction. Fifty microliter of caspase reaction mix containing 10 mM DTT was added to each well. N-Acetyl-Asp-Glu-Val-Asp p-nitroanilide (DEVD-p-NA) (200µM) substrate was added, then after 120 min incubation at 37ºC the plate was measured at 405 nm. Fold-increase in caspase 3 activity can be determined by comparing treated cells results with the level of the untreated control.

Real time RT-PCR
Quantitative real-time PCR reaction was carried out to evaluate the expression levels of GAPDH, IL-10, PI3K, AKT, Bcl2, BAX, caspase9 and caspase3. Total RNA from MDA-MB-231 and MCF10A cells were extracted using Axygen total RNA extraction kit (Axygen, Corning GmbH, Germany) according to the manufacturer's instruction. The concentration and purity of the extracted total RNA were determined by Nanodrop (Thermo Scientific, USA) in triplicates. The extracted RNA had a 260/280 ratio of 1.9-2.1. Total RNA concentration was then adjusted to 250 µg/μl before storing at -80 o C immediately after extraction. The cDNA was synthesized from 1 µg RNA using high capacity cDNA reverse transcription kit (life technology, Applied biosystem, USA) according to the manufacturer's instruction. Quantitative real-time PCR reaction was used 1μl of cDNA, 300 nM of forward and reverse primers Table 1 in 20 ul of 1X PCR master mix with SYBR Green (Applied biosystem, life technology, USA). All reactions were run in triplicate with reaction profile as pre-denaturation for 2 min at 95°C followed by 40 cycles with 95°C for 10 secs and 60°C for one min. The gene expression results were analyzed using 2 -ΔΔCT method using MCF10A as a control and GAPDH as endogenous control (Hafez et al., 2014). The data were expressed as mean fold changes ± standard error for three independent amplifications.

Statistical analyses
Each experiment was performed in triplicat on three independent cluture. Data were reported as means ± SEM. Student's t-test (two-tailed) was used to compare levels of PI3K in non-transfected and transfected MDA-231 cell line was shown in Figure 2A. The silencing of IL-10 induced by siRNA resulted in a significant downregulation of PI3K by 4-fold compared to the non-transfected cells (P<0.002) and by 5-fold compared to MCF10A cells.
The effect of IL-10 knockdown on the AKT expression levels in non-transfected and transfected MDA-231 cell line compared to MCF10A was shown in Figure 2B. The siRNA silencing of IL-10 resulted in significant down regulation of AKT by 5.44-fold compared to the non-transfected cells and by 4-fold compared to MCF10A cells (P<0.002). Whereas, the AKT expression levels was significantly increased in the untreated cells compared to the MCF10A cells (P<0.005).
differences between groups. P < 0.05 was considered statistically significant. Using Graph pad prism 5 software (GraphPad Software, CA, USA) The differences were considered statistically significant when p < 0.05.

Transfection efficiency
To determine the transfection efficiency and the successful delivery of siRNA in MDA-MB-231 cells we used GAPDH as a positive control for gene silencing. The results showed a significant inhibition of GAPDH and IL-10 mRNA expression levels after transfection compared to the non-transfected cells (P<0.05) Figure 1.
The effect of IL-10 gene silencing on the expression Caspase-9 5'-ATTGCACAGCACGTTCACAC-3' 5'-TATCCCATCCCAGGAAGGCA-3'   The effect of IL-10 gene silencing on the caspase 9 expression levels in non-transfected and transfected MDA-231 cell line was shown in Figure 3B.
The silencing of IL-10 resulted in up regulation of caspase 9 by 12-fold compared to the non-transfected cells and by 3.6-fold compared to the MCF10A cells (P<0.005). In the non-transfected cells, the expression levels of caspase 9 was decreased by 70% compared to the MCF10A cells (P<0.001).
The effect of IL-10 silencing on the expression levels of caspase 3 and caspase 3 cleavages were shown in Figure 4 A and B. Gene silencing induced by siRNA lead to increase in the expression levels of caspase 3 mRNA by 31-fold compared to the non-transfected cells and by 6.3-fold compared to the MCF10A cells (P<0.001). The   increase in the caspase3 gene expression was associated with the significant increase in its cleavages in transfected cells compared to non-transfected cells. Figure 5 (A and B) shows the effect of silencing IL-10 on the Bbc3 and BAX genes expression levels. The silencing of IL-10 increased the Bbc3 and BAX expression levels in the transfected cells compared to the non-transfected cells. The knockdown of IL-10 was resulted in increasing in the BbC3 expression levels by 5.4-fold compared to both the MCF10A cells and to the non-transfected cells (P<0.002). In the same manner, the knockdown of IL-10 lead to significant increase in the BAX expression levels by 4.2-fold compared to MCF10A and by 9.3-fold compared to the non-transfected cells (P<0.005). On the other hand, the expression levels of BAX gene in the non-transfected cells was significantly decreased by 2-fold compared to the MCF10A cells (P<0.05).

Discussion
The expression of cytokines within tumor microenvironment plays an important role in the cancer development and progression. Most tumor cells produce immunosuppressive cytokines which lead to their escape from the host immune response (Yanaihara et al., 2012), therefore the regulation of apoptosis in malignant cells is a vital area in cancer research. Also different cytokines play an important role in the breast cancer initiation and progression.
Anti-inflammatory cytokine such as Interleukin-10 induces immunosuppression and assists the escape mechanism of tumor cells from tumor immune surveillance. Human cancer cells can produce IL-10 but it is mainly produced by monocytes (Heckel et al., 2011). Like several other cytokines, IL-10 can exert dual proliferative and inhibitory effect on breast cancer cells showing its complex role in the cancer initiation and progression. Some studies reported the high IL-10 expression levels in breast cancer paraffin section and its expression is correlated with worse outcome in patients with malignant tumor (Li et al., 2014;Zhao et al., 2015). The IL-10 suppresses the production of other cytokines and inhibits IFN-γ synthesis. It also inhibits nuclear factor-κB (NF-κB) translocation by activating Th-cells and peripheral blood mononuclear cells that considered as a mechanism for inhibiting immediate-early pro-inflammatory response (Hamidullah et al., 2012).
In the present study, IL-10 gene silencing induced by siRNA lead to down regulation of PI3K/AKT gene expression. The PI3K activity is associated with a variety of human tumors as breast cancer (Fry, 2001), lung cancer (Lin et al., 2001), melanomas (Krasilnikov et al., 1999), and leukemia (Martinez-Lorenzo et al., 2000). The PI3K can be activated after the cytokine activation for a specific receptor. The inhibition of PI3K/AKT pathway can be used as new chemotherapeutic targets for different types of cancer. The Akt (protein kinase B, PKB), a downstream kinase of PI3K, is involved in the cancer transformation and has some downstream substrates that may also contribute to malignant transformation (Nicholson and Anderson, 2002). Some of these substrates are Bad, procaspase-9, I-kB kinase (IKK), cAMP response element binding protein (CREB), the fork head family of transcription factors (FKHR/AFX/FOX), glycogen synthase kinase-3 (GSK-3), cyclin-dependent kinase inhibitor 1 (p21Cip1) and Rapidly Accelerated Fibrosarcoma (Raf) (Nicholson and Anderson, 2002).
The Bcl2 is a family of proteins includes the anti-apoptotic and pro-apoptotic molecules. Its members are important regulators for apoptosis and inflammation. The Bcl2 family is classified into three subfamilies (depending on the homology and the function of each protein: The first subfamily has anti-cell death activity e.g. Bcl2, B-cell lymphoma-extra-large (Bcl-xL) and B-cell lymphoma-2-like protein 2 (Bcl2L2). The second subfamily has pro-apoptotic activity e.g. Bax and B-cell lymphoma -2 antagonist killer 1 (Bak). The third subfamily includes pro-apoptotic protein e.g. Bcl2-Interacting Killer (Bik) and BH3 interacting-domain death agonist (Bid) (Tsujimoto, 1998). In the current study, the silencing of IL-10 lead to downregulation of the Bcl2 gene expression. The IL-10 is a known promoter of Bcl-2 expression in the hematopoietic cells (Levy and Brouet, 1994;Cohen et al., 1997). Therefore, the down-regulation of Bcl-2 expression may serve as a possible mechanism for the sensitization of tumor cells to a variety of chemotherapeutic drugs.
The Bcl-2-binding component 3 (Bbc3), also known as p53 upregulated modulator of apoptosis (PUMA), gene encodes a member of the Bcl-2 family of proteins. It can bind to the anti-apoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation (Nakano and Vousden, 2001;Gomez-Lazaro et al., 2005;Belle et al., 2016). In the current study, silencing the IL-10 gene increased the Bbc3 and BAX expression levels in the transfected cells compared to the non-transfected cells. Similarly, PUMA can promote apoptosis either by the direct inhibition of the anti-apoptotic molecules or the direct activation of Bax and Bak (Nakano and Vousden, 2001). The PUMA is involved in various apoptotic responses (Akhtar et al., 2006;Hikisz and Kilianska, 2012), and can play a major role in the control of memory T-and B-lymphocyte survival (Akhtar et al., 2006). Because of Bbc3 gene pro-apoptotic role, it may be a possible drug target for tumor treatment.
The phosphorylation of several substrates by Akt increases the cell resistance to apoptosis. The Akt activation enhances the expression of Bcl-2 anti-apoptotic gene via phosphorylation of cyclic AMP response binding protein (Pugazhenthi et al., 2000). Synergy or additive effects between PI3K/AKT pathway and Bcl-xL in controlling apoptosis were reported in lung cancer (Qian J1, 2009). The mechanism by which IL-10 induces its protective effects against apoptosis are still unknown, but it may be involved in common pathways of diverse drugs use to induce apoptosis. The Bcl-2 plays an important role in the cancer cells survival via increasing their ability to resist different apoptotic stimuli (Alas et al., 2001).
The caspases are a family of genes broadly classified by their known roles in apoptosis (caspase-3, -6,-7, -8, and -9 in mammals), and in inflammation (caspase-1, -4, -5, -12 in humans and caspase-1, -11, and -12 in mice) (McIlwain et al., 2015). The caspases involved in apoptosis can be sub-classified by their mechanism of action as initiator (caspase-8 and -9) or killer caspases (caspase-3, -6, and -7). In the present study, caspase3 (and its cleavage) and caspase9 were up-regulated resulted in silencing of IL-10. Similarly, other study found that the increase levels of IL-10 leads to caspase 3 inactivation via NF-kB inactivation (Bachis et al., 2001). Thus, IL-10 has an intrinsic ability to inhibit directly or indirectly caspase-3 activity, and this mechanism can explain the protective effect of IL-10 on neurons cells (Grilli et al., 2000). Caspase-9 is activated by the dimerization induced when the caspase-9 CARD domain binds to the adapter protein apoptotic protease-activating factor-1 (APAF1) (Shiozaki et al., 2002). Both APAF1 and caspase-9 exist in an inactive form that could be activated by cytochrome c released from the mitochondria after the cells exposed to the stress.
In conclusion, the production of IL-10 may represent a new escape mechanism by breast cancer cells to evade the destruction of cancer cells by the immune system. The IL-10 gene silencing causes down regulation of both PI3K/ AKT and Bcl2 genes expression and also increases the Bbc3, BAX, caspase3, and caspase 3 cleavage expression levels. Therefore, IL-10 may be used as a good target for breast cancer treatment.


s2fieldsofstudy: Biology Psychology

text:
Anxiolytic Effects of Acanthopanax senticosus HARMS Occur via Regulation of Autonomic Function and Activate Hippocampal BDNF–TrkB Signaling

Mental stress, such as anxiety and conflict, causes physiological changes, such as changes in autonomic nervous activity and gastric ulcers. In addition, stress induces glucocorticoids and changes the hippocampal brain-derived neurotrophic factor (BDNF) expression levels. We previously reported that Acanthopanax senticosus HARM (ASH) prevents stress-induced gastric ulcers. Thus, we investigated the potential anxiolytic effect and influence of ASH on the hippocampus BDNF-related protein in male Sprague-Dawley rats fed 1% and 5% ASH extract-containing food for one week using novelty suppressed feeding (NSF) and improved elevated beam walking (IEBW) tests. ASH treatment significantly decreased latency to eat in the NSF test and increased the time spent on the open arm in the IEBW test. ASH5% treatment showed a significant decrease in LFnu, indicative of sympathetic nervous activity, and a significant increase in HFnu, indicative of parasympathetic nervous activity, in the NSF test. In addition, ASH1% and ASH5% treatments significantly decreased LFnu and significantly increased HFnu in the IEBW test. ASH5% treatment significantly increased hippocampal BDNF protein expression in both Western blotting and immunohistochemistry experiments. Our findings suggest that anxiolytic effects of ASH occur via the regulation of autonomic function and increased hippocampal BDNF signaling.

Introduction
Acanthopanax senticosus HARMS (ASH) is a deciduous shrub and a member of the Araliaceae family that grows abundantly in various regions of Russia, China, Korea, Southeast Asia, and north Japan. In China, ASH root bark is traditionally used to treat high blood pressure, mental disorders, and rheumatoid arthralgia [1][2][3][4]. In Western countries, ASH is widely used as an alternative medicine. A recent study was conducted on the importance of ASH root and stem barks, as well as ASH fruit and leaf. The ASH fruit improves insulin resistance and hepatic lipid accumulation [5]. ASH leaves have

Behavior in the NSF Test
In this study, in rats housed under home cage (HC) housing conditions, we found no differences in the latency time to start eating 1% and 5% ASH extract-containing feed in the ASH1% and ASH5% groups compared with that in the Cont. group ( Figure 1A). In contrast, under novel cage (NC) housing conditions, we observed a significant decrease in the latency time to start eating feed in the ASH1% and ASH5% groups compared with that in the Cont. group (p < 0.05; Figure 1A). In addition, when comparing HC and NC housing conditions, we found a significant increase in the latency time to start eating feed in both ASH1% and ASH5% groups under NC housing conditions (p < 0.05; Figure 1A).
However, under HC housing conditions, there were no differences in food intake over 30 min in both groups ( Figure 1B). Therefore, when we examined the frequency of pellet touches before the first continuous eating period exceeding 1 min under HC housing conditions, we found no change in frequency in the ASH1% and ASH5% groups ( Figure 1C). In addition, when comparing HC and NC under HC housing conditions, the frequencies of the Cont. and ASH1% groups were significantly increased under NC housing conditions (p < 0.05; Figure 1C).
Molecules 2019, 24, 132 3 of 15 to start eating feed in both ASH1% and ASH5% groups under NC housing conditions (p < 0.05; Figure  1A). However, under HC housing conditions, there were no differences in food intake over 30 min in both groups ( Figure 1B). Therefore, when we examined the frequency of pellet touches before the first continuous eating period exceeding 1 min under HC housing conditions, we found no change in frequency in the ASH1% and ASH5% groups ( Figure 1C). In addition, when comparing HC and NC under HC housing conditions, the frequencies of the Cont. and ASH1% groups were significantly increased under NC housing conditions (p < 0.05; Figure 1C).

ANS Activity (Heart Rate Variability)
Under HC housing conditions, we observed no differences in the LFnu (normalized unit of low frequency components), indicative of sympathetic nervous activity; HFnu (normalized unito of low frequency components), indicative of parasympathetic nervous activity; and LF/HF index of the cardiac sympathetic-parasympathetic tone balance values for all groups ( Figure 2). In contrast, under NC housing conditions, the ASH5% group demonstrated significantly increased HFnu and significantly decreased LF/HF and LFnu values (p < 0.05; Figure 2A-C).

Figure 2.
Effects of ASH on the autonomic nervous control of heart rate in the novelty suppressed feeding (NSF) test. Power spectrum analysis of heart rate variability in the NSF test. (A) LF/HF indicates the balance of the sympathetic and parasympathetic nervous systems. (B) LFnu (normalized unit of the LF value) indicates the sympathetic nervous system activity. (C) HFnu (normalized unit of the HF value) indicates the parasympathetic nervous system activity. Data are presented as mean ± SE; n = 6; statistical significances are denoted by * p < 0.05 and ** p < 0.01 compared with the NC Cont. group (unpaired t-test).

Comparison of Three Conditions in Heart Rate Variability
On the EPM test, the LF/HF and LFnu values tended to increase, but this autonomic change was unstable or statistically insignificant compared to HC housing conditions ( Figure 3A-C). In contrast, Data are presented as mean ± SE; n = 6; statistical significances are denoted by * p < 0.05 and ** p < 0.01 compared with the novel cage (NC) Cont. group (Dunnett's t-test), # p < 0.05 and ## p < 0.01 compared with the home cage (HC) group (paired t-test).

ANS Activity (Heart Rate Variability)
Under HC housing conditions, we observed no differences in the LFnu (normalized unit of low frequency components), indicative of sympathetic nervous activity; HFnu (normalized unito of low frequency components), indicative of parasympathetic nervous activity; and LF/HF index of the cardiac sympathetic-parasympathetic tone balance values for all groups ( Figure 2). In contrast, under NC housing conditions, the ASH5% group demonstrated significantly increased HFnu and significantly decreased LF/HF and LFnu values (p < 0.05; Figure 2A-C). to start eating feed in both ASH1% and ASH5% groups under NC housing conditions (p < 0.05; Figure  1A). However, under HC housing conditions, there were no differences in food intake over 30 min in both groups ( Figure 1B). Therefore, when we examined the frequency of pellet touches before the first continuous eating period exceeding 1 min under HC housing conditions, we found no change in frequency in the ASH1% and ASH5% groups ( Figure 1C). In addition, when comparing HC and NC under HC housing conditions, the frequencies of the Cont. and ASH1% groups were significantly increased under NC housing conditions (p < 0.05; Figure 1C).

ANS Activity (Heart Rate Variability)
Under HC housing conditions, we observed no differences in the LFnu (normalized unit of low frequency components), indicative of sympathetic nervous activity; HFnu (normalized unito of low frequency components), indicative of parasympathetic nervous activity; and LF/HF index of the cardiac sympathetic-parasympathetic tone balance values for all groups ( Figure 2). In contrast, under NC housing conditions, the ASH5% group demonstrated significantly increased HFnu and significantly decreased LF/HF and LFnu values (p < 0.05; Figure 2A-C).

Figure 2.
Effects of ASH on the autonomic nervous control of heart rate in the novelty suppressed feeding (NSF) test. Power spectrum analysis of heart rate variability in the NSF test. (A) LF/HF indicates the balance of the sympathetic and parasympathetic nervous systems. (B) LFnu (normalized unit of the LF value) indicates the sympathetic nervous system activity. (C) HFnu (normalized unit of the HF value) indicates the parasympathetic nervous system activity. Data are presented as mean ± SE; n = 6; statistical significances are denoted by * p < 0.05 and ** p < 0.01 compared with the NC Cont. group (unpaired t-test).

Comparison of Three Conditions in Heart Rate Variability
On the EPM test, the LF/HF and LFnu values tended to increase, but this autonomic change was unstable or statistically insignificant compared to HC housing conditions ( Figure 3A-C). In contrast, Figure 2. Effects of ASH on the autonomic nervous control of heart rate in the novelty suppressed feeding (NSF) test. Power spectrum analysis of heart rate variability in the NSF test. (A) LF/HF indicates the balance of the sympathetic and parasympathetic nervous systems. (B) LFnu (normalized unit of the LF value) indicates the sympathetic nervous system activity. (C) HFnu (normalized unit of the HF value) indicates the parasympathetic nervous system activity. Data are presented as mean ± SE; n = 6; statistical significances are denoted by * p < 0.05 and ** p < 0.01 compared with the NC Cont. group (unpaired t-test).

Comparison of Three Conditions in Heart Rate Variability
On the EPM test, the LF/HF and LFnu values tended to increase, but this autonomic change was unstable or statistically insignificant compared to HC housing conditions ( Figure 3A-C). In contrast,

Time Spent in the Open Arm of the IEBW
In this study, the staying time in the open area of the IEBW significantly increased in the ASH1% and ASH5% groups compared to the Cont. group (p < 0.05; Figure 4).

ANS Activity (Heart Rate Variability)
In this study, we placed rats implanted with the telemetry radio under HC housing conditions and measured the ANS activity at rest. As a result, in each conditioned rat, we observed no effects of the treatment by each reagent on the ANS and LF/HF values were low. Subsequently, we measured the ANS activity under IEBW conditions. As we observed under HC housing conditions, we found no effects of the treatment by each reagent on the ANS in each rat. However, under IEBW conditions, LFnu values were higher compared to HC housing conditions. Accordingly, we also observed an increase in LF/HF values ( Figure 5A). Thereafter, the rats were orally administered the corresponding reagents for each treatment group and the ANS activity at rest measured. As a result, HFnu and LFnu values of the ASH1% and ASH5% groups significantly increased and decreased, respectively,

Time Spent in the Open Arm of the IEBW
In this study, the staying time in the open area of the IEBW significantly increased in the ASH1% and ASH5% groups compared to the Cont. group (p < 0.05; Figure 4).

Time Spent in the Open Arm of the IEBW
In this study, the staying time in the open area of the IEBW significantly increased in the ASH1% and ASH5% groups compared to the Cont. group (p < 0.05; Figure 4).

ANS Activity (Heart Rate Variability)
In this study, we placed rats implanted with the telemetry radio under HC housing conditions and measured the ANS activity at rest. As a result, in each conditioned rat, we observed no effects of the treatment by each reagent on the ANS and LF/HF values were low. Subsequently, we measured the ANS activity under IEBW conditions. As we observed under HC housing conditions, we found no effects of the treatment by each reagent on the ANS in each rat. However, under IEBW conditions, LFnu values were higher compared to HC housing conditions. Accordingly, we also observed an increase in LF/HF values ( Figure 5A). Thereafter, the rats were orally administered the corresponding reagents for each treatment group and the ANS activity at rest measured. As a result, HFnu and LFnu values of the ASH1% and ASH5% groups significantly increased and decreased, respectively,

ANS Activity (Heart Rate Variability)
In this study, we placed rats implanted with the telemetry radio under HC housing conditions and measured the ANS activity at rest. As a result, in each conditioned rat, we observed no effects of the treatment by each reagent on the ANS and LF/HF values were low. Subsequently, we measured the ANS activity under IEBW conditions. As we observed under HC housing conditions, we found no effects of the treatment by each reagent on the ANS in each rat. However, under IEBW conditions, LFnu values were higher compared to HC housing conditions. Accordingly, we also observed an increase in LF/HF values ( Figure 5A). Thereafter, the rats were orally administered the corresponding reagents for each treatment group and the ANS activity at rest measured. As a result, HFnu and LFnu values of the ASH1% and ASH5% groups significantly increased and decreased, respectively, compared to the Cont. group ( Figure 5B). Furthermore, LF/HF values of the ASH1% and ASH5% groups significantly decreased compared to the Cont. group ( Figure 5). compared to the Cont. group ( Figure 5B). Furthermore, LF/HF values of the ASH1% and ASH5% groups significantly decreased compared to the Cont. group ( Figure 5).

Western Blotting
In ASH1% or ASH5% groups, we dissected the hippocampus from the brain of each rat and detected BDNF signaling by Western blotting. The hippocampal BDNF protein expression levels in the ASH1% group were the same as those observed in the Cont. group, but significantly increased in the ASH5% group ( Figure 6A). Moreover, in the ASH5% group, the phosphorylation of tropomyosin receptor kinase B (TrkB) as a BDNF receptor was significantly elevated, followed by a significant increase in the downstream phosphorylation of cAMP response element binding protein (CREB) of the TrkB cascade ( Figure 6B,C). indicates the sympathetic nervous system activity. (C) HFnu (normalized unit of the HF value) indicates the parasympathetic nervous system activity. Data are presented as the mean ± SE; n = 8; statistical significances are denoted by * p < 0.05 and ** p < 0.01 compared with IEBW Cont. (Dunnett's t-test ( # p < 0.05 compared with the HC group (paired t-test).

Western Blotting
In ASH1% or ASH5% groups, we dissected the hippocampus from the brain of each rat and detected BDNF signaling by Western blotting. The hippocampal BDNF protein expression levels in the ASH1% group were the same as those observed in the Cont. group, but significantly increased in the ASH5% group ( Figure 6A). Moreover, in the ASH5% group, the phosphorylation of tropomyosin receptor kinase B (TrkB) as a BDNF receptor was significantly elevated, followed by a significant increase in the downstream phosphorylation of cAMP response element binding protein (CREB) of the TrkB cascade ( Figure 6B,C).

Immunohistochemistry
Next, we examined potential immunohistological changes in the BDNF protein in the hippocampus of the ASH5% group. We observed a marked increase in BDNF-positive protein in the rat hippocampus in the case of ASH5% oral administration (Figure 7).

Discussion
Previous studies showed that ASH administration has a protective effect against gastric ulcers induced by cold-water restraint [12]. In addition, studies showed that ASH administration prevents depressive symptoms induced by tail suspension and the forced swimming test, with decreased immobility time [13][14][15]. Consequently, ASH is presumed to have an anti-stress effect that prevents gastric ulcers and depression. It is generally known that excessive stress causes or exacerbates gastric ulcers and depression. According to the general adaptation syndrome postulated by Hans Selye, stress affects the hypothalamus, and both sympathetic-adrenergic and pituitary-adrenal cortex systems induce gastric ulcers and depression [28]. Stress-induced diseases develop with stress stimulation to the CNS as the basic axis. In addition, ASH appears to prevent stress-induced disease states, so we can infer the possibility of ASH also affecting the CNS. However, the potential effects of ASH on the CNS have not been sufficiently studied.
On the other hand, stress-induced gastric ulcers develop because of stress and its associated anxiety [46]. This mental anxiety is known to increase SNS activity, while decreasing PNS activity

Immunohistochemistry
Next, we examined potential immunohistological changes in the BDNF protein in the hippocampus of the ASH5% group. We observed a marked increase in BDNF-positive protein in the rat hippocampus in the case of ASH5% oral administration (Figure 7).

Immunohistochemistry
Next, we examined potential immunohistological changes in the BDNF protein in the hippocampus of the ASH5% group. We observed a marked increase in BDNF-positive protein in the rat hippocampus in the case of ASH5% oral administration (Figure 7).

Discussion
Previous studies showed that ASH administration has a protective effect against gastric ulcers induced by cold-water restraint [12]. In addition, studies showed that ASH administration prevents depressive symptoms induced by tail suspension and the forced swimming test, with decreased immobility time [13][14][15]. Consequently, ASH is presumed to have an anti-stress effect that prevents gastric ulcers and depression. It is generally known that excessive stress causes or exacerbates gastric ulcers and depression. According to the general adaptation syndrome postulated by Hans Selye, stress affects the hypothalamus, and both sympathetic-adrenergic and pituitary-adrenal cortex systems induce gastric ulcers and depression [28]. Stress-induced diseases develop with stress stimulation to the CNS as the basic axis. In addition, ASH appears to prevent stress-induced disease states, so we can infer the possibility of ASH also affecting the CNS. However, the potential effects of ASH on the CNS have not been sufficiently studied.
On the other hand, stress-induced gastric ulcers develop because of stress and its associated anxiety [46]. This mental anxiety is known to increase SNS activity, while decreasing PNS activity

Discussion
Previous studies showed that ASH administration has a protective effect against gastric ulcers induced by cold-water restraint [12]. In addition, studies showed that ASH administration prevents depressive symptoms induced by tail suspension and the forced swimming test, with decreased immobility time [13][14][15]. Consequently, ASH is presumed to have an anti-stress effect that prevents gastric ulcers and depression. It is generally known that excessive stress causes or exacerbates gastric ulcers and depression. According to the general adaptation syndrome postulated by Hans Selye, stress affects the hypothalamus, and both sympathetic-adrenergic and pituitary-adrenal cortex systems induce gastric ulcers and depression [28]. Stress-induced diseases develop with stress stimulation to the CNS as the basic axis. In addition, ASH appears to prevent stress-induced disease states, so we can infer the possibility of ASH also affecting the CNS. However, the potential effects of ASH on the CNS have not been sufficiently studied.
On the other hand, stress-induced gastric ulcers develop because of stress and its associated anxiety [46]. This mental anxiety is known to increase SNS activity, while decreasing PNS activity [32]. Therefore, it is important to analyze the effects of ASH on the ANS. In this study, we investigated the effects of ASH on the ANS in rats under both novel environmental stress and high-fear stress conditions, as well as on the CNS in rats.
Rats feel anxiety in novel environments. Therefore, even if rats are fed, the confirmation work of the bait becomes longer and, as a result, the latency time to start eating becomes longer. As such, as the latency time is shortened by the chronic administration of antidepressants, the chronic administration of antidepressants is then assessed by the NSF test [47]. Therefore, in this study, the NSF test was conducted to evaluate the anti-anxiety and/or anti-stress effects of ASH. Before starting the NSF test, to normalize the NSF test results, ASH-administrated rats were placed under HC conditions. As a result, even when ASH was administered under HC housing conditions, no changes were observed in any of the consideration items examined, including the latency time to start eating, food intake, or the number of pellet touches before the first continuous eating of >1 min (Figure 1). In addition, ASH administration did not affect the ANS. We also found that ASH does not affect the ANS and causes no behavioral changes under normal conditions without stress from these events. Thus, by administering ASH to rats under stressed conditions, it was possible for us to examine the anti-anxiety and anti-stress effects of ASH. Subsequently, we examined the effects of ASH under NC conditions, and found that the latency time to start eating and the number of pellet touches before the first continuous eating period of >1 min significantly increased by the novel environmental change, but significantly decreased by ASH5% administration. In addition, the latency time to start eating significantly decreased, even with ASH1% administration ( Figure 1A). It is known that the latency time to start eating in rats becomes significantly longer when the rats are in novel environments, which is consistent with our findings [47]. Concomitantly, we confirmed that the number of pellet touches before the first continuous eating period of >1 min significantly increased ( Figure 1C). These behaviors are attributed to the increased alertness caused by anxiety. Thus, our results confirmed that this alertness is relieved by ASH administration, which may suppress anxiety-induced alertness (Figure 1). In addition, because ASH decreased SNS activity and increased PNS activity under NC housing conditions, the anxiolytic action of ASH is supported (Figure 2). Benzodiazepines have already been reported to reduce the latency time to start eating in novel environments [47].
Next, we examined the effects of ASH on novel environmental stress and high-fear stress. It is known that environmental factors, such as increased fear of heights, activate the SNS and inhibit the PNS, resulting in various anxiety-related behaviors and impaired physical condition [46,48,49]. In addition, patients suffering from anxiety disorders have excessive SNS activity and PNS inactivation, and similar symptoms are observed even in rodents [31][32][33]. Thus, rats undergoing high-fear stress can become animal models of anxiety disorders. Therefore, we analyzed the ANS of the rats by ECG under a high-fear stress environment. To this end, we first conducted an EPM test, which is generally used as an anxiety-related behavior assessment method. We found that the ANS score showed a tendency to increase, but not significantly, under HC housing conditions compared to the Cont. group (Figure 3). We then performed an IEBW test, in which we increased the height to induce a stronger fear stress. As a result, LFnu values, which are a parameter of SNS activation, significantly increased and the balance state of LF/HF became excited (Figure 3). This correlated with the autonomic balance observed in typical anxiety disorders [32]. Therefore, in this study, we used IEBW as an assessment method of anxiety. The staying time in the open arm of the IEBW, which strengthened the height fear stress, is a parameter used to evaluate the degree of anxiety. Then, when the Cont. and ASH groups were each placed in the IEBW, a significant extension of the staying time in the open arm was observed in the ASH group ( Figure 4). Next, the effects on the ANS were examined. Under HC conditions, we confirmed that HFnu values, an indicator of PNS activity, remained stable at a high level in all the rats in each treatment group. In addition, the ANS was also stable from the LF/HF index ( Figure 5). However, when rats were placed in the IEBW environment, LFnu values, an index parameter of SNS activity, increased and the rats became more excited. This was also confirmed from the LF/HF index. Thereafter, when ASH was administered orally and the same IEBW test was carried out, the rat SNS activity, which had been rising, significantly decreased by the administration of ASH1% or ASH5%. Accordingly, the rat PNS activity, which had decreased, increased with ASH1% or ASH5% administration ( Figure 5). This process led to significant reductions (to a state close to rest) in the LF/HF index of rats that had been rising because of IEBW. These results suggest that ASH has an anxiolytic effect against not only mild anxiety, but also anxiety due to higher levels of stress, such as when the ANS is severely affected.
Recent studies reported that BDNF/TrkB signaling activation is important for ameliorating the depression-like pathology observed by the NSF test, and it can be improved by increasing BDNF expression in the hippocampus [50,51]. In addition, studies have reported that increased BDNF activity in the hippocampus has an anxiolytic effect [52]. Moreover, hippocampal BDNF mRNA levels reportedly decrease as a result of both acute and chronic stress [53]. These findings suggest that stress-and anxiety-related behaviors caused by stress are closely related to changes in hippocampal BDNF expression. In addition, CREB phosphorylation plays an important role in mediating BDNF responses in neurons [54]. Furthermore, a previous study reported the inducement of a neuroprotective effect through BDNF mRNA levels in PC12 cells [41]. It is possible that the anxiolytic effects due to ASH administration under high-level stress conditions may be due to changes in BDNF expression. Therefore, we conducted Western blot analyses of BDNF-/TrkB-related proteins in the hippocampus, along with an immunostaining analysis using a specific anti-BDNF antibody from ASH-administered rat brains from the IEBW test. Our results revealed that when ASH5% is administered to rats under IEBW conditions, the protein expression levels of BDNF in the rat hippocampus significantly increase and the phosphorylation of TrkB, and CREB also significantly increases. Furthermore, immunohistochemical analysis revealed that BDNF expression in the rat hippocampus is markedly elevated under IEBW conditions when treated with ASH5%. These behavioral and pharmacological experimental results obtained by ASH administration, including the resultant changes in BDNF expression, suggest that the effects of ASH on ANS activity occur via the hippocampus. This is strongly supported by the observation that the ventral part of the hippocampus is known to be involved in the defense reaction against anxiety [55].
Anxiety is also reported to be related to emotional eating in obese individuals [56,57]. In addition, hippocampal BDNF plays important role in food intake and body weight regulation [58]. Furthermore, the harmful effect of high-fat diet on learning and memory is linked to decrease in the hippocampal BDNF expression [58]. This study suggests that the anxiolytic effect and activation of the BDNF signaling by ASH extract improve the exacerbation in obesity-related pathological changes.

Plant Extract
A dried 2-5 cm root tip of ASH from Heilongjiang, China, was cut and extracted with hot water. The extract was concentrated under reduced pressure and dried using a spray dryer. The herb specimen was authenticated by Emeritus Professor Sansei Nishibe of the Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Health Science University of Hokkaido. The extract powder (Lot No. 8142) was provided by Sun Chlorella Co., Ltd. (Kyoto, Japan). We measured each major component in the extract using HPLC and an ODS column. ASH extracts primarily comprised isofraxidine (101.4 mg/100 g), eleutheroside B (325.2 mg/100 g), eleutheroside E (625.2 mg/100 g), eleutheroside B1 (95.2 mg/100 g), and chlorogenic acid (829.5 mg/100 g).

Ethics Statement
This study was conducted according to the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 85-23, revised in 1996). All experimental protocols were approved by the Ethics Committee on Animal Use of the Suzuka University of Medical Science (No. 1 of April 1, 2016).

Animals
Male Sprague-Dawley (SD) rats (6 weeks old) were purchased from SLC, Inc. (Shizuoka, Japan), individually housed in standard polycarbonate cages for 7 days, and subjected to serial 7-day handling. A normal diet (ND: MF, Oriental Yeast Manufacturing Co., Ltd., Tokyo, Japan) and water were available ad libitum. Rats were kept in a room maintained at 23 ± 2 • C and 50-65% humidity under a 12 h light/dark cycle (lights on at 7:00).

Surgery
After a 1-week acclimatization, rats were treated with a local anesthetic (ropivacaine hydrochloride hydrate) and general anesthesia (sodium pentobarbital, 40 mg/kg, i.p.), and a wireless telemeter (model TR50BB; Biotelemetry Research, Phoenix, AZ, USA) was implanted in the abdomen. Animals were then intramuscularly injected with antibiotics (imipenem hydrate and cilastatin sodium, 8.3 mg/kg) to prevent postoperative infections. After the operation, rats were allowed to recover for 7 days.

Administration
Rats were divided into three groups [control (Cont.), ASH1%, and ASH5%] based on their body weight before administration. Test foods were prepared by mixing ND with the ASH extract and were provided ad libitum for 5-7 days. In addition, 1 mL of distilled water, or ASH1% and ASH5% dissolved in distilled water, was administered through a probe only once 30 min before each behavioral test.

NSF Test
The NSF test is often used as a measure of depression/anxiety-like behaviors and was conducted as described in Bodnoff et al. [47], with slight modifications. The NSF test schedule is described in Figure 8A. For the NC, we used a cage where the bottom and sides were covered with a black plastic garbage bag without bedding (NC; Figure 8B). Food-deprived (24 h) animals were subjected to a 30-min HC ( Figure 8B, HC: 27 × 43 × 20 cm 3 ) test after placing pellets on the wire netting of the cage in the housing room and in the NC test by the same method after a 1-day interval. Pellets were given to rats during the interval period. We assessed latency time to start eating, the number of pellet touches before the first continuous eating period of >1 min, and food intake. HRV analysis was conducted in Cont. and ASH5% groups. Male Sprague-Dawley (SD) rats (6 weeks old) were purchased from SLC, Inc. (Shizuoka, Japan), individually housed in standard polycarbonate cages for 7 days, and subjected to serial 7-day handling. A normal diet (ND: MF, Oriental Yeast Manufacturing Co., Ltd., Tokyo, Japan) and water were available ad libitum. Rats were kept in a room maintained at 23 ± 2 °C and 50-65% humidity under a 12 h light/dark cycle (lights on at 7:00).

Surgery
After a 1-week acclimatization, rats were treated with a local anesthetic (ropivacaine hydrochloride hydrate) and general anesthesia (sodium pentobarbital, 40 mg/kg, i.p.), and a wireless telemeter (model TR50BB; Biotelemetry Research, Phoenix, AZ, USA) was implanted in the abdomen. Animals were then intramuscularly injected with antibiotics (imipenem hydrate and cilastatin sodium, 8.3 mg/kg) to prevent postoperative infections. After the operation, rats were allowed to recover for 7 days.

Administration
Rats were divided into three groups [control (Cont.), ASH1%, and ASH5%] based on their body weight before administration. Test foods were prepared by mixing ND with the ASH extract and were provided ad libitum for 5-7 days. In addition, 1 mL of distilled water, or ASH1% and ASH5% dissolved in distilled water, was administered through a probe only once 30 min before each behavioral test.

NSF Test
The NSF test is often used as a measure of depression/anxiety-like behaviors and was conducted as described in Bodnoff et al. [47], with slight modifications. The NSF test schedule is described in Figure 8A. For the NC, we used a cage where the bottom and sides were covered with a black plastic garbage bag without bedding (NC; Figure 8B). Food-deprived (24 h) animals were subjected to a 30min HC ( Figure 8B, HC: 27 × 43 × 20 cm 3 ) test after placing pellets on the wire netting of the cage in the housing room and in the NC test by the same method after a 1-day interval. Pellets were given to rats during the interval period. We assessed latency time to start eating, the number of pellet touches before the first continuous eating period of >1 min, and food intake. HRV analysis was conducted in Cont. and ASH5% groups.

IEBW Test
In order to examine the anxiolytic effect of ASH, we carried out behavioral experiments using high-fear stress. In generally, the EPM test was conducted to evaluate animal anxiety [59]. We observed stable ANS activity in the HC ( Figure 9A). Only those rats in which the ANS was not disturbed by the experimenter under normal conditions were included. However, in the EPM apparatus 50 cm above the floor ( Figure 9B) for 3 min, the LF/HF and LFnu values tended to increase, but this ANS change was unstable or statistically insignificant compared to HC housing conditions ( Figure 3A-C). In contrast, LF/HF and LFnu values significantly increased compared to HC housing conditions on the IEBW ( Figure 3A,B). Thus, we used the IEBW apparatus ( Figure 9C) by installing a 2 × 4 timber (180 × 8.9 cm 2 ) 190 cm above the floor level and an open (140 × 8.9 cm 2 ) and closed (40 × 8.9 × 28.5 cm 3 ) arms, which improved the EPM apparatus. The IEBW test schedule is described in Figure 9D. After a 7-day administration, they were placed in the tip of the open arm 140 cm away from the closed arm, and their behavior and HRV were examined for 3 min from 0.5 h after oral administration (IEBW test). We then assessed the time spent in the open arm, LF/HF, LFnu, and HFnu. In order to examine the anxiolytic effect of ASH, we carried out behavioral experiments using high-fear stress. In generally, the EPM test was conducted to evaluate animal anxiety [59]. We observed stable ANS activity in the HC ( Figure 9A). Only those rats in which the ANS was not disturbed by the experimenter under normal conditions were included. However, in the EPM apparatus 50 cm above the floor ( Figure 9B) for 3 min, the LF/HF and LFnu values tended to increase, but this ANS change was unstable or statistically insignificant compared to HC housing conditions ( Figure 3A-C). In contrast, LF/HF and LFnu values significantly increased compared to HC housing conditions on the IEBW (Figure 3A,B). Thus, we used the IEBW apparatus ( Figure 9C) by installing a 2 × 4 timber (180 × 8.9 cm 2 ) 190 cm above the floor level and an open (140 × 8.9 cm 2 ) and closed (40 × 8.9 × 28.5 cm 3 ) arms, which improved the EPM apparatus. The IEBW test schedule is described in Figure 9D. After a 7-day administration, they were placed in the tip of the open arm 140 cm away from the closed arm, and their behavior and HRV were examined for 3 min from 0.5 h after oral administration (IEBW test). We then assessed the time spent in the open arm, LF/HF, LFnu, and HFnu.

Brain Tissue Preparation
After behavioral tests, rats were euthanized, and their brains were rapidly excised. Hippocampal tissues were stored at −70 °C until Western blotting.

Assessment of Cardiac Autonomic Activity (Heart Rate Variability)
After the operation, ECG data were continuously recorded using the radio-telemetry system, comprising the implanted device, a battery-charging and telemetry-receiving device underneath the cage, and a data acquisition system (PowerLab16/35, PL3516, AD Instruments, Castle Hill, New South Wales, Australia) interfaced with a computer. Cardiac autonomic activity was assessed by spectral analysis of R-R interval variability. Data were stored and analyzed using LabChart Pro (ver. 8.0, AD Instruments). Frequency domain analysis and power spectra of R-R interval variability were obtained

Brain Tissue Preparation
After behavioral tests, rats were euthanized, and their brains were rapidly excised. Hippocampal tissues were stored at −70 • C until Western blotting.

Assessment of Cardiac Autonomic Activity (Heart Rate Variability)
After the operation, ECG data were continuously recorded using the radio-telemetry system, comprising the implanted device, a battery-charging and telemetry-receiving device underneath the cage, and a data acquisition system (PowerLab16/35, PL3516, AD Instruments, Castle Hill, New South Wales, Australia) interfaced with a computer. Cardiac autonomic activity was assessed by spectral analysis of R-R interval variability. Data were stored and analyzed using LabChart Pro (ver. 8.0, AD Instruments). Frequency domain analysis and power spectra of R-R interval variability were obtained using the fast Fourier transform algorithm. High (HF: 0.6-3.0 Hz), low (LF: 0.2-0.6 Hz), and very low (VLF: ≤0.2 Hz) frequencies were determined. LF and HF components were expressed in normalized units (LFnu and HFnu). The power of the HF component indicates cardiac vagal activity, whereas the LF component indicates the sympathetic activity with vagal modulation. LF/HF is an index of the cardiac sympathetic-parasympathetic tone balance.

Immunohistochemistry
Rat brains were fixed overnight by 4% paraformaldehyde, followed by immersion in graded sucrose solutions for cryopreservation (10%, 20%, and 30% sucrose in phosphate-buffered saline [PBS]) and freezing with dry ice. Frozen brains were mounted using Tissue-Tek ® O.C.T. compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and stored at −80 • C. Brains were then sectioned into 10-µm thick sections using a cryostat at −15 • C. Sections were collected in PBS and incubated with 10% BSA in PBS for 1 h, followed by incubation with anti-BDNF antibody (sc-20981, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 days at 4 • C. Incubation with the secondary antibody (#4413S, 1:1000, Cell Signaling Technology Japan) proceeded for 2 h, after which sections were mounted onto MAS-coated glass slides and cover-slipped using Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA). Images were obtained with a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan).

Statistics Analysis
Values are expressed as means ± standard errors and were derived from measurements of 4-8 rats (NSF test, n = 6; IEBW test, n = 8, Western Blotting, n = 4-5). Statistical analysis was performed using SPSS statistics 24 (IBM, Tokyo, Japan). Homogeneity of variances was checked with the Levene's test. One-way ANOVA was used for inter-group comparisons. When ANOVA revealed significant differences, Dunnett's t or T3 post hoc tests were used to identify significant differences from the Cont. group. Differences between conditions were analyzed using repeated measures ANOVA. Differences between two groups were analyzed by paired Student's t-test. P-values < 0.05 were considered significant.

Conclusions
Our novel findings revealed a unique regulation of the balance of the ANS based on parasympathetic dominance and the anxiolytic actions of ASH. In addition, our results suggested that an increase in hippocampal BDNF signaling is involved in the actions of ASH. Collectively, our findings demonstrated that ASH extract with the anxiolytic effects might be a beneficial supplement or preventive medicine for the maintenance of mental health or against obesity associated with emotional eating.