Protective effects of linalool against amyloid beta-induced cognitive deficits and damages in mice

Accepted Manuscript Protective effects of linalool against amyloid beta-induced cognitive deficits and damages in mice

Pan Xu, Kezhu Wang, Cong Lu, Liming Dong, Li Gao, Ming Yan, Silafu Aibai, Yanyan Yang, Xinmin Liu PII: DOI: Reference:

S0024-3205(17)30066-8 doi: 10.1016/j.lfs.2017.02.010 LFS 15141

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Life Sciences

Received date: Revised date: Accepted date:

7 January 2017 17 February 2017 19 February 2017

Please cite this article as: Pan Xu, Kezhu Wang, Cong Lu, Liming Dong, Li Gao, Ming Yan, Silafu Aibai, Yanyan Yang, Xinmin Liu , Protective effects of linalool against amyloid beta-induced cognitive deficits and damages in mice. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.02.010

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ACCEPTED MANUSCRIPT Protective effects of Linalool against Amyloid beta-induced cognitive deficits and damages in mice Pan Xua*, Kezhu Wanga, Cong Lua, Liming Donga, Li Gaob, Ming Yanb, Silafu Aibaib, Yanyan Yangc, Xinmin Liua,c*1 a

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Research Center of Pharmacology and Toxicology, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China b Department of Pharmacology and Toxicology Laboratory, Xinjiang Institute of Traditional Uighur Medicine, Urumqi, Xinjiang, 830049, China c China Astronaut Research and Training Center, Yuanmingyuan West Road No. 1, Beijing 100094, China,

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Abstract Aim: Amyloid-beta (Aβ)-mediated neurotoxicity plays a pivotal role in the pathogenesis of Alzheimer's disease (AD), which induces oxidative stress and apoptosis. Linalool (LI) is a volatile monoterpene showing positive effect in AD treatment. This study was designed to research the protective effect of LI against neurotoxicity and cognitive deficits induced by Aβ1-40 in mice. Main methods: Aβ1-40 (4 g) solution was injected in the bilateral hippocampus to induce cognitive deficits of mice. The protective effects of LI were evaluated by behavioral tests and the related mechanism was further explored by observing the apoptosis and oxidative stress changes in the hippocampus of mice. Key fingdings: LI (100 mg/kg, i.p.) administration significantly improved the cognitive performance of model mice in Morris water maze test and step-through test. Meanwhile, LI effectively reversed the Aβ1-40 induced hippocampal cell injury in histological examination, apoptosis in TUNEL assay, changes of oxidative stress indicators (SOD, GPX, AChE). Besides, the activated cleaved caspase (caspase-3, caspase-9) was suppressed and Nrf2, HO-1 expression was elevated by LI treatment. Significance: LI could attenuate cognitive deficits induced by Aβ, and the neuroprotective effect of LI might be mediated by alleviation of apoptosis, oxidative stress depending on activation of Nrf2/HO-1 signaling. We could assume that LI has the potential to be a neuroprotective substance for AD therapy. Abbreviations: AD, Alzheimer's disease; Aβ, Amyloid-beta; AChE, acetylcholinesterase; GPX, glutathione peroxidase; HO-1, heme oxygenase-1; LI, linalool; LDH, lactate dehydrogenase; MDA, malondialdehyde; MMP, mitochondrial membrane potential; MWM, Morris water maze; NO, nitric oxide; Nrf2, Nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase.

*Corresponding authors at: Research Center of Pharmacology and Toxicology, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China. Tel: +86 13331169087. E-mail addresses: [email protected] (X.M. Liu), [email protected] (P. Xu).

ACCEPTED MANUSCRIPT Keywords: Linalool; Alzheimer’s disease; cognitive improvement; oxidative stress; apoptosis

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1. Introduction Alzheimer’s disease (AD), the most common form of dementia affecting more than 26.6 million people worldwide, which is clinically characterized by a progressive decline in memory and cognitive function [1]. The key neuropathological hallmarks of the AD brain are extracellular senile plaques induced by accumulation of amyloid β (Aβ) protein and intracellular neurofibrillary tangles [2]. Though the complicated mechanisms underlying AD remain unclear, evidences have indicated that the Aβ induced neurotoxicity and oxidative stress play a key role in its pathogenesis [3-5]. Oxidative stress promotes the production of Aβ, alternatively, the augmentation of Aβ make neurons more susceptible to free radicals, particularly for mitochondrial in neuron [6]. Mitochondrial dysfunction induce energy store exhaustion and ROS overproduction, which contribute to DNA cleavage, protein oxidation and lipid peroxidation, eventually leading to caspase activation and apoptosis [7-9]. Apoptosis is known as programmed cell death, which aggravate the memory and cognitive decline in AD [10]. Thus, it is an important strategy for AD treatment to attenuate Aβ induced oxidative stress and apoptotic neuron death. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is one of the most important transcription factors having protective response against oxidative stress, [11]. In normal conditions, Nrf2 is sequestered in the cytosol by kelch-like ECH-associated protein 1 (Keap1). Under oxidative stress or regulation, Nrf2 is translocate to bind with antioxidant response element (ARE), activating the expression of defensive genes [12]. Of those genes, heme oxygenase-1 (HO-1) is a vital antioxidant, which exert beneficial effects in the protection against oxidative injury and regulation of apoptosis in AD [13]. Thus Nrf-2 and HO-1 are considered as important targets for the treatment of AD. (-)-Linalool (LI) is a major volatile monoterpene component of essential oils from several aromatic plants, such as Lavandula angustifolia Mill., Rosmarinus officinalis L. and Coriandrum sativum L. which were used in traditional medicine [14-16]. LI possesses a variety of bioactivities including anti-inflammatory, antioxidant, anti-tumor, antidepressant, anticonvulsant and antimicrobial [17-21]. Increasing evidences demonstrated that the antioxidant activity of LI is obvious and bring marked benefits for central nervous system (CNS). For example, Linalool exhibited antioxidant properties in H2O2 treated guinea pig brain and neuroprotective effect against acrylamide induced neurotoxicity. [22,23]. Besides, linalool also modulates glutamatergic neurotransmission in vitro and in vivo, by interaction with NMDA receptors [24,25]. Thus, we speculated that LI should show neuroprotective effect in AD models. Recent evidences suggested that LI reverses neuropathological and behavioral impairments in old triple transgenic AD mice and Silexan, which mainly contains LI, has potent neuroprotective effects in scopolamine induced AD mice [26,27]. However, the direct effect of LI on AD model and its mechanism were researched insufficiently.

ACCEPTED MANUSCRIPT The intrahippocampal Aβ1-42 infusion model could induce oxidative damage and neuronal apoptosis in mice to mimic some pathology of AD [28-30]. Therefore, the study was designed to investigate the improvement effect of LI on mice cognitive deficits induced by Aβ1-40 and the related mechanism. 2. Materials and methods

Fig. 1. Molecular structure of linalool

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2.1 Drug and Aβ preparation (-)- Linalool (LI, Fig. 1) was obtained from the National institutes for Food and Drug Control (Beijing, China). The enantiomer present in lavender is (R)-linalool, which is more woody and lavender-like than (S)-form. Amyloid β-protein Fragment 1-40 (Aβ1-40) were purchased from Sigma-Aldrich (Lot# SLBL0744V, St. Louis, MO, USA). For intrahippocampal injection, Aβ1-40 was dissolved in sterile 0.1 M phosphate-buffered saline (PBS) to get the solution of 1μg/μL and then incubated at 37 °C for 7 days to obtain the aggregated form of Aβ [31,32].

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2.2 Animals 60 male C57BL/6J mice (8 weeks) were provided by the Vital River Laboratories (Qualified No.: SCXK 2012-0001, Beijing, China). All animal were housed in a temperature controlled (25C) condition with alternating light/dark cycle (lights, 8:00 AM-8:00 PM), and were given free access to water and diet. All experiments were performed under the approval and supervision of the Academy of Experimental Animal Center of the Institute of Medicinal Plant Development and in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

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2.3 Groups and drug administration The mice were divided randomly into five experimental groups including a control groups, a sham operated group and four Aβ1-40-treated group divided into a model group, two LI treated groups (50, 100 mg/kg/d). All mice in Aβ1-40-treated group were anesthetized and stereotactically injected aggregated Aβ1-40 (4 L) into the bilateral hippocampus of mice (anterior-posterior position -2.0 mm, medial-lateral position  1.6 mm, dorsoventral 1.5 mm from bregma). As the previous reports and previous model pretest showed, more plentiful Aβ deposits were observed in model mice through the immunohistochemical staining test, which verified the successful preparation of the Aβ1-40 injection AD model [33-35]. The sham group was operated like model preparation but injected with PBS in the hippocampus. LI was dissolved in a normal saline solution with 2% Tween-80 and 1% DMSO (vehicle) based on the dose of 50 and 100 mg/kg. Drugs were administered intraperitoneally (i.p.) once per day for 7 days before surgery and then for the

ACCEPTED MANUSCRIPT subsequent 14 days after surgery. The control, sham and model groups were received the same volume of vehicle for 3 weeks.

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2.4 Behavioral tests 2.4.1 locomotor activity test 14 days after surgical operation, the locomotor activity of mice was assessed to preclude the interference of locomotor activity change in the parameters of cognitive function. An open-field computer-aided controlling system was used which consists of four metal tanks (diameter 30 cm, height 40 cm) with a 120 Lux light source and a video camera fixed at the top [36]. 30 minutes after dosing, each mouse was adapted to the tank for 3 min freely, then the distances travelled in the following 10 min were recorded automatically as the index. 2.4.2 Morris water maze test The Morris water maze (MWM) test was performed after locomotor activity test to evaluate the spatial learning and memory. The apparatus contains a circular pool filled with water (24-26 °C) and divided into four equal quadrant. A hyaline platform (6 cm diameter, 15 cm height) was submerged 1 cm below the surface in one quadrant (e.g. SE). In navigation experiment which contains four test sessions per day for five days. Each mouse was placed at one quadrants and allowed to find the platform in 60 s. Before and after the swimming, mice were left on platform for 10 s. The escape latency and the escape rate were analyzed by a tracking and image analyzer system. Probing test was conducted with the platform removed the next day after navigation. Mice were released from the quadrant (e.g. NW) opposite from the previous platform location (target quadrant) to receive 90 s memory retention test. The time in target quadrant and crossing number were recorded and analyzed. 2.4.3 Passive avoidance task The passive avoidance test was performed as previous method with modification. The apparatus consisted of a white illuminated camber and a dark camber (17 cm×13.5 cm×25 cm, respectively) in trough-shape. In training trial, following 180 s habitation each mouse was put into the light chamber to explore with the door opened for 300 s. When it entered the dark chamber, a 0.5 mA electric foot shock (5 s) was delivered. Then mouse was removed from the dark chamber and put back to its home cage. 24 h later, the consolidation trial was performed in the same way as training, and latency to enter the dark chamber and error time were recorded. The latency was recorded up to 300 s. 2.5 Brain sample preparation After the last behavioral test, all mice were anesthetized and decapitated, and their brains were removed rapidly. 3 whole mice brains in each group was fixed in 10% formalin at 4 °C for histopathological and TUNEL assays. The hippocampus and cortex of the other mice were isolated out on ice respectively, and stored at -80 °C. For the biochemical detection, 6 hippocampus and 6 cortex of each group were sonicated with cold normal saline (1:10). The homogenate was centrifuged at 3,500 rpm (10 min, 4 °C) and the supernatants was collected for assay. The left hippocampus in each group were used for protein analysis.

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2.6 Histopathological study and TUNEL assay For histopathology, the brains were immersed and fixed in 4% paraformaldehyde (PFA) for 48 h, and then embedded in paraffin. Serial (neighboring) sections of 5 μm were cut, which represented distinct antero-posterior levels of the hippocampus. After stained with hematoxylin and eosin, sections were observed under a light microscope [37]. TUNEL assay of apoptotic cells was also performed in formalin-fixed, paraffin-embedded brain tissue sections. According to the manufacturer's protocol (Beyotime Institute of Biotechnology, Jiangsu, China). Sections were observed under a confocal microscopy (Olympus, Japan). The apoptosis rate of each group was also calculated to evaluate the effect of LI.

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2.7 Biochemical parameter assay of the hippocampus The acetylcholinesterase (AChE), superoxide dismutase (SOD), glutathione peroxidase (GPX) activities and content of malondialdehyde (MDA) were measured using commercially available assay kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) respectively, according to the manufacturer's protocols. Briefly, the AChE activity was determined using thiol agent to form trinitrobenzene at 412 nm. The measurement of SOD activity was based on its ability to inhibit the oxidation by superoxide anion free radical produced from the xanthine–xanthine oxidase system. GPX activity was assayed by measuring the decline of triphosphopyridine nucleotide (NADPH) in a coupled system at 340 nm. Levels of MDA were measured using the thiobarbituric acid reactive substance (TBARS) method as previously described in 535 nm.

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2.8 Western blotting Total proteins from hippocampus tissues were isolated from according to the instructions of the protein extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China), and protein concentrations were determined using a BCA protein assay kit (Bioworld, USA). Protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking in a 5% nonfat dry milk-Tris buffered saline with Tween-20 (TBST) for 3 hours, the membranes were incubated overnight at 4 °C with different antibodies, including anti-HO-1 (1:1000, Abcam, USA), anti-Nrf2 (1:1000, Abcam, USA), anti-cleaved caspase-9 (1:1000, Cell Signaling, USA), anti-cleaved caspase-3 (1:1000, Cell Signaling, USA) and anti-β-actin (1:5000, Bioworld, USA). After rinsing three times with TBST, the membranes were incubated for 2 h with a horseradish peroxidase-conjugated secondary antibody at room temperature, then visualized with chemiluminescence reagents using an ECL kit (Beyotime Institute of Biotechnology, Jiangsu, China). The intensities of bands was scanned and determined using a Quantity One Software (Bio-Rad, Hercules, USA). 2.9 Statistical analysis Data were analyzed using the SPSS 17.0 software package and expressed as means  standard error mean (SEM). Indexes in acquisition of MWM trials including escape latency and escape rate test were analyzed by repeated-measure two-way ANOVA. Data of the other determinations were analyzed by one-way ANOVA followed by

ACCEPTED MANUSCRIPT Tukey’s post hoc test among groups. For all statistical tests, P< 0.05 was regarded as significant. 3. Results 3.1 LI attenuated the Aβ1-40 induced cognitive impairment in mice

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3.1.1 LI improved Aβ1-40 induced spatial memory impairment in MWM test In training phase, like normal mice, sham-operated mice rapidly learned the location of platform, indicated by lower escape latency and higher escape rate. As shown in Fig. 2A, longer escape latency was observed in Aβ1-40 treated mice on all testing days (P< 0.05,). However, LI (100 mg/kg) treatment could significantly shorten this escape latency prolongation form day 3 to day 5 (P<0.05). For the escape rate (Fig. 2B), Aβ1-40 treated mice maintained lower escape rates than sham mice with from the first day (P<0.05). The escape rate of model mice was elevated by LI (100 mg/kg) treatment significantly on the first four training days (P<0.05), and LI (50 mg/kg) also showed effect in the fourth day (P<0.05). In probing trial which aimed to determine whether they remembered the platform position. The time in target quadrant (P<0.05, Fig. 2C) and the crossing number (P<0.05, Fig. 2D) in Aβ1-40 treated mice were decreased significantly compared with sham group. However, LI treatment (100 mg/kg) increased the time in target quadrant and crossing number of model group with significance (P<0.05)

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Fig. 2. Effect of LI on Aβ1-40 induced cognitive deficits. In MWM test, escape latency to find platform (A) and escape rate (B) were measured for five consecutive training days. Time spent in the target quadrant (C) and crossing number (D) were recorded during the probe trial. In step-through passive avoidance tests, error times (E) and time in dark chamber (F) were detected in consolidation test. Values are presented as mean ± SEM (𝑛=10-12 in each group). #𝑃< 0.05 compared with the sham group, *𝑃< 0.05 compared with the Aβ1-40 treated group.

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3.1.2 LI improved Aβ1-40 induced cognitive deficits in step-through passive avoidance task In step-though test, the Aβ1-40 induced cognitive deficits of mice were indicated as longer time in dark chamber (P<0.05) and more error times than that of sham-operated mice. LI (100 mg/kg) treatment decreased the prolonged time in dark chamber of model mice significantly (Fig. 2E). Though there were no significance, LI also showed the tendency to decline error times (Fig. 2F). 3.1.3 The locomotor activities Open-field test was performed to evaluate the locomotor activity. As shown in Fig. 3, there were no significant changes in total distance among all groups, though LI (100 mg/kg) and had shorter total distance compared with model group. The results guaranteed that locomotor activity change had no interference in the evaluation of

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Fig. 3. Effect of LI on locomotor activities of mice expressed by total distance travelled. Data are expressed as means ± SEM. n=10-12 in each group.

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3.2 Effects of LI on pathological damage in the Aβ1-40 treated mice hippocampus Hippocampus plays an important role in learning and memory. The damage or apoptosis of neuronal cells in hippocampus, especially dentate gyrus, were observed with Aβ aggregated. To explore the effect of LI on the Aβ induced injuries, we used histological methods and the TUNEL assay to evaluate. Compared to the sham-operated group, the hippocampus of Aβ1-40 injected mice displayed pathological features including nucleoli ambiguity and circumscription (between nuclei and cytoplasm) obfuscation. With treatment of LI, the Aβ1-40-induced changes was alleviated and the pathological lesions got close to a normal range (Fig. 4A). Meanwhile, compared with sham group, a significant increased number of TUNEL positive nuclei was observed (59.3%, P<0.01) when Aβ1-40 exposure. LI treatment showed a marked inhibitory effect on cell apoptosis of Aβ1-40 group (P<0.01, Fig. 4B).

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Fig. 4. Effects of LI on Aβ1-40 induced histological changes and apoptosis in the hippocampus of mice. Mice were treated as described in the text and examined by HE staining (A, in the first row black bar stands for 100 μm; in the second row black bar stands for 400 μm) and TUNEL assay (B, black bar stands for 40 μm). The neuronal apoptosis rate(C) was indicated by the percentage of TUNEL positive cell. Data are shown as mean ± SEM, ##P<0.01 compared with the sham group, **𝑃< 0.01 compared with Aβ1-40 treated group.

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3.3 LI attenuated the oxidative stress in the hippocampus and cortex of Aβ1-40 treated mice As shown in Fig. 5A, 5B, Aβ1-40 injection brought obvious oxidant stress to the mice brain, indicated by significant decreased SOD and GPX activities in the hippocampus and cortex (P<0.05 and P<0.01). Meanwhile, the level of MDA in model group was much higher than the sham group (P<0.05, Fig. 5C). However, the administration of LI (100 mg/kg) resulted in the significant increase of SOD and GPX activities (the hippocampus and cortex) compared with the model group. Beside, with the LI treatment (100 mg/kg), the MDA level in the cortex of Aβ1-40 treated mice was decreased close to the sham group (P<0.05). AChE activity which has close relation with the oxidative stress (Fig. 5D), was found to be increased significantly in model group compared with the sham group in the hippocampus and cortex (P<0.05). LI treatment (100 mg/kg) markedly reduced the AChE level in Aβ1-40 treated mice (P<0.05).

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Fig. 5. Effect of LI on Aβ induced oxidative stress. Mice were treated as described in the text. The activity of SOD (C), GPX (D) and level of MDA (E) in the hippocampus and cortex were measured by assay kits. The AChE activity (D) was also determined. Data are presented as the mean value ±SEM, #P<0.05, ##P<0.01, compared with control group; *P<0.05, **P<0.01, compared with Aβ treated group.

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3.4 LI depressed the Aβ1-40 induced activation of caspases in the mice hippocampus Caspase-9 and caspase-3 are known as biomarkers of oxidative stress-induced cell death which is mediated by mitochondria-dependent apoptotic pathway. The cleaved proteins are activated form and their levels in the hippocampus were detected by Western blot. As shown in Fig. 6A, the expressions of cytosolic cleaved caspase-9 and cleaved caspase-3 were increased significantly in the Aβ treated group, when compared to the sham group. However, LI treatment was shown to be effective at inhibiting activation of caspases induced by Aβ in the treatment groups. 3.5 LI activates the Nrf2/HO-1 signaling pathway in Aβ1-40 treated mice The Nrf2/HO-1 signaling pathway plays an important role in modulating oxidative stress. To investigate whether LI has effect on the signaling pathway, the expressions of Nrf2 and HO-1 were detected by Western blot. Fig. 6C showed that Aβ1-40 exposure caused a decrease in Nrf2 (60 %) and HO-1 (50%) expression compared to that of the sham group. With the treatment of LI (100 g/mL), the decrease of Nrf2 expression in Aβ1-40 treated mice was significantly reversed. Meanwhile, level of the HO-1 was reversed obviously closed to the normal level by LI.

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Fig. 6. Effects of LI on caspases activation, Nrf2 and HO-1 level in mice hippocampus. Mice were treated as described in the text. The expression of cleaved caspase-9, cleaved caspase-3(A), Nrf2 and HO-1 (C) were measured by Western blot. The density values of bands were quantified and expressed as the ratio to β-actin (B & D, n=3 per group). Values are shown as mean ± SEM., # P<0.05 compared with sham group, *𝑃< 0.05 compared with Aβ1-40 treated group.

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4. Discussion Aβ induced oxidative stress mediates apoptotic cell death, which play a vital role in the pathogenesis of cognitive deficits and neural damages in AD [38,39]. Therefore, a promising therapeutic agent should be effective to reduce oxidative stress and alleviate apoptosis. LI is a monoterpene component of essential oils, which showed bioactivities for CNS diseases [18,40]. In the present study, LI was able to ameliorate the cognitive deficits of mice induced by Aβ1-40, and its neuroprotective effect might be related to the role against Aβ induced oxidative stress and apoptosis depending on Nrf2/HO-1 signaling. It is well known that Aβ accumulation in the brain leads to the production of senile plaques, and its neurotoxicity disrupt the synaptic transmission between neurons [41]. During the progression of AD, it is also noteworthy that Aβ induced oxidative stress may play a key role. Except for aggravating the neurotoxicity of Aβ alternatively, the overproduced ROS initiate disruption of mitochondrial membrane potential, lipid peroxidation, synaptic dysfunction, activation of mitochondrial apoptotic pathway and finally cell death [42]. The hippocampus is vulnerable to the series of damages, which is necessary for LTP and plays an important role in spatial memory, working memory and long-time memory. Its damages could exacerbate the cognitive deficits in AD. Cognitive impairment is one of the most typical characteristics of AD. In this study, Morris water maze tasks was used to evaluate the spatial memory ability of mice, while step-though test assess the passive avoidance ability which depends on ability to retain and recall information [43,44]. Our results demonstrated that intra-hippocampal injection of Aβ1-40 damaged the normal performance of mice. However, compared with the model group, treatment of LI (100 mg/kg) effectively shortened escape

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latencies, increased escape rate, extended time spend exploring the previous platform in MWM test, and significantly reduced error times and the time in dark chamber in step-though test. The cognitive improvement effect of LI was dose-dependent and consistent with its inhibition of AChE activity in the hippocampus. Considering LI did not influence the locomotor activity of mice, it suggests that the cognitive effect of LI is to be mnemonic in origin, rather not induced by sensorimotor effects. Apoptosis is closely related to the memory and cognitive decline in AD (Obulesu and Lakshmi, 2014), which is known as programmed cell death involving mitochondrial dysfunction, caspase activation and fragmentation [45]. In the present study, the anti-apoptotic efficiency of LI was determined by staining and western blot. Results demonstrated that the hippocampal injury and cell apoptosis rate in model mice were markedly reversed by LI. Meanwhile, the Aβ1-40 activated cleaved caspase-3 and cleaved caspase-9 expression were also decreased with LI treatment. It suggested the neuroprotective effects of LI against apoptosis by inhibiting activation of caspase. Previous evidences indicated that oxidative stress injury generated in the progression of AD, contributes to apoptotic neuronal cell death. Oxidative stress can cause an imbalance between ROS production and removal in mitochondria, resulting in biological molecules damage and apoptotic cell death ultimately. To suppress oxidative stress could be beneficial for neurons in AD. Our results demonstrated that LI enhanced activity of antioxidant enzyme SOD, GPX and inhibited production of oxidative stress indicator MDA in the hippocampus. These suggested that the LI could attenuate the Aβ induced oxidative stress effectively. Endogenous antioxidant enzymes would be developed to alleviate oxidative insults in AD [46]. Overexpression of HO-1 or pharmacological induction of HO is able to confer an adaptive survival response against oxidative insults [47]. In the study, LI could significantly enhance the expression of HO-1 in the hippocampus of Aβ1-40 treated AD mice. The induction of HO-1 gene and many other antioxidant responses are primarily regulated by Nrf2 [48]. Our results showed that the inhibitory effect of Aβ on Nrf2 expression was reversed markedly by treatment with LI. It seems that neuroprotective effect of LI against oxidative stress might rely on the Nrf2/HO-1 pathway. 5. Conclusion In summary, our study provided evidence that LI effectively improved the cognitive impairment induced by Aβ1-40 in mice, and its neuroprotective effects of LI may be mediated by alleviation of apoptosis and oxidative stress induced by Aβ, depending on activation of Nrf2/HO-1 pathway. With previous reports and our results, we could assume that LI has a potential to be developed as a neuroprotective drug for AD therapy. Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments This work was supported by the Xinjiang Science and Technology Aid Projects

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Graphical abstract