Foxg-1 pathway in mice

Accepted Manuscript Title: Saikosaponin-d-mediated downregulation of neurogenesis results in cognitive dysfunction by inhibiting Akt/Foxg-1 pathwayin mice Authors: Xu Lixing, Ji zhouye, Guo Liting, Zhang Ruyi, Qu Rong, Ma Shiping PII: DOI: Reference:

S0378-4274(17)31457-1 https://doi.org/10.1016/j.toxlet.2017.11.009 TOXLET 10000

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

12-6-2017 30-10-2017 7-11-2017

Please cite this article as: Lixing, Xu, zhouye, Ji, Liting, Guo, Ruyi, Zhang, Rong, Qu, Shiping, Ma, Saikosaponin-d-mediated downregulation of neurogenesis results in cognitive dysfunction by inhibiting Akt/Foxg-1 pathwayin mice.Toxicology Letters https://doi.org/10.1016/j.toxlet.2017.11.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Saikosaponin-d-mediated downregulation of neurogenesis results in cognitive dysfunction by inhibiting Akt/Foxg-1 pathwayin mice Xu Lixinga1, Ji zhouye a1, Guo Litinga, Zhang Ruyia, Qu Rongb*, Ma Shipinga* a

Department of Pharmacology of Chinese MateriaMedica, China Pharmaceutical University,

b

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Nanjing 210009, PR China Department of Pharmacology of Traditional Chinese Medical Formulae, Nanjing University

of Chinese Medicine, Nanjing 210046, PR China

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*Corresponding authors. Addresses: Department of Pharmacology of Traditional Chinese Medical Formulae, Nanjing University of Chinese Medicine, Hanzhong Road 282,

Nanjing210046, PR China, Tel: +86 25 85811929; fax: +86 25 85811929(Qu Rong);

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Department of Pharmacology ofChinese Materia Medica, China Pharmaceutical University,

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Tongjiaxiang 24, Nanjing, Jiangsu210009, PR China, Tel.:+86 25 3271507; fax: +86 25

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3271505 (Ma Shiping).

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E-mail addresses: [email protected] (Qu Rong), [email protected] (Ma Shiping).

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Graphical Abstract

Highlights 

SSd impaired learning and memory in mice.

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SSd decreased hippocampal neurogenesis.

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The Akt/Foxg1 signaling pathway was involved in SSd-induced neurotoxicity.

Abstract

Saikosaponin-d (SSd), one of the main constituents of the total saikosaponins extracted from Bupleurum falcatum L, possesses anti-inflammatory and anti-apoptosis effect. Recently, SSd was proved to improve depressive symptoms although exhibit hepatotoxicity in animals, but the central nervous system (CNS) toxicity of SSd remains unclear. The present study investigated the SSd-induced impairment in hippocampal cognitive function and explored the possible mechanisms involved.

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After intragastric administration of SSd (4mg/kg, 8mg/kg) for 7 days, the learning and memory abilities of mice were evaluated by behavioral experiments. In the step-down

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passive avoidance test, we found that the mice treated with SSd showed a significant decrease of step-down latency and increase of the frequency of errors. In the Morris water maze task, both the escape latency and swimming distance of the mice treated

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with SSd were increased, correspondingly, both the time of mice staying in the target

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zone and the frequency of crossing platform were decreased. These neurobehavioral

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changes were accompanied by the reduction of the expression of 5-bromo-2'deoxyuridine (BrdU), nestin, doublecortin (Dcx) and microtubule associated protein 2

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(MAP2). Moreover, SSd significantly inhibited the expression of p-Akt, Foxg-1 and

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fibroblast growth factor 2 (FGF2) in the hippocampus of mice. These results indicated that SSd had a toxic effect on cognitive function in mice, which was associated with

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inhibiting the hippocampal neurogenesis via Akt/Foxg1 pathway. Keywords: Saikosaponin-d; learning and memory; neurogenesis; Akt; Foxg-1

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1. Introduction

Saikosaponin-d (SSd) is a triterpene saponin isolated from Bupleurum falcatum

L, which is usually used in traditional Chinese medicine (TCM) to treat liver injury

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for more than one thousand years (Chen et al., 2016). SSd has a common steroid-like structure and its chemical formula is shown in Fig. 1. It has also been reported that SSd had some pharmacological activities such as pain-relieving, anti-inflammation, immunoregulation, anti-viralinfection, and hepatocyte protection (Cai et al., 2017). In animal experiments, SSd remarkably alleviated the oxidative stress damage and decreased the apoptosis rate in lung tissues (Wang et al., 2015). At the molecular

level, SSd exhibited an anti-apoptotic efficacy in PC12 cell treated with H2O2 by scavenging ROS and inhibiting MAPK-dependent pathway (Lin et al., 2016). Moreover, SSd reversed the corticosterone-induced physiological changes via regulation of mitochondrial function in neuronal cells (Li, et al., 2014). Recent study have shown that SSd possessed neuroprotective effects in rats exposed to chronic unpredictable mild stress (Li et al., 2017). Nevertheless, SSd has been

neurotoxicity is unclear and accordingly deserves further studies.

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reported to cause hepatotoxicity (Zhang et al., 2015). While whether SSd causes

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Neurogenesis occurs in the adult mammalian central nervous system (CNS)

which contains neural stem cells (NSCs) (Taupin, 2006). NSCs residing in the adult brain generate neurons, astrocytes, and oligodendrocytes in the nervous system

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(Taupin andGage, 2002). On the one hand, NSCs can divide symmetrically, followed

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by forming two daughter stem cells and on the other hand, NSCs also underdo an

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asymmetric division which produce a daughter stem cell and a rapidly amplifying

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progenitor cell (Sierra et al., 2015). In the stable surrounding microenvironment, the NSCs or neural progenitor cells (NPCs) also gradually mature and become neurons

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which integrate into existing neuronal networks (Rotheneichner et al., 2014). In mammals, adult neurogenesis is mostly restricted to two main neurogenic regions: the

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dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle (Farinetti et al., 2015). It is already known that adult hippocampal

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neurogenesis participates in regulating the physiological and affective states including fear/anxiety, learning/memory, and synaptic plasticity (Ueda et al., 2017). Previous study has shown that learning and memory impairment is involved in the inhibition of

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neurogenesis in thiamine deficiency mouse model by feeding a thiamine-depleted diet (Zhao et al., 2008). Enhancing hippocampal neurogenesis ameliorates memory impairment and cognitive deficits (Park et al., 2016). Foxg1 (formerly BF-1), as a member of the winged-helix or forkhead family of transcription factors, positively regulates neurogenesis by dramatically stimulating neurite outgrowth (Brancaccio et al., 2010). Foxg1 is expressed in neurogenic areas of

the postnatal brain such as the SVZ and the hippocampal DG. Researches have shown that Foxg1+/- mice reduced survival of postnatally generated cell population, followed by the loss of newly produced DG neurons (Shen et al., 2006). Suppression of Foxg1 expression induces cell death in healthy neurons, which was suggested to be mediated by the PI-3kinase/Akt signaling pathway (Dastidar et al., 2011). A recent study found that SSd inhibited phosphorylation of Akt in hepatocarcinoma cells (Li et al., 2017).

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Moreover, to our knowledge there are no reports showing that SSd could cause

neurotoxicity. Based on these findings, we designed this experiment to evaluate

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whether SSd cause downregulation of neurogenesis and cognitive dysfunction through Akt/Foxg1 pathways in mice. 2. Materials and methods

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2.1. Animals

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Four-week-old male ICR mice were purchased from Yangzhou University

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Comparative Medicine Center in Jiangsu Province (Yangzhou, China) and were

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housed under conditions of controlled temperature (25±2℃) with a 12 h/12 h light/dark cycle. The animals were provided with enough water and food which may

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be consumed ad libitum. Experimental procedure was approved by Animal Ethics

2.2. Drugs

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Committee of school of Chinese Materia Medica, China Pharmaceutical University.

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SSd (purity ≥98%) was purchased from Xian Kailai Medical Technology Co., Ltd (Xian, PR China). LY294002 (purity ≥98%) was purchased from Apexbio (Boston, MA, USA). BrdU was purchased from Sigma-Aldrich (St Louis, MO, USA).

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All reagents used were of analytical grade. 2.3. Experiment design Forty mice were randomly divided into four groups: Control, SSd (4.0 mg/kg, 8.0 mg/kg) and LY294002 (30 mg/kg). SSd groups received daily intragastric administration of SSd. LY294002 group received administration via i.p. injection. The

Control group received same volume of saline by intragastric administration. After administration for 7 consecutive days, mice underwent behavioral evaluation. The Control group received same volume of saline.LY294002 was dissolved in DMSO and diluted 100 times with PBS to a final concentration of 1.5 mg/ml. To analyze the cell proliferation after repeated treatments, the mice received three i.p. injections of BrdU (50 mg/kg, i.p.) for 3 consecutive days before sacrifice. The experimental

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schedule was in Fig. 2.

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2.4. Behavioral evaluation 2.4.1. Morris water maze test

Animal spatial learning and memory ability were examined by Morris water

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maze test. In brief, a 100 cm diameter swimming pool was divided into four parts.

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Mice were put into the water in one of the virtual quadrants. During the training trails,

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mice were first trained to find a randomly positioned visible platform submerged under water, which was 2 cm beneath the surface of the water. When the animal

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reached the platform, it was allowed to stay for 15 s. If the mouse failed to find the

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platform in 120 s, it was guided to the target and also allowed to stay for 15 s. All mice were subjected to 2 trials per day for 5 consecutive days. The behaviors were recorded by a video-tracking program. On the testing day, the platform was removed,

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escape latency, swimming distance, time in target zone and platform crossing

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frequency were analyzed.

2.4.2. Step-down passive avoidance test This test relates to learning and memory. The animals do not step down from a

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platform in order to avoid a mild footshock. The avoidance apparatus consisted of six plastic boxes (15×15×40 cm3) which had a high platform with a diameter of 4.5 cm and a height of 4.5 cm. In the floor of plastic box, the parallel caliber stainless steel bars were installed to produce a mild footshocks. Before test the mice received training. At the first 2 min, mice were allowed to acclimation. Mice were then placed on the platform, and received footshocks (36 V) continuously for 4 min once stepped

down on the grid floor. When the mice received footshocks, they stepped upon the platform. After 24 h, the mice received the same experimental procedure as the procedure employed in the training day. The time of first step down from platform and the frequency of step down onto the grid floor within 180 s were recorded. 2.5. Immunohistochemistry

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Three mice per group were euthanised by injecting chloral hydrate. Brains were removed on ice and immersed in fixative of 10% formalin. Coronal sections were cut at the level of the dorsal hippocampus and processed for staining with primary

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antibody (BrdU, nestin, Dcx and MAP2) by the standard method of

immunohistochemistry. BrdU is generally used in detecting S-phase cells, providing the information on the aggressiveness of neurons and is used to label proliferating

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cells. Nestin is an intermediate filament protein, which is considered as a marker of

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nerve stem cell with self-renewal capacity. Dcx is crucial for neuronal interaction

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before and during migration, and highly expressed in neuronal cells. MAP2 has

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historically been perceived primarily as a static, structural protein, necessary along with other cytoskeletal proteins to maintain neuroarchitecture and also used as a

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clinically useful marker for neurons. The positive expression of target proteins in DG region of hippocampus were visualised under a light microscope at 20×

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magnification. Quantitative image analysis was processed by Image-Pro Plus 6. 2.6.Western blot

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The brain tissues from three mice per group were used for western blot. The

hippocampus were thawed on ice, weighed rapidly and homogenized in ice-cold tissue lysis buffer (w:v 1:5). The lysed tissue was then centrifuged 30 min after

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pyrolysis at 12,000 rpm for 20 min at 4 ℃. The total protein concentration of the supernatant was determined using bicinchoninic acid (BCA) protein assay kit (Beyotime, Haimen, China) according to the manufacturer's instructions. After heating at 100 ℃ for 5 min, the proteins were separated on SDS–PAGE gels and transferred to the polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Laboratories, Inc., California) for immune blotting. The membrane was then blocked

with blocking solution (5% skimmed milk) at room temperature for 2 h and followed by an overnight incubation at 4 ℃ with anti-Akt, p-Akt, Foxg1 monoclonal antibodies(1:1000 dilution, Abcam Inc., Cambridge, MA, USA). After wash in phosphate-buffered saline (PBS)-Tween, the membranes were incubated with secondary antibodies (1:5000 dilution, Cell Signaling Technology, MA, USA) , followed by three washing in TBS-T. Immunoreactivity was detected with enhanced

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chemiluminescence detection system (Beyotime, Haimen, China) and visualized in an imaging system (Bio-Rad, Hercules, CA, USA). β-actin (1: 1000; Cell Signaling

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Technology, MA, USA) was used as the control. The images were quantified using Image-Pro Plus software. 2.7. Measurement of FGF2 protein level

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The brain tissues from six mice per group were used for measurement of FGF2

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protein level. The protein level of FGF2 was determined using commercially available

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enzyme linked immunosorbent assay (ELISA) kit, following the instructions provided

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by the manufacturer (Elabscience, Wuhan, China)and quantified by a microplatereader (450 nm). The results were shown as picograms per mg protein

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(pg/mgprot). 2.8. Statistical analysis

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The data were expressed as mean ± SEM. The statistical differences were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s test. A p-

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value less than 0.05 was considered statistically significant. 3. Result

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3.1. SSd impaired learning and memory performance In the Morris water maze task, all mice showed a modest but progressive

reduction of the escape latency to find the platform during the training period of 4 consecutive days. As shown in Fig. 3, on the test day, the escape latency and swimming distance of SSd groups were increased (p < 0.01), conversely, time in the target zone and platform crossing frequency were reduced compared to the control

group (p < 0.05, p < 0.01). The group injected with Akt inhibitor LY294002 exhibited the similar behavior changes (p < 0.05, p < 0.01). In the step down passive avoidance test(Fig. 4), the latency of the step down after receiving footshock was dosedependently decreased (p < 0.05, p < 0.01), and the error frequency to step down from a platform was dose-dependently increased compared with the control group (p < 0.05). LY294002 group also presented a decrease of latency and an increase of error

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frequency (p < 0.05, p < 0.01). These results demonstrated that SSd exerted an impairment in learning and memory performance.

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3.2. SSd inhibited hippocampal neurogenesis

To determine whether SSd affected neurogenesis, we detected the expression of

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Brdu, nestin, Dcx and MAP2 by immunohistochemistry. As shown in Fig. 5, SSd significantly decreased the expression of BrdU-, nestin-, Dcx and MAP2 (shown as

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brown granules) in the DG region of hippocampus as compared to the control group

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(p < 0.05, p < 0.01). Similarly, LY294002 group decreased the expression of BrdU,

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nestin, Dcx and MAP2 (p < 0.05, p < 0.01). Therefore, SSd inhibited the hippocampal

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neurogenesis, which is related to the impairment of learning and memory. 3.3. SSd regulated Akt/Foxg1 signaling pathway

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The results of western blot showed that there was an obvious expression of p-Akt and Foxg1 in the hippocampus of mice. As shown in Fig. 6A-C, SSd and LY294002

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groups exhibited a remarkable decrease in the protein level of p-Akt and Foxg1 as compared to the control group (p < 0.05, p < 0.01). The expression of p-Akt and Foxg1 were inhibited by SSd, especially at the high dose level. According to these

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results, SSd influenced the Akt/Foxg1 signaling pathway. 3.4. SSd decreased protein level of FGF2 in hippocampus Fibroblast growth factor 2 (FGF2) is a well-known survival factor. As shown in Fig. 6D, the protein level of FGF2 in SSd and LY294002 groups were obviously decreased as compared to the control group (p < 0.01). It indicated that the

impairment of in hippocampal cognitive function caused by SSd was involved in FGF signaling pathway. 4. Discussion In the present study, we showed that SSd impaired learning and memory in mice by observing the behavior changes in Morris water maze and step-down passive

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avoidance tests. The immunohistochemistry test provided experimental evidence that SSd decreased hippocampal neurogenesis by affecting the level of Brdu, nestin, Dcx and MAP2. Western blot and ELISA test showed that SSd downregulated the

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expression of p-Akt, Foxg1 and FGF2. In addition, the Akt inhibitor LY294002

showed consistent results as SSd. These results suggested that SSd had toxic effect on cognitive function, which was associated with inhibiting hippocampal neurogenesis

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by Akt/Foxg1 pathway.

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Adult neurogenesis, a complex and dynamic phenomenon that continues several

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weeks, involves a switch of neural stem and progenitor cells from proliferation to

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differentiation. Neural progenitors go through different stages before becoming mature granule cells integrated in functional circuits (De Pietri Tonelli et al., 2008;

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Lopez-Atalaya et al., 2011). In our experiments, the number of BrdU-positive cells in the hippocampal DG region was decreased after BrdU administration in SSd groups

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as well as in LY294002 group. SSd affected the cell proliferation in hippocampus, which was in connection with Akt pathway. Similarly to previous results, we proved

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that SSd regulated adult neurogenesis by examining the expression of nestinexpressing stem cells. Likewise, SSd had a potentially deleterious effect on NSCs survival and self-renewal. To define more precisely the impairment of SSd associated

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to neurogenesis in the dentate gyrus of adult rodents, we examined the protein levels of Dcx and MAP2. SSd inhibited the expression of these proteins, which marked the migration and differentiation of neural cells. Previous researches have clarified that newborn neurons at different maturation stages may make distinct contributions to learning and memory (Deng et al., 2010). And the reduction of neurogenesis was always concomitant with impairments in cognitive functions (Villeda et al., 2011).

These findings indicated that impairing neurogenesis and the integration of newborn neurons in functional circuits resulted in decrease in learning and memory deficits. Therefore, these results proved that SSd had a toxic effect on cognitive function by inhibiting hippocampal neurogenesis. The Akt signaling pathway is important in the potential activation of

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neuroprotective and repair pathways during critical time windows of hippocampal development (Jiang et al., 2012). Increased hippocampal Akt phosphorylation

contributed to improving diabetes-induced cognitive deficit in rats (Zhou et al., 2017).

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In our experiment, SSd (4.0 mg/kg, 8.0 mg/kg) significantly decreased the protein

levels of p-Akt, which followed by hippocampal cognitive impairment. These results indicated that PI3K/Akt signal pathway participated in hippocampal learning and

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memory ability. It has been reported that the phosphorylation of Akt is upregulated by

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insulin growth factor1 (IGF1) in hippocampal neurons (Zheng and Quirion, 2004).

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And IGF1 induces an increase of cell proliferation in the dentate gyrus by acting

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directly on IGF-I receptors expressed by newly born cells (Mudò et al., 2009). Activating the signaling pathway of PI3K/Akt promoted NSC proliferation, increased

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the number of neurons differentiated from NSCs and subsequently repaired damaged neural systems in AD mice (Zheng et al., 2017). Consistent with previous studies, our

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results showed that SSd activated PI3K/Akt signaling pathway, which involved in impairing hippocampal neurogenesis. LY294002, an inhibitor of Akt signaling, has

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been documented to abolish Sildenafil-induced phosphorylated Akt and blocked Sildenafil-increased cell proliferation in hippocampus of the adult rats (Wang et al., 2005). Our study indicated that LY294002 inhibited Akt signaling pathway and

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damaged hippocampal neurogenesis, suggesting that SSd decreased the number of newborn neuron and induced cognitive deficits by regulating Akt signaling pathway. Separately and simultaneously, SSd decreased the protein level of Foxg1 in hippocampus. We speculated that SSd impaired hippocampal neurogenesis by inhibiting Akt/Foxg1 signaling pathway. These findings were strongly supported by that PI3K/Akt pathways in the neurogenesis-promotion were inhibited in Foxg1+/-

mice (Kinsler et al., 2010). In addition, elevating Foxg1 expression inhibited cellular death caused by neurotoxity in cultured cerebellar granule neurons stimulated with low potassium treatment (Dastidar et al., 2012). Neurotrophic factors, such as basic fibroblast growth factor (FGF2), epidermal growth factor, and the diffusible gas nitric oxide have been shown to play important

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roles in NSC differentiation in animal models (Woodbury and Ikezu, 2014). It is suggested that cerebral ischemia in adult rats rapidly and robustly induced

neurogenesis by increasing FGF2 protein level (Grabiec et al. 2016). Besides, FGF2

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increased the number of proliferating cells in the SVZ and induced the recruitment of new neurons from the SVZ into the neostriatum and cerebral cortex in a transgenic mouse model of Huntington's disease (Jin et al., 2005). Promoting the expression of

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FGF2 also induces neurogenesis in the hippocampus and enhances the proliferation,

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migration and differentiation of oligodendrocyte progenitor cells (OPCs) in LPC-

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treated rats (Azin et al., 2015). In cultured adult hippocampal neural stem cells, FGF2

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is required for generating new neurons for neuronal survival (Kang and Hébert, 2015). Besides, Foxg1 has been shown to be involved in modulating FGF signaling pathway

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in the development of neural stem cells of the olfactory epithelium (Kawauchi et al., 2009). We also detected the protein level of FGF2 by ELISA, and the results showed

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that SSd decreased the expression of FGF2 as compared to the control group. Together with previous studies, we suggested that SSd impaired hippocampual

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neurogenesis by decreasing the expression of Foxg1 and FGF2. Altogether, this study proved the toxicity of SSd in hippocampus and the

impairment in learning and memory caused by SSd. Hippocampal neurogenesis, as a

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critical regulator in diverse neurologic disease, plays an important role in cognitive function. Furthermore, enhancing Akt/Foxg1 signaling pathway to restore neurogenesis makes it a potential target for the prevention or reversal of cognitive deficits in hippocampal function. Disclosure

The authors have declared no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No.81573701,No.81703735), Chinese Postdoctoral Science Foundation（ 2017M611958）, Priority Academic Program Development of Jiangsu Higher

Training Programs for Innovation and Entrepreneurship (206).

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Fig. 1. Chemical structure of SSd. Fig.2. Experimental design of SSd in mice.

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Fig. 3. Effect of SSd on learning and memory performance in Morris water maze task. (A) The escape latency to reach the platform. (B) Swimming distance in testing time.

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(C) Platform crossing frequency in testing time. (D) Time in target zone in testing

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time. Behavioral data showed that SSd induced cognitive disorders. Data were

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expressed as mean ± SEM (n = 10). #p < 0.05 and ##p < 0.01 vs. the control.

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Fig. 4. Effect of SSd on learning and memory performance in Step-down passive

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avoidance test. (A) The latency of the step-down response onto the grid floor 24 h after the training. (B) The error frequency to step down from a platform after electric shock on the test day. Behavioral data showed that SSd induced cognitive

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the control.

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disorders. Data were expressed as mean ± SEM (n = 10). #p < 0.05 and ##p < 0.01 vs.

Fig. 5. Effect of SSd on hippocampal neurogenesis. (A) Proliferating cells in rat hippocampus which labeled by BrdU. (B) Neural stem cells in rat hippocampus which

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labeled by nestin. (C) Differentiation of neural stem cells in rat hipacampus which labeled by Dcx. (D) Neurons in rat hippocampus which labeled by MAP2. These results showed that SSd impaired hippocampal neurogenesis. Data were expressed as mean ± SEM (n = 3). #p < 0.05 and ##p < 0.01 vs. the control. Fig. 6. Effect of SSd on Akt/Foxg1 signaling pathway. (A) Protein levels of p-Akt and Foxg1 by western blot test. (B) p-Akt protein level were quantified using Image-Pro

Plus software. (C) Foxg1 protein level were quantified using Image-Pro Plus software. (D) FGF2 protein level were detected by ELISA kit. These results showed that SSd regulated the Akt/Foxg1 signaling pathway. Data were expressed as mean ±

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SEM (n≥ 3). #p < 0.05 and ##p < 0.01 vs. the control.

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