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Neurochem Res (2017) 42:1299–1307 DOI 10.1007/s11064-016-2171-y

ORIGINAL PAPER

Ameliorative Effect of Ginsenoside Rg1 on Lipopolysaccharide-Induced Cognitive Impairment: Role of Cholinergic System

Yang Jin1 · Jian Peng1 · Xiaona Wang2 · Dong Zhang3 · Tianyin Wang4 

Received: 8 October 2016 / Revised: 23 December 2016 / Accepted: 28 December 2016 / Published online: 11 January 2017 © Springer Science+Business Media New York 2017

cortex (PFC) and hippocampus of LPS-treated rats. These findings suggest that ginsenoside Rg1 has protective effect against LPS-induced cognitive deficit and that prevention of LPS-induced changes in cholinergic system is crucial to this ameliorating effect.

Keywords Ginsenoside Rg1 · Lipopolysaccharide · Cognitive impairment · Acetylcholine · Alpha7 nicotinic acetylcholine receptor

Introduction

Lipopolysaccharide (LPS), an endotoxin isolated from Gram-negative bacteria, has been shown to trigger exces-sive production of multiple inflammatory cytokines in the brain, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6, that are accom-panied with neuroinflammation, neuronal death and mem-ory deficit [1]. Even systemic injection LPS could impair spatial learning and memory function in the Morris water maze (MWM) [1] and produce reduced retention of pas-sive avoidance learning in rodents [2]. As neuroinflamma-tion is considered to have a critical role in multiple sclero-sis and Alzheimer’s disease [3], animal model of LPS has been repeatedly used to investigate the biochemical mecha-nisms of cognitive dysfunction due to inflammation and to develop targeted therapeutics in neurological disorders.

Ginsenoside Rg1 is a main bioactive constitute of extracts of ginseng. It has been established that ginsenoside Rg1 exerts neuroprotective [4] and anti-inflammatory prop-erties [5]. Ginsenoside Rg1 has been documented to inhibit LPS-induced cytokine production in vitro [6, 7] and acute lung injury in  vivo [8]. Converging evidence indicates that ginsenoside Rg1 enhances cognitive function. Recent

Abstract Bacterial endotoxin lipopolysaccharide (LPS) can induce systemic inflammation, and therefore dis-rupt learning and memory processes. Ginsenoside Rg1, a major bioactive component of ginseng, is shown to greatly improve cognitive function. The present study was designed to further investigate whether administration of ginsenoside Rg1 can ameliorate LPS-induced cognitive impairment in the Y-maze and Morris water maze (MWM) task, and to explore the underlying mechanisms. Results showed that exposure to LPS (500  μg/kg) significantly impaired working and spatial memory and that repeated treatment with ginsenoside Rg1 (200  mg/kg/day, for 30 days) could effectively alleviate the LPS-induced cogni-tive decline as indicated by increased working and spatial memory in the Y-maze and MWM tests. Furthermore, gin-senoside Rg1 treatment prevented LPS-induced decrease of acetylcholine (ACh) levels and increase of acetylcho-linesterase (AChE) activity. Ginsenoside Rg1 treatment also reverted the decrease of alpha7 nicotinic acetylcholine receptor (α7 nAChR) protein expression in the prefrontal

* Tianyin Wang tianyinwang001@126.com

1 Hepatobiliary and Enteric Surgery Research Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan, People’s Republic of China

2 Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA

3 Minimally Invasive Urology Center, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250012, Shandong, People’s Republic of China

4 Health and Family Planning Commission, Shandong Province Medical Guidance Center, Jinan 250012, Shandong, People’s Republic of China

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efforts have shown that ginsenoside Rg1 alleviates spatial memory impairment induced by okadaic acid [9] and in Alzheimer’s disease [10]. Also, ginsenoside Rg1 treatment promotes hippocampal long-term potentiation in middle-aged mice [11]. Drawing on these findings, we therefore hypothesized that ginsenoside Rg1 might exert beneficial effect against LPS-induced impairment in working and spa-tial memory.

Acetylcholine (ACh), the major excitatory neurotrans-mitter in the adult mammalian brain, has long been impli-cated in cognitive control processes [12, 13]. Report sug-gests that enhancement of AChergic activity correlates with improved learning and memory [14]. Evidence has also confirmed that the ACh levels are rapidly degraded by the hydrolytic enzyme acetyl cholinesterase (AChE), by which cholinergic function is regulated and escape latency is shortened [15]. Moreover, ACh acting through ligand-gated nicotinic ACh receptor, can regulate neuronal excitabil-ity, synaptic communication and cognitive function [16]. In particular, alpha7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the prefrontal cortex (PFC) and hippocampus [17] and involved in regulating cognitive function in mice [18]. Mice lacking α7 nAChR displayed learning and memory deficit [19]. In addition, ACh has been demonstrated to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines [20, 21]. Administration of LPS could decrease α7 nACh receptor density in the brain of mice [22]. Despite these observations, it remains presently unknown whether ACh, AChE and α7 nACh receptor are involved in ginseno-side Rg1-induced cognitive enhancement in LPS-exposed rats.

In the present study, we were prompted to see the pro-tective effect of ginsenoside Rg1 on LPS-induced cognitive decline in the Y-maze and MWM task. To further examine the underlying mechanisms, ACh concentrations, AChE activity and α7 nACh receptor in the PFC and hippocam-pus of rats, involved in the cognitive processes, were also assessed by high-performance liquid chromatography-mass spectrometry (HPLC-MS), Ellman’s method and western blot analysis.

Materials and Methods

Animals

Wistar male rats (200–250  g) were procured from Shan-dong University Animal Services. All animals were fed standard laboratory chow, housed in wire cages at 20–22 °C and 50 ± 5% humidity, and allowed water ad  libitum. All experiments were approved by the Shandong Univer-sity Animal Care and Use Committee and performed in

compliance with the National Institutes of Health guide for the care and use of Laboratory animals (Publications No. 8023, revised 1978).

Drug Administration and Experimental Groups

LPS (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline (0.9%) and injected intraperitoneally (i.p.) at a dose of 500 μg/kg. This dose was used for induction of moderate inflammation and did not affect motor activ-ity [2]. Ginsenoside Rg1 (purity >98%, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in pathogen-free phos-phate buffer saline (PBS) and injected intragastrically at a dose of 200  mg/kg, as previously described [8]. Whereas the control groups were administered the same amount of physiological saline or ginsenoside Rg1 [2].

Rats were randomly divided into four groups (n = 8) as follows:

Group I (Saline): The rats received saline by oral gav-age (200 mg/kg) for 30 days followed by injection (i.p.) of saline (500 μg/kg).

Group II (Rg1): The rats received Rg1 by oral gavage (200  mg/kg) for 30  days followed by injection (i.p.) of saline (500 μg/kg).

Group III (LPS): The rats received saline by oral gavage (200 mg/kg) for 30 days followed by injection (i.p.) of LPS (500 μg/kg). Group IV (LPS plus Rg1): The rats received Rg1 by oral gavage (200  mg/kg) for 30  days followed by injection (i.p.) of LPS (500 μg/kg).

The interval between intragastric administration and intraperitoneal injection was 30 min. Drugs were adminis-tered between 9:00 a.m. and 11:00 a.m.

A cohort of rats were submitted to Y-maze and MWM task 120  min after the last drug administration. One day interval was given between tests for adaptation of new cir-cumstances [23]. At 24 h after completion of the behavio-ral test, rats were sacrificed by cervical decapitation. Brain tissues were immediately removed, and the PFC (from bregma, anterioposterior, +4.2 to + 2.2) [24] and hip-pocampus were dissected for further neurochemical and Western blot analyses.

Y-Maze Task

Short-term working memory was assessed by recording spontaneous alternation behavior in Y-maze, as described elsewhere [25]. The walls of each arm had geometric shapes that provided spatial cues. Each rat was placed at the distal end of one arm and allowed to explore the appara-tus during an 8-min session. The number and the sequence of arm entries were recorded manually. Alternation was defined as successive entries into the three arms on overlap-ping triplet sets. The percentage of spontaneous alternation

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behavior was calculated according to the following equa-tion: alternation (%) = [(number of alternations)/(total arm entries − 2)] × 100. The number of arm entries served as an indicator of locomotor activity [26].

Morris Water Maze Test

Morris water maze was used to evaluate spatial learn-ing and memory. Briefly, it consists of a circular water tank (120 cm in diameter and 60 cm in height), filled with 23 ± 1 °C water. A camera was fixed to the ceiling above the water maze and connected to the computer-based program (Viewer 2 Tracking Software, Ji Liang Instruments, China). During acquisition, rats were trained over 4 days (4 trials/day) to find the hidden escape platform in the middle of 3rd quadrant. The rats had a maximum of 60 s to find the plat-form. If animals were unable to locate the platform, they were gently guided to the platform and required to stay on the platform for 20 s before being removed. Escape latency to find the platform were to indicate the learning perfor-mance. On the 5th day, rats were subjected to a probe trial by removing the platform. The number rats crossed the tar-get quadrant was used as measures of spatial memory.

HPLC-MS for ACh Quantification

As described before [27], samples were chromatographi-cally separated on a hydrophilic interaction chromatogra-phy mode column (Supelco, Bellefonte, PA, USA; mobile phase A: ammonium formate, B: acetonitrile) with a flow rate of 200 μl/min and detected and quantified by a linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Multiple reaction monitoring (MRM) was in positive ionization mode and electrospray ioniza-tion (ESI) source parameters were as follows: nebulising and curtain gas (N2) 50 psi, capillary temperature 200 °C, ion spray voltage 5000  V. Collision-induced dissociation (CID) product-ion MS/MS spectra were collected using 1.5 Th parent ion isolation width and 30% relative colli-sion energy. The parent ions of ACh (m/z 148) and d4-ACh (m/z 150) were mass-selected for CID and, their principal product ions m/z 85 and 93, respectively, were monitored. XcaliburTM 3.0.63 software (Thermo Fisher Scientific, San Jose, CA, USA) was used for instrument control, data acquisition and processing.

AChE Activity Assay

The activity of AChE was carried out according to the method of Ellman et al. [28], using an AChE activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). Brain samples (30 μg protein) were added to a reaction mixture contain-ing 150 μl of 0.1 M phosphate buffer (pH 8.0), 1 μl of the

substrate 0.075  M acetylthiocholine iodide and 5  μl of 0.01  M 5–5 Dithiobis (2-Nitrobenzoic acid). The absorb-ance at 410 nm was measured using a UV spectrophotom-eter (Bio-Rad, Hercules, CA, USA).

Western Blot Analysis

The protocol for western blotting was described previ-ously [29]. Briefly, samples of homogenates were centri-fuged for 20  min at 12,000  g at 4 °C. The equal amounts (50 μg) of proteins were subjected by sodium dodecyl sul-fate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Milli-pore, Milford, MA, USA). The membranes were blocked with 5% non-fat milk in Tris buffered saline containing 0.1% Tween 20 for 2 h at room temperature. Subsequently, the membranes were probed with the primary antibodies overnight at 4 °C: anti-α7 nACh receptor antibody (1:5000) (Abcam, Cam-bridge, UK) and anti-β-actin (1:1000) (ZSGB-Bio, Beijing, China). The filters were incubated with the secondary anti-body horseradish peroxidase conjugated to goat anti-rabbit/mouse IgG (1:5000) (ZSGB-Bio, Beijing, China). Mem-brane were visualized by using a super enhanced chemilu-minescence reagent (Thermo Fisher Scientific, Rockford, IL, USA) and images were captured by luminescent image analysis system (Fujifilm, LAS-4000 mini, Japan). Immu-nodetection of bands was determined by Image J software program. A ratio between α7 nACh receptor and β-actin corresponding bands was used to quantify the levels of each protein (normalized value).

Statistical Analysis

Statistical procedures were performed on SPSS version 21.0. Data are expressed as mean ± SEM. Values from the acquisition days in MWM were analyzed using repeated measures ANOVA. If the interaction between the treat-ment and day was significant, then one-way ANOVA using post hoc Tukey’s test for the treatment effect for each day was carried out. Other data were conducted statistically using two-way ANOVA for multiple comparisons with LPS or ginsenoside Rg1 and post hoc Tukey’s test was to determine differences between specific groups. Values of p < 0.05 were considered statistically significant.

Results

Effect of Ginsenoside Rg1 on Working Memory

To examine whether ginsenoside Rg1 could improve work-ing memory in LPS-treated rats, we firstly analyzed spon-taneous alternation percentage in the Y-maze. Results of

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memory measurement were analyzed by two-way ANOVA (LPS × ginsenoside Rg1). As shown in Fig.  1a, ANOVA revealed the significant effect of LPS [F (1, 28) = 36.475, p < 0.001], ginsenoside Rg1 [F (1, 28) = 10.288, p < 0.01], and the LPS × ginsenoside Rg1 interaction [F (1, 28) = 6.033, p < 0.05] on spontaneous alternation percent-age. Post-hoc analysis indicated that administrations of LPS produced the significant decrease in spontaneous alter-nation percentage (p < 0.001) compared with the control rats. Nevertheless, we observed the remarkable increase of working memory in ginsenoside Rg1-treated groups exposed to LPS (p < 0.01), indicated by the increase of spontaneous alternation percentage compared to LPS-treated groups.

Additionally, the number of arm entries was also recorded and used as an indicator of locomotor activity.

As shown in Fig.  1b, no significant differences in num-ber of arm entries were detected between groups [F(3, 28) = 1.213, p > 0.05, Fig. 1b].

Effect of Ginsenoside Rg1 on Spatial Learning and Memory

To assess spatial learning acquisition, animals were trained with 4 trials per day for 4 consecutive days on the MWM task. The escape latency gradually declined over the train-ing period for all groups [F (3, 28) = 30.507, p < 0.001, repeated measures ANOVA] (Fig.  2a), indicating a grad-ual spatial memory acquisition in all experimental ani-mals. Repeated measures ANOVA revealed no interac-tion between training days and groups [F (3, 28) = 0.955, p > 0.05], suggesting that all rats effectively learned the

Fig. 1 Effect of administration of ginsenoside Rg1 on LPS-induced working memory impairment of spontaneous alternation behavior (a) and number of arm entries (b) in the Y-maze test. Values rep-resent the mean ± SEM (n = 8 per group). ***p < 0.001 compared

with saline groups; ##p < 0.01 compared with LPS groups (one-way ANOVA followed by Tukey’s post hoc test). No overall significant differences were obtained between groups for number of arm entries

Fig. 2 Effect of LPS and ginsenoside Rg1 administration on spa-tial learning and memory in the Morris water maze test. a Escape latency to locate the hidden platform during the 4 days of acquisition training. b Number of crossing in the target quadrant in the spatial

exploratory test. Data represent the mean ± SEM, n = 8, ***p < 0.001, **p < 0.01 compared with saline groups; ##p < 0.01,###p < 0.001, #p < 0.05 compared with LPS groups

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task. Furthermore, the escape latencies of the LPS groups were significantly higher than those of the saline groups on days 2–4 (p < 0.05). Whereas the animals of treatment with ginsenoside Rg1 groups spent the less time to reach the platform than LPS alone (p < 0.05).

During the spatial probe test, with the platform removed, the number of crossings in the target quad-rant was recorded. ANOVA revealed the effect of LPS [F (1, 28) = 28.527, p < 0.001], ginsenoside Rg1 [F (1, 28) = 8.927, p < 0.01], and the LPS × ginsenoside Rg1 interaction [F (1, 28) = 6.443, p < 0.05] on the number of crossing. Post-hoc analysis indicated that rats treated with LPS markedly exhibited lower crossings number when compared to saline groups (p < 0.001). However, ginseno-side Rg1 plus LPS groups significantly increase the number of crossings in the target quadrant as compared with LPS groups (p < 0.01) (Fig. 2b). These results showed that gin-senoside Rg1 markedly improved the spatial learning and memory of LPS-treated rats as indicated in the MWM test.

Effect of Ginsenoside Rg1 on ACh Levels

ACh was suggested to be implicated in the inflammation of LPS-exposed rats [7]. Next, we measured ACh to investi-gate the ability of ginsenoside Rg1 to modulate learning and

memory in LPS-treated rats. The results obtained in ACh concentrations are shown in Fig.  3a, b. ANOVA revealed the effect of LPS [PFC: F (1, 28) = 83.283, p < 0.001; hip-pocampus: F (1, 28) = 120.313, p < 0.001], ginsenoside Rg1 [PFC: F (1, 28) = 30.682, p < 0.001; hippocampus: F (1, 28) = 30.009, p < 0.001], and the LPS × ginsenoside Rg1 interaction [PFC: F (1, 28) = 47.460, p < 0.001; hip-pocampus: F (1, 28) = 40.179, p < 0.001] on ACh content. Post-hoc analysis indicated that ACh levels in the PFC (p < 0.001) and hippocampus (p < 0.001) of LPS-treated rats were remarkably decreased compared with in the saline groups. Whereas the decrease was significantly attenuated by treatment with ginsenoside Rg1 in ACh levels in the PFC (p < 0.001) and hippocampus (p < 0.001) compared to LPS-treated rats.

Effect of Ginsenoside Rg1 on AChE Activity

To further illuminate the potential mechanism of Gin-senoside Rg1 in ameliorating cognition deficiency caused by LPS, the AChE activity which is responsi-ble for degradation of ACh was detected. As shown in Fig.  4, ANOVA revealed the effect of LPS [PFC: F (1, 28) = 68.350, p < 0.001; hippocampus: F (1, 28) = 5.475, p < 0.05], ginsenoside Rg1 [PFC: F (1, 28) = 32.728,

Fig. 3 Effect of ginsenoside Rg1 administration on LPS-induced changes of ACh levels (pmol/μl) in the prefrontal cortex (PFC) (a) and hippocam-pus (b). Values are represented as Mean ± SEM; n = 8, One way ANOVA followed by post hoc Tukey’s test analysis. ***p < 0.001 compared with saline groups; ###p < 0.001 com-pared with LPS groups

Fig. 4 Effect of ginsenoside Rg1 on LPS-induced changes in AChE activity in the prefrontal cortex (PFC) (a) and hippocam-pus (b). Data are expressed as Mean ± SEM; n = 8, One way ANOVA followed by post hoc Tukey’s test analysis. ***p < 0.001 compared with saline groups; ###p < 0.001 com-pared with LPS groups

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p < 0.001; hippocampus: F (1, 28) = 10.072, p < 0.01], and the LPS × ginsenoside Rg1 interaction [PFC: F (1, 28) = 21.242, p < 0.001; hippocampus: F (1, 28) = 15.208, p < 0.01] on AChE activity. Post-hoc anal-ysis indicated that AChE activity in the PFC (p < 0.001) and hippocampus (p < 0.001) of LPS-treated rats was significantly increased as compared to saline groups. However, ginsenoside Rg1 treatment markedly nor-malized the alteration in AChE activity in the PFC (p < 0.001) and hippocampus (p < 0.001) as compared with LPS alone.

Effect of Ginsenoside Rg1 on α7 nACh Receptor Expression

The α7 nACh receptor, expressed on the surface of inflammatory cell, plays a significant role in cogni-tive function. Thus, we determined whether α7 nACh receptor in the PFC and hippocampus is involved in cognition-facilitating effect in the action of ginseno-side Rg1. ANOVA revealed the effect of LPS [PFC: F (1, 28) = 31.376, p < 0.001; hippocampus: F (1, 28) = 68.350, p < 0.001], ginsenoside Rg1 [PFC: F (1, 28) = 14.364, p < 0.05; hippocampus: F (1, 28) = 32.728, p < 0.001], and the LPS × ginsenoside Rg1 interaction [PFC: F (1, 28) = 13.327, p < 0.01; hippocampus: F (1, 28) = 21.242, p < 0.001] on α7 nACh receptor protein expression. The results showed that exposure of rats to LPS significantly decreased α7 nACh receptor expres-sion in the PFC (p < 0.001) and hippocampus (p < 0.001) compared with the corresponding controls. However, prolonged administration of ginsenoside Rg1 effectively reversed LPS-induced the reduction of α7 nACh recep-tor expression in the PFC (p < 0.001) and hippocampus (p < 0.001) (Fig. 5a, b).

Discussion

Our study demonstrated that prolonged administration of ginsenoside Rg1 could ameliorate the deleterious effect of LPS on working and spatial memory in the Y-maze and MWM task. Furthermore, ginsenoside Rg1 treatment could reversed LPS-induced the reduction of ACh levels and increase in AChE activity. As well, the decrease of α7 nACh receptor protein expression was alleviated by ginse-noside Rg1 in the PFC and hippocampus of LPS-treated rats.

In this study, ginsenoside Rg1 was tested in LPS-impaired cognition using two paradigms indicative of dif-ferent forms of memory, the Y-maze and MWM tests. LPS-induced memory decline was tested using Y-maze task, a specific and sensitive test for spatial short-term working memory [30], in rats administered ginsenoside Rg1 for 1 month. Consistent with previous studies [31, 32], our results showed that LPS-induced inflammation demon-strated decreased spontaneous alternation compared with saline-treated groups, suggesting that LPS exposure sig-nificantly produced working memory impairment in rats. In addition, LPS did not alter the locomotor activity of rats in this test in terms of total arm entries. Ginsenoside Rg1 also did not cause changes in the locomotor activity in LPS-treated rats, similar to that of earlier reports [31]. There-fore, it is of high importance that ginsenoside Rg1 treat-ment could reversed the lowered spontaneous alteration in LPS-exposed rats, indicative of the protective function of ginsenoside Rg1 in short-time working memory deficit induced by LPS.

Of note, LPS-induced cognitive decline was also tested by the MWM test, which tests whether treatment groups remarkably differ from control in efficiency to find the hid-den platform by observing escape latency [33]. The pre-sent data showed that rats exposed to LPS demonstrated

Fig. 5 Effect of ginsenoside Rg1 on the α7 nACh receptor expression in the prefrontal cortex (PFC) (a) and hippocam-pus (b). The expression of α7 nACh receptor was determined by western blot, and β-actin was used to evaluate protein loading. Normalized intensity bands of α7 nACh receptor are expressed as the mean ± SEM of at least three separate experiments; n = 8. Results showed the percentage changes from saline groups. ***p < 0.001 compared with saline groups; ###p < 0.001 compared with LPS groups (one-way ANOVA followed by Tukey’s post hoc test)

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the prolonged escape latency in the place navigation task and reduced number of crossings in the platform quadrant in the probe test, suggesting that administration of LPS produced remarkable long-term impairment in learning and memory. Of particular noteworthy, ginsenoside Rg1 treatment mitigated the deficit as shown by the decreased escaping time and the increased number of crossings in the platform quadrant in LPS-treated rats, indicating that ginsenoside Rg1 effectively prevented the deficit in spa-tial learning and memory induced by LPS. Our data were in accordance with studies showing that ginsenoside Rg1 could alleviate okadaic acid induced spatial memory deficit in rats [9]. Taken together, the present study demonstrated, ameliorative effect of prolonged treatment with ginsenoside Rg1 on LPS-induced impairment in spatial learning and memory. However, further researches are required to more specifically target whether acute ginsenoside Rg1 treatment is involved in cognitive facilitation in LPS-treated rats.

The cholinergic system has been considered as a medi-ator in the neuroinflammation [34]. In particular, ACh has been shown to exert an anti-inflammatory function by diminishing TNF-α concentrations [35]. Tyagi et  al. reported that ACh attenuated LPS-induced neuroinflamma-tion with exposure to LPS in rat brain [36]. It was dem-onstrated that administration of IL-1β led to pronounced reduction in ACh release in rat hippocampus [37]. Our findings confirm these reports, showing that LPS markedly decreased ACh levels in the PFC and hippocampus. Previ-ous studies also revealed that ginsenoside Rg1 could allevi-ate LPS-induced inflammatory responses in murine BV-2 microglial cells [38] and elevate ACh levels in the hip-pocampus of scopolamine-treated mice [39]. In accordance with these findings, we observe that long-term administra-tion of ginsenoside Rg1 prevented the ACh levels, which had been decreased by LPS.

AChergic system has a well-documented relationship to cognitive function [40–43]. It was shown that the increase of ACh levels in the hippocampus reversed scopolamine-induced impairment in social and object recognition mem-ory in rats [41]. In contrast, the reduction of ACh release was correlated with the memory deficit after IL-1β admin-istration in rats [37]. Impairment of cognitive function can be consistently observed by decreased ACh levels of dia-betic mice using Y-maze and Morris water maze tests [40]. Noticeably, numerous studies have demonstrated that PFC and hippocampus are involved in working and spatial mem-ory processes [44, 45]. In line with published evidence, we also found that administration of ginsenoside Rg1 sig-nificantly attenuated the LPS-induced deficit in working and spatial memory. Collectively, this raises the intrigu-ing possibility that long-term treatment with ginsenoside Rg1 could reverse cognitive dysfunction produced by LPS was in part, due to rebounded ACh levels in the PFC and

hippocampus, which attributed to the protective effect of prolonged ginsenoside Rg1 treatment.

Another important regulator of cholinergic signaling is AChE, responsible for degradation of ACh. AChE activity has been shown to increase in response to IL-1β [46] and LPS exposure [47]. Similar to previous reports, our results showed the elevated AChE activity in the PFC and hip-pocampus of inflammation induced rats. Moreover, mount-ing evidence suggests that AChE has a fundamental role in learning and memory [48, 49]. Altered AChE activity is neurochemically associated with cognitive decline observed in the neuroinflammatory response [50, 51]. Particularly, AChE inhibitors such as donepezil, which increase synap-tic availability of ACh by reducing the rate of catabolism, improve memory function in rodent models and Alzhei-mer’s patients [41]. A recent study found that decreasing the AChE activity while also increasing the ACh levels in the brain ameliorated the cognitive dysfunction in Y-maze of scopolamine treated rats [52]. Importantly, we found that increased AChE activity caused by inflammation, pos-sibly leads to reduction of cholinergic neurotransmission efficiency due to decrease in ACh levels in the synaptic cleft, thus contributing to cognitive impairment. Whereas ginsenoside Rg1 treatment impeded the elevation of AChE activity following LPS exposure. Accordingly, our findings suggest that alteration in AChE activity might be responsi-ble for the facilitation of cognitive function by ginsenoside Rg1 treatment in LPS-exposed rats.

Accumulating evidence indicates that α7 nACh recep-tor is participating in inflammation [43]. Furthermore, activation of α7 nACh receptor was shown to attenu-ate the release of pro-inflammatory cytokines in response to LPS [53, 54]. Lykhmus et  al. found administration of LPS decreased α7 nACh receptor density in brain of mice [22]. In accordance with these previous views, the present data revealed the significant decrease in α7 nACh receptor expression in the PFC and hippocampus of LPS-exposed rats. Moreover, α7 nACh receptor is involved in cognitive function, such as learning and memory both in humans and animals model [55]. Specifically, the cognitive deficit associated with Alzheimer’s disease may be related to α7 nACh receptor dysfunction [43]. Likewise, mice lacking the α7 nAChR displayed impaired learning and memory performance in the passive avoidance test [19]. In con-trast, up-regulation of α7 nACh receptor expression led to enhancement of long-term potentiation and spatial cogni-tion in mice [56]. It is worthwhile noting that the reduction in α7 nACh receptor expression may be related to cognitive decline in LPS-treated rats. However, prolonged treatment with ginsenoside Rg1 significantly reverted the down-regu-lation of α7 nACh receptor produced by LPS, accompanied by the enhancement of working and spatial memory. Con-sequently, it can be assumed that capacity of ginsenoside

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Rg1 to ameliorate cognitive deficit induced by LPS was in part due to the restoration of α7 nACh receptor in the PFC and hippocampus of rats.

We conclude that repeated treatment with ginsenoside Rg1 was capable of ameliorating LPS-induced deficit in working and spatial memory, and the beneficial effects are, at least partially, mediated through alterations in ACh levels, AChE activity and α7 nACh receptor expression. Furthermore, the data presented here provide confirmatory evidence for the neuroprotective role of ginsenoside Rg1 and pave way for the better management of inflammation-mediated cognitive impairment.

Acknowledgements This work was supported by the Provin-cial Natural Science Foundation of Shandong [Grant Number ZR2015HQ002], the National Natural Science Foundation of China [Grant Numbers 81572534 and 81602226], the Provincial Projects of Medical and Technology Development Program of Shandong [Grant Numbers 2014WS0091 and 2014WS0347], the Key Research and Development Program of Shandong Province [Grant Number 2015GSF118096], and a China Postdoctoral Science Foundation Funded Project [Grant Number 2016M590641].

Compliance with Ethical Standards

Conflict of interest The authors have no conflict of interest.

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