Curcumin attenuates cognitive impairment by enhancing autophagy in chemotherapy

Journal Pre-proof Curcumin attenuates cognitive impairment by enhancing autophagy in chemotherapy

Li-Tao Yi, Shu-Qi Dong, Shuang-Shuang Wang, Min Chen, Cheng-Fu Li, Di Geng, Ji-Xiao Zhu, Qing Liu, Jie Cheng PII:

S0969-9961(19)30390-0

DOI:

https://doi.org/10.1016/j.nbd.2019.104715

Reference:

YNBDI 104715

To appear in:

Neurobiology of Disease

Received date:

3 August 2019

Revised date:

13 November 2019

Accepted date:

13 December 2019

Please cite this article as: L.-T. Yi, S.-Q. Dong, S.-S. Wang, et al., Curcumin attenuates cognitive impairment by enhancing autophagy in chemotherapy, Neurobiology of Disease(2019), https://doi.org/10.1016/j.nbd.2019.104715

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© 2019 Published by Elsevier.

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Curcumin attenuates cognitive impairment by enhancing autophagy in chemotherapy Li-Tao Yia,b,c,* , Shu-Qi Donga, Shuang-Shuang Wanga, Min Chena, Cheng-Fu Lid, Di Genga,b,c, Ji-Xiao Zhue, Qing Liua,b,c, Jie Chenga,b,c

a

Department of Chemical and Pharmaceutical Engineering, College of Chemical

Institute of Pharmaceutical Engineering, Huaqiao University, Xiamen 361021, Fujian,

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b

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Engineering, Huaqiao University, Xiamen 361021, Fujian, People's Republic of China

Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, Xiamen,

d

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361021, Fujian, People's Republic of China

Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361009, Fujian, People's

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Republic of China

Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine ，

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c

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People's Republic of China

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Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, People's Republic of China

*Corresponding authors: Dr. Li- Tao Yi. Tel./Fax.: 86-592-6162302; E-mail address: [email protected]

Running title: Enhancement of autophagy by curcumin

Abstract 1

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Cisplatin, a commonly used chemotherapy drug, can increase the survival rate of cancer patients. However, it often causes various side effects, including neuronal deficit- induced cognitive impairment. Considering that curcumin is effective in neuronal protection, the action of curcumin on cognitive improvement was evaluated in cisplatin-treated C57BL/6

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mice in the present study. Our results first showed that curcumin restored impaired cognitive

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behaviors. Consistent with this, neurogenesis and synaptogenesis were improved by curcumin.

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In addition, cisplatin- induced dysfunction of apoptosis-related proteins was partly reversed by

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curcumin. Moreover, cisplatin- induced autophagy was enhanced by curcumin. Our results

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also indicated that cisplatin induced autophagy through the endoplasmic reticulum (ER) stress-mediated ATF4-Akt- mTOR signaling pathway. Curcumin activated AMPK-JNK

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signaling, which mediated both mTOR inhibition and Bcl-2 upregulation and in turn enhanced

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autophagy and suppressed apoptosis, respectively. In contrast, pretreatment with the

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autophagy inhibitor 3- methyladenine (3-MA) completely abolished the effects of curcumin on cognitive improvement and improved neurogenesis, synaptogenesis and autophagy. Our results show that cognitive improvement ind uced by curcumin during chemotherapy is mediated by the enhancement of hippocampal autophagy.

Keywords: Cisplatin; Autophagy; Cognition; Neurogenesis; Curcumin.

1. Introduction 2

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Over the past few decades, chemotherapeutic agents have significantly increased survival rates in the treatment of cancer (de Moor et al., 2013). However, chemotherapy causes undesirable and even debilitating side effects, such as neuropathy, fatigue, motivational deficit and cognitive impairment. Chemotherapy- induced cognitive impairment results from

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alterations in the functional network architecture of the brain, decreasing gray matter volume

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in frontal, temporal, and cerebellar regions in breast cancer patients (Piccirillo et al., 2015).

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Cisplatin, a widely used chemotherapy drug, is part of the standard treatment for a myriad of

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solid tumors. Despite its efficacy in treating numerous malignancies, the use of cisplatin has

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been limited due to the associated cognitive impairments, which severely affect quality of life and hinder occupational goals(O'Farrell et al., 2013; Selamat et al., 2014; Vardy and Tannock,

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2007).

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Curcumin is a natural product derived from the root of the plant Curcuma longa and has

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attracted much attention in drug development. There is evidence suggesting that curcumin is active against a wide range of human diseases, including diabetes, obesity, Alzheimer ’s disease, Parkinson’s disease, and neurological and psychiatric disorders. Curcumin can attenuate cognitive deficits in clinical studies and appears to be safe, well tolerated, and efficacious (Cox et al., 2015; Mythri and Bharath, 2012). Furthermore, curcumin can reverse cognitive impairments induced by chronic mild stress and induce the proliferation of astrocytes in the hippocampus and striatum (Esatbeyoglu et al., 2012; Gupta et al., 2012). Epidemiological findings also suggest that long-term use of curcumin can ameliorate cognitive dysfunction in elderly people(Ng et al., 2006). 3

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Recently, mitochondria, the energy factories of the cell, have received increasing attention as a target for the treatment of many diseases. Damaged mitochondria can trigger inflammation, apoptosis and autophagy, which are related to cognitive impairment(Ejlerskov et al., 2015). The side effects induced by cisplatin are associated with mitochondrial gene alteration(Vichaya et al., 2016). Mitochondrial dysfunction in neurons is an underlying cause

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of cisplatin- induced cognitive impairment(Chiu et al., 2017). In addition, a previous study

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showed that curcumin decreased cisplatin- induced toxicity in the brain through the

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enhancement of mitochondrial lipid peroxidation levels and protein carbonyl content(Waseem

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and Parvez, 2013). However, little is known about the autophagy-related mechanisms in

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chemotherapy-induced cognitive impairment.

During chemotherapy, cisplatin could promote both apoptosis and autophagy in ce lls

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(Pisanu et al., 2017). This induced apoptosis plays a dominant role in inhibiting cell

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proliferation. Autophagy is inhibited and thus cannot provides sufficient protein turnover,

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which might restrict neurogenesis. Therefore, in the present study, we established a mouse model of cisplatin- induced cognitive impairment and assessed the cognitive changes after curcumin administration. Moreover, to evaluate whether autophagy enhancement was required for the curcumin- induced cognitive improvement of mice receiving chemotherapy, mice were pretreated with the autophagy inhibitor 3- methyladenine (3-MA) prior to curcumin administration. Given that autophagy and apoptosis usually have opposing functional physiological roles, it is rational to speculate that they produce complementary effects to modulate the homeostatic balance of neurogenesis and synaptogenesis. In fact, there is accumulating evidence that autophagy is required for adult neurogenesis (Wu et al., 2016; 4

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Yazdankhah et al., 2014). In addition, induction of synaptic proteins and synaptic plasticity is highly dependent on protein turnover during autophagy under abnormal conditions (Nikoletopoulou et al., 2017). Therefore, an adequate protein supply for cellular components (the complex biomolecules and structures of cells) from autophagy may be one of the best coping strategies for neuronal activity and function. In the present study, our results also

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suggested that an increased number of impaired cells were degraded and that their cellular

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components were recycled for neurogenesis and synaptogenesis during curcumin treatment.

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2. Materials and methods

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2.1. Animals

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Male SPF C57BL/6 mice (24±2 g; 8 weeks old) were purchased from Shanghai SLAC

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Animal Center (Shanghai, PR China) and were housed in barrier facilities of Animal center in Huaqiao University. Five animals were housed per cage (320×180×160 mm) with a normal 12-h/12-h light/dark cycle with the lights on at 07:00 a.m. The animals were allowed one week to acclimatize to the housing conditions before beginning the experiments. Ambient temperature and relative humidity were maintained at 22±2 °C and 55±5%, respectively, and these animals were given standard chow and water ad libitum for the duration of the study. All procedures were approved by the Committee on Animal Research of Huaqiao University (HQU-CE/2017003) and performed in accordance with the published guidelines of the China Council on Animal Care (Regulations for the Administration of Affairs Concerning 5

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Experimental Animals, approved by the State Council on 31 October 1988 and promulgated by Decree No. 2 of the State Science and Technology Commis sion on 14 November 1988). All the behavioral tests were performed between 9:00 a.m. and 1:00 p.m. during the light cycle to avoid the circadian-related fluctuation.

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2.2. Reagents

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Curcumin (SC-200509), 3-MA (SC-205596), DAPI (sc-3598) and anti-JNK (sc-7345)

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were purchased from Santa Cruz Biotech (Santa Cruz, USA). Cisplatin (HY-17394) was

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purchased from MedChem Express (New Jersey, USA). Anti- LC3B (L7543) and anti-β-actin (A3854) antibodies were purchased from Sigma (St. Louis, USA). The primary antibodies (ab56416),

anti-Bcl-2

(ab692),

anti- Bax (ab32503),

anti-Bim

(ab32158),

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anti-p62

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anti-doublecortin (ab18723), anti-Iba1 (ab5076), anti-p-AMPK (ab133448), anti-AMPK

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(ab80039), and anti-ATF4 (ab184909) and the secondary antibodies donkey anti- mouse lgG (ab150108) and donkey anti-goat lgG (ab175704) were purchased from Abcam Biotech (London, UK). The primary antibodies anti-p-Akt (4060), anti-Akt (4691), anti-p-mTOR (5536), anti- mTOR (2983), and anti-p-JNK (9255) were purchased from Cell Signaling Technology (Beverly, USA). The donkey anti-rabbit lgG (711-545-152) was purchased from Jackson ImmunoResearch Laboratories (West Grove, USA).

2.3. Drug administration

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The mice were divided into four groups by computer based randomization (n=15): the control- vehicle group, the control-curcumin group, the cisplatin-vehicle group, and the cisplatin-curcumin group. To further determine whether curcumin improved cognitive impairment by promoting autophagy, we conducted follow-up experiments. The mice were randomly divided into five groups by computer based randomization (n=15): the

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control- vehicle group, the cisplatin- vehicle group, the cisplatin-curcumin group, the

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3-MA-cisplatin group and the 3-MA-cisplatin-curcumin group. Cisplatin (2.3 mg/kg,

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dissolved in PBS) or vehicle (PBS) was intraperitoneally (i.p.) injected for 3 cycles consisting

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of 5 daily injections followed by 5 days of rest according to a previous study (Zhou et al.,

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2016). The total cumulative dose of cisplatin was 34.5mg/kg. Curcumin was dissolved in PBS containing 0.5% carboxymethylcellulose. PBS containing 0.5% carboxymethylcellulose was

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used as a vehicle. Curcumin was orally administered at a dose of 100 mg/kg 1 h prior to

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cisplatin treatment. Intracerebral ventricle (icv, 0.6 mm posterior, 1.5 mm right lateral, 1.5

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mm ventral to the bregma) injection (5 μL) of 3-MA (dissolved in artificial cerebrospinal fluid, at a dosage of 100 nmol/day) or vehicle (artificial cerebrospinal fluid) was performed 1 h prior to curcumin and cisplatin treatment. The ICV injection was performed under light isoflurane inhalation. The dose of curcumin was selected as 100 mg/kg curcumin since that alleviated cognitive deficits in mice (Wu et al., 2017). In the present study, we mainly evaluated cognitive impairments including deficits in exploration, recognition memory and spatial memory in cisplatin- induced mice.

2.4. Open-field test 7

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Open field test was used to assess exploration between 9:00 a.m. and 1:00 p.m. during the light cycle to avoid the circadian-related fluctuation. All the animals are transferred to the testing room in their home cages and left there to habituate for 60 min. Mice were placed in the corner of a wooden box (40 ×40 × 30 cm, Jiliang Software Technology, Shanghai, PR

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China), and the number of instances of crossing (squares crossed with all paws, assessment of

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the drive to explore a novel environment) and rearing (raising the forepaws, assessment of the

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presence of potential environmental dangers) was recorded in a 5 min session. After each test

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session, 70% ethanol was used to clean the box thoroughly. The test session was recorded by

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2.5. Novel object recognition test

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video and was scored by an observer blind to treatment.

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Novel object recognition test was used to assess recognition memory and was performed in the experimental arena (44× 40×40 cm, Jiliang Software Technology, Shanghai, PR China) in a sound-attenuated room with dimmed illumination. The test was performed between 9:00 a.m. and 1:00 p.m. during the light cycle to avoid the circadian-related fluctuation. All the animals are transferred to the testing room in their home cages and left there to habituate for 60 min. The experiment was divided into three parts: adaptation period, familiarization period and test period. Mice were individually habituated to the arena for 5 min during three consecutive days after the last cisplatin injection. On the fourth day, two identical objects (Colorful cube Lego bricks) were placed onto the floor of the arena, and each mouse was 8

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allowed to explore the arena for 5 min. The fifth day was the test period; one object was replaced with a new object of a different color and shape (Grey pyramid Lego bricks) in the same position, and then, mice were placed into the arena for 5 min to explore. The recognition index was calculated as the exploration time of the novel obje ct divided by the total exploration time of both objects. After each test session, 70% ethanol was used to clean the

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arena thoroughly. The test session was recorded by video and was scored by an observer blind

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to treatment.

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2.6. Morris Water Maze test

Morris Water Maze test was used to assess spatial memory between 9:00 a.m. and 1:00

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p.m. during the light cycle to avoid the circadian-related fluctuation. All the animals are

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transferred to the testing room in their home cages and left there to habituate for 60 min. Mice

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were placed in an apparatus (Jiliang Software Technology, Shanghai, PR China) consisting of a circular pool (120 cm diameter and 60 cm deep) filled with water to a depth of 30 cm. Visual cues (posters, door, shelf and table) outside the pool were held constant. The Morris water maze test lasted five days, including 4 days of place navigation training and a spatial probe test on day 5. A platform (10 cm in diameter) was submerged 1 cm under the water in the midpoint of one of four identical quadrants. For the first 4 days, the mouse was placed in the water in the middle of the quadrant and allowed to freely explore for 90 seconds. The time when the mouse successfully landed on the hidden underwater platform, i.e., the escape latency, was recorded. Boarding the hidden platform for 10 seconds within 90 seconds was 9

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regarded as a successful boarding of the platform; if the mouse failed to successfully board the platform, it was manually guided to the platform for 10 seconds, and the latency was recorded as 90 seconds (Xiong et al., 2008). The fifth day was a space exploration experiment: the underwater quadrant was removed from the target quadrant. The mice were placed in the pool at the midpoint of the contralateral quadrant, and the number of mice crossing the

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original hidden platform position within 90 seconds was recorded. The test session was

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recorded by video and was scored by an observer blind to treatment.

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2.7. Autophagy Assays

Three mice were randomly selected for autophagy assays. Mice were immediately

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anesthetized with chloral hydrate (0.35 g/kg) and then sacrificed by intracardial perfusion

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with PBS followed by an ice-cold 4% polyoxymethylene mixture. The hippocampus was

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carefully dissected from each hemisphere, and a section approximately 1 mm3 was cut and then quickly fixed in 2.5% glutaraldehyde for 4 h. The hippocampal tissue was washed twice for 15 min with 0.1 M phosphate buffer, fixed with 1% osmic acid for 1 h, flushed with 0.1 M phosphate buffer, acetone dehydrated three times, soaked in epoxy resin 618 for 2-3 h after embedding, and finally subjected to oven polymerization. Thin sections (50 nm) were cut on a Reichert Ultracut E microtome and stained with uranyl acetate and lead citrate for observation at 80 KV under a JEM-1010 transmission electron microscope. Four sections from each mouse were randomly chosen for observation.

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2.8. Western blotting

Six mice were randomly selected for hippocampus dissection. The tissues of hippocampus in each hemisphere were carefully dissected and frozen in liquid nitrogen. One hemisphere was stored for PCR and the other hemisphere was stored for western blotting.

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Four from the six stored hemispheres were used for protein detection. Briefly, hippocampus

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samples were homogenized in lysis buffer and incubated on ice for 15 min. The homogenates

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were centrifuged at 12,000 × g for 5 min at 4 °C, and the supernatants were collected. The

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protein concentration was determined by a BCA assay. The proteins were separated by

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SDS-PAGE and transferred to a PVDF membrane. Following blocking in 5% skimmed milk powder in TBST at room temperature for 1 h, the membranes were incubated with the

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appropriate primary antibodies at 4 °C overnight (anti- LC3B: 1:1000, anti-p62: 1:2000,

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anti-Bcl-2: 1:500, anti- Bax: 1:2000, anti-Bim: 1:1000, and anti-β-actin: 1:5000). After the

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membranes were washed three times with TBST, the membranes were incubated with an HRP- labeled secondary antibody (1:2000). The blots were again washed three times with TBST buffer, and the immunoreactive bands were detected

using an enhanced

chemiluminescence method. The relative intensity of target protein normalized to β-actin was analyzed by ImageJ software.

2.9. Immunofluorescence

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Three mice were randomly selected and were immediately anesthetized with chloral hydrate (0.35 g/kg) and then sacrificed by intracardial perfusion with heparinized 0.9% saline followed by ice-cold 4% paraformaldehyde. The brains were removed, postfixed with 4% paraformaldehyde for 24 h and incubated with 10%, 20%, and 30% sucrose solution at 4 °C until the brain lost buoyancy Then, the brain was embedded in OCT and stored at -80 °C.

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Briefly, 15-μm-thick sections were incubated with a blocking buffer (1× PBS/5% normal goat

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serum/0.3% Triton X-100) for 1 h. Then, the sections were incubated in 4% PFA for 48 h at

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4 °C. After washing with PBS, the sections were immersed in 0.3% PBST (Triton-X 100) for

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10 min. The sections were incubated with the following primary antibodies overnight in PBS

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at a 1:500 dilution factor at 4 °C: doublecortin (DCX), p62 and LC3B. After washing with PBS, DAPI was used for nuclear staining. Later, the sections were observed under a Leica

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(Wetzlar, Germany) TCS SP5 confocal microscope. Examination was performed in dentate

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gyrus. Four images were randomly chosen for each mouse and their average was used for

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analysis. Every DCX+, p62+ and LC3B+ cells within the image was counted. The quantitative evaluation was performed by an observer blind to treatment.

2.10. Golgi staining

Three mice were randomly selected for Golgi staining. The brains were removed and stored for 14 days in Golgi-Cox solution, followed by 3 days in 30% sucrose solution. Coronal sections 50 mm thick of regions to be studied were obtained using a vibratome. Sections were then treated with ammonium hydroxide for 30 min, followed by 30 min in 12

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Kodak Film Fixer, and finally rinsed with distilled water, dehydrated and mounted in a resinous medium. During morphological analysis, three pyramidal neurons in CA3 subregion of each hemisphere were selected on three slices at A/P levels (Bregma -2.06 mm, -2.46 mm, -2.80 mm approximately) according to the brain diagram of mouse (Franklin and Paxinos, 2008). All protrusions were considered as spines only if they were in direct continuity with

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the dendritic shaft, so the numbers of branch from the dendritic trees were quantified tracing.

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The dendritic spine density in the CA3 subregion of the hippocampus was expressed as the

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number of spines per 10 micrometer of dendritic length. The quantitative evaluation was

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performed by an observer blind to treatment. Hippocampus CA3 subregion was selected for

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analysis because the number of dendritic spines in CA3 subregion are the most vulnerable in cognitive impairment mice induced by stress and corticosterone, as compared with that in

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2.11. Statistical analyses

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CA1 and DG subregions (Sousa et al., 2000).

All data are expressed as the mean ± SEM. Data were verified as normally distributed using the Kolmogorov-Smirnov test prior to ANOVA. Data were analyzed using a two-way ANOVA followed by Tukey's post hoc test to compare differences between any two groups using GraphPad Prism 7. A value of P<0.05 was considered statistically significant.

3. Results

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3.1. The effects of curcumin on mouse behavior

As shown in Fig. 1A, B, cisplatin significantly decreased the crossing number [p<0.01] and rearing number [p<0.05] in the open- field test. Curcumin significantly increased the crossing number [p<0.01] and rearing number [p<0.05] in mice treated with cisplatin.

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Additionally, neither crossing number nor rearing number was altered by curcumin in the

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control groups.

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As shown in Fig. 1C, cisplatin significantly decreased the recognition index [p<0.01] in

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mice. Curcumin significantly increased the recognition index induced by cisplatin [p<0.05].

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Additionally, curcumin did not significantly alter the recognition index in the control animals. The effects of curcumin on spatial learning and memory were evaluated in the Morris

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water maze test. During the navigation training, the escape latency was defined as the time

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spent by the mice to find the hidden platform. As shown in Fig. 1D, from the second day to

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the fourth day, the mice in the cisplatin group required more time to find the hidden platform as compared with that in the control group [Day 2: p<0.01, Day 3: p<0.01, Day 4: p<0.01]. Compared to the mice in the cisplatin- vehicle group, the mice in the cisplatin-curcumin group required a shorter amount of time to find the platform from the third and fourth days [p<0.01, p<0.01, respectively]. Additionally, compared to the control-vehicle group, no significant changes in escape latency were observed in the control-curcumin group. As shown in Fig. 1E, cisplatin significantly decreased the crossing number [p<0.01], which was reversed by curcumin treatment [p<0.05]. Consistently, curcumin did not significantly alter the crossing number in the control group. 14

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3.2. The effects of curcumin on hippocampal neurogenesis and synaptogenesis in mice receiving chemotherapy

As shown in Fig. 2, cisplatin induced the inhibition of DCX positive cells [p<0.01] and

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dendritic spine density [p<0.01] in the hippocampus. The post hoc test revealed that curcumin

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significantly increased the number of hippocampal DCX positive cells [p<0.05] and dendritic

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spine density [p<0.01] in mice treated with cisplatin. However, the number of Iba1 positive

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cells was not changed by either cisplatin or curcumin.

3.3. The effects of curcumin on hippocampal apoptosis-related proteins in mice receiving

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chemotherapy

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As shown in Fig. 3, western blotting confirmed that cisplatin induced the levels of apoptosis related proteins. Curcumin significantly increased the hippocampal levels of the antiapoptotic Bcl-2 [p<0.05] in mice treated with cisplatin [p<0.01]. In addition, the levels of Bax and Bim, two proapoptotic members, which were increased by cisplatin [p<0.01, p<0.01, respectively], were inhibited by the administration of curcumin [p<0.05, p<0.05, respectively].

3.4. The effects of curcumin on hippocampal autophagy in mice

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As shown in Fig. 4A, transmission electron microscopy revealed that cisplatin increased the formation of autophagic vesicles in the hippocampal neurons. However, compared with the control-cisplatin group, the formation of autophagic vesicles was further increased in the cisplatin-curcumin group. In addition, compared with the control- vehicle group, no significant changes in autophagic vesicle number were observed in the control-curcumin group.

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Then, western blotting confirmed that cisplatin induced the levels of autophagy related

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proteins. Fig. 4B shows that curcumin significantly reversed the reduction of hippocampal

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LC3BII/LC3BI ratio [p<0.05] induced by cisplatin [p<0.05]. Consistently, as shown in Fig.

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4C, curcumin [p<0.05] also reversed the increase in p62 levels induced by cisplatin [p<0.01].

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Additionally, the hippocampal LC3BII/LC3BI ratio and p62 levels were unchanged by curcumin in the control group.

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In parallel to protein levels, as shown in Fig. 4D, E, the immunofluorescent images

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showed that cisplatin induced a decrease of LC3B [p<0.01] and increase of p62 [p<0.01] in

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mice, which were reversed by curcumin [LC3B: p<0.05; p62: p<0.05].

3.5. The effects of curcumin on the Akt-mTOR and AMPK-JNK signaling pathways

As the endoplasmic reticulum (ER) stress signaling pathway mediates the regulation of autophagy, we thus analyzed the proteins related to ER stress. As shown in Fig. 5, cisplatin caused the upregulation of ATF4 [p<0.05], which blocked the phosphorylation of its downstream effectors Akt [p<0.05] and mTOR [p<0.05] levels. Curcumin administration increased the phosphorylation of AMPK and JNK [p<0.05, p<0.01, respectively], which 16

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further enhanced the levels of ATF4 [p<0.01] and inhibited the levels of p-Akt and p- mTOR [p<0.01, p<0.01, respectively].

3.6. The effects of 3-MA pretreatment on mouse behaviors

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As shown in Fig. 6, 3-MA pretreatment significantly blocked the curcumin- induced

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improvement of the crossing number (Fig. 6A) and rearing number (Fig. 6B) in the open- field

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test [p<0.05, p<0.05, respectively], the recognition index (Fig. 6C) in the novel object

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recognition test [p<0.05], and escape latency (Fig. 6D) [Day 3: p<0.05, Day 4: p<0.05] and

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crossing number (Fig. 6E) [p<0.05] in the Morris water maze test.

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3.7. The effects of 3-MA on hippocampal neurogenesis and synaptogenesis in mice

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As shown in Fig. 7, 3-MA abolished the effects of curcumin on the hippocampal immature neurons [p<0.01] and dendritic spines [p<0.05] in mice treated with cisplatin.

3.8. The effects of 3-MA on hippocampal apoptosis-related proteins in mice

As shown in Fig. 8, 3-MA significantly prevented the curcumin- induced increased hippocampal levels of the antiapoptotic protein Bcl-2 [p<0.05] and decreased levels of the proapoptotic proteins Bax and Bim [p<0.05, p<0.05, respectively].

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3.9. The effects of 3-MA on hippocampal neuron autophagy in mice

As shown in Fig. 9A, compared with the cisplatin-curcumin group, the number of autophagic vesicles significantly decreased

in the 3-MA-cisplatin-curcumin

group,

confirming the effects of 3-MA in the present experiment.

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The western blot indicated that pretreatment with 3-MA significantly decreased the

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hippocampal LC3BII/LC3BI levels (Fig. 9B) [p<0.01] and increased the p62 levels (Fig. 9C)

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[p<0.01] in the cisplatin-curcumin mice.

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In parallel to protein levels, as shown in Fig. 9D, the immunofluorescent images showed

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that pretreatment with 3-MA significantly blocked the increase in LC3B [p<0.01] and

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4. Discussion

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decrease in p62 [p<0.01] in cisplatin- mice treated with curcumin.

In the present study, our results not only demonstrated that curcumin could reverse the cognitive impairment induced by cisplatin but also demonstrated the effects of curcumin on autophagy during the treatment. To the best of our knowledge, this is the first study investigating the mechanism of autophagy in cognitive improvement during chemotherapy. Autophagy is a process of digestion and degradation of cellular organelles and proteins that are damaged, misfolded or aging. Generally, autophagy is active at basal levels in the brain, and it is beneficial for neurons to remain stable under normal physiological conditions. In the present study, cisplatin inhibited neurogenesis and decreased synaptogenesis in the 18

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hippocampus, which caused cognitive impairment in mice receiving chemotherapy. The results suggested that the lack of neurogenesis and synaptogenesis during cisplatin treatment was induced by excessive apoptosis, which resulted in neuronal death. This finding is consistent with previous studies showing that cisplatin promotes cell death by increasing the levels of several proapoptotic proteins and inhibits neurogenesis by decreasing the levels of

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several antiapoptotic proteins in the hippocampus (Lei et al., 2012; Lindqvist et al., 2014;

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Manohar et al., 2014; Zhang et al., 2006). In contrast, consistent with previous studies

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showing that cisplatin induced autophagy in several cancer cells(He et al., 2015; Lin and Li,

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2015; Zhang et al., 2017), our present study also indicated that cisplatin partially increased

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autophagy and thus allowed degradation and recycling of cellular components; however, this effect could not compensate for the loss by apoptosis. On the other hand, curcumin partly

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inhibited apoptosis levels by increasing Bcl-2 levels and decreasing Bax and Bim levels, and

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further promoted autophagy (evidenced by mitochondrial morphology, LC3, and p62) in the

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hippocampus, consistent with previous studies showing curcumin- induced autophagy (Aoki et al., 2007; Han et al., 2012; Jiang et al., 2013). Thus, the results of our study indicate that curcumin maintains neurogenesis and synaptogenesis by enhancing autophagy, which can mediate the improvement of cognitive function. A growing amount of evidence indicates that the ER stress signaling pathway is involved in the regulation of autophagy (Ahumada-Castro et al., 2018; Yang et al., 2018). We therefore investigated changes in the ER stress- mediated ATF4-Akt- mTOR signaling pathway in the hippocampus. The results demonstrated that cisplatin caused ER stress and then upregulated ATF4 levels, which in turn inhibited Akt- mTOR phosphorylation and finally increased 19

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autophosphorylation. In addition, activation of ER stress can also induce apoptosis. In contrast, our results showed that curcumin activated the AMPK-JNK signaling pathway, which enhanced autophagy through further inhibition of Akt- mTOR signaling and inhibited apoptosis by modulating Bcl-2 levels. These results highlight this pathway as a crucial mediator of curcumin-induced autophagy in the hippocampus during chemotherapy.

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There is evidence that inflammation and autophagy are interrelated. The autophagic

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degradation of cellular constituents eliminates dysfunctional or damaged mitochondria, thus

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counteracting cellular degeneration and dampening inflammation. A previous study indicated

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that the removal of intracellular endogenous damage-associated molecular patterns (DAMPs)

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by autophagy can reduce inflammasome and cytokine synthesis and subsequently inhibit inflammation activation(Sun et al., 2017). In contrast, inflammation can also stimulate

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autophagosome formation directly or indirectly. Autophagy proteins directly interact with

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nucleotide oligomerization domain- like receptor (NLR) domains and trigger a mechanism for

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direct NLR regulation of autophagy(Sun et al., 2017). To test whether cisplatin- induced autophagy was induced by inflammation, we measured inflammation-related gene expression in the hippocampus. However, we could not find any evidence that inflammation was induced by either cisplatin or curcumin in the hippocampus, as the expression of IL-1β, IL-6 and TNF-α was not changed (Fig.S1). Notably, these results are consistent with a recent study showing that cisplatin does not cause neuroinflammation in the brain(Chiu et al., 2017). To further confirm the independence of inflammation in cisplatin- induced cognitive impairment, we further detected microglial activation by using the same slice during neurogenesis evaluation. The results showed that although curcumin reversed the reduction of 20

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DCX-positive cells in the dentate gyrus induced by cisplatin, administration of cisplatin or curcumin did not exert any effects on the number of Iba1-positive cells. In this respect, it is a rational interpretation that the increase in autophagy induced by both cisplatin and curcumin was neuroinflammation independent. It can also be speculated that neuroinflammation is not the key factor in cisplatin-induced cognitive impairment.

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To further prove that curcumin improves cognitive impairment in cisplatin- treated mice

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by enhancing autophagy, mice were pretreated with the autophagy inhibitor 3-MA to

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investigate whether autophagy inhibition abolished the effects of curcumin on neurogenesis

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and thus affected the curcumin- induced cognitive improvement. First, we verified that 3-MA

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had no influence on the levels of Bcl-2, Bax and Bim, which was consistent with the finding that 3-MA did not interfere with the apoptotic process(Li et al., 2018; Wang et al., 2016). Next,

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we found that 3-MA completely prevented the curcumin- induced improvement in the

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cognitive-related behavioral tests. In addition, we observed that curcumin administration

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increased autophagy in the hippocampus of cisplatin- treated mice, whereas 3-MA inhibited this phenomenon. In accordance with the changes in autophagy, 3-MA also abolished the curcumin- induced enhancement of neuronal proliferation and synapse development in the hippocampus. Taken together, these results indicate that autophagy enhancement is involved in curcumin- induced cognitive recovery. Collectively, our results provide new insight into the mechanism underlying the cognitive improvement role of curcumin. Cisplatin induces autophagy through the ER stress-mediated ATF4-Akt- mTOR signaling pathway in the hippocampus. Curcumin activates AMPK-JNK signaling, which mediates both Bcl-2 upregulation and mTOR inhibition and in 21

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turn suppresses apoptosis and enhances autophagy, respectively (Fig. 10). The enhancement of autophagy in turn promotes neurogenesis and synaptogenesis in the hippocampus, which can explain the cognitive improvement. The findings of this study imply a new strategy of autophagy enhancement for alleviating side effects during chemotherapy and provide information on the clinical application of curcumin in cisplatin- induced learning and memory

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impairment.

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Acknowledgement

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This work was supported by Huaqiao University [grant number ZQN-PY218]; the Outstanding Youth Scientific Research Training Program in Colleges and Universities of

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Fujian Province [grant number JA14015]; and the Program for Innovative Research Team in

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Science and Technology in Fujian Province University. We would like to thank Dr. Shi- Bin

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Wang for the help during the experimental design, and thank Instrumental Analysis Center of Huaqiao University for the help of confocal testing.

Author contributions

LY designed the experiments; LY, SD, SW and MC performed the research; CL, DG and JZ assisted in animal models and data analysis; JZ, QL and JC contributed to the study design; and LY and SD prepared and revised the manuscript. All authors approved the final paper.

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Conflict of interest

The authors declared no conflict of interest.

References:

f

Ahumada-Castro, U., Silva-Pavez, E., Lovy, A., Pardo, E., Molgo, J., Cardenas, C., 2018.

oo

MTOR- independent autophagy induced by interrupted endoplasmic reticulum- mitochondrial

pr

Ca(2+) communication: a dead end in cancer cells. Autophagy, 1-4.

e-

Aoki, H., Takada, Y., Kondo, S., Sawaya, R., Aggarwal, B.B., Kondo, Y., 2007. Evidence that

Pr

curcumin suppresses the growth of malignant gliomas in vitro a nd in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways.

al

Molecular pharmacology 72, 29-39.

rn

Chiu, G.S., Maj, M.A., Rizvi, S., Dantzer, R., Vichaya, E.G., Laumet, G., Kavelaars, A.,

Jo u

Heijnen, C.J., 2017. Pifithrin- mu Prevents Cisplatin-Induced Chemobrain by Preserving Neuronal Mitochondrial Function. Cancer Res 77, 742-752. Cox, K.H., Pipingas, A., Scholey, A.B., 2015. Investigation of the effects of solid lipid curcumin on cognition and mood in a healthy older population. J Psychopharmacol 29, 642-651. de Moor, J.S., Mariotto, A.B., Parry, C., Alfano, C.M., Padgett, L., Kent, E.E., Forsythe, L., Scoppa, S., Hachey, M., Rowland, J.H., 2013. Cancer survivors in the United States: prevalence across the survivorship trajectory and implications for care. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, 23

Journal Pre-proof

cosponsored by the American Society of Preventive Oncology 22, 561-570. Ejlerskov, P., Hultberg, J.G., Wang, J., Carlsson, R., Ambjorn, M., Kuss, M., Liu, Y., Porcu, G., Kolkova, K., Friis Rundsten, C., Ruscher, K., Pakkenberg, B., Goldmann, T., Loreth, D., Prinz, M., Rubinsztein, D.C., Issazadeh-Navikas, S., 2015. Lack of Neuronal IFN-beta-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia. Cell 163, 324-339.

f

Esatbeyoglu, T., Huebbe, P., Ernst, I.M., Chin, D., Wagner, A.E., Rimbach, G., 2012.

oo

Curcumin--from molecule to biological function. Angewandte Chemie 51, 5308-5332.

pr

Franklin, K.B., Paxinos, G., 2008. The mouse brain in stereotaxic coordinates. Academic

e-

press New York:.

Pr

Gupta, S.C., Patchva, S., Koh, W., Aggarwal, B.B., 2012. Discovery of curcumin, a component of golden spice, and its miraculous biological activities. Clinical and experimental

al

pharmacology & physiology 39, 283-299.

rn

Han, J., Pan, X.Y., Xu, Y., Xiao, Y., An, Y., Tie, L., Pan, Y., Li, X.J., 2012. Curcumin induces

Jo u

autophagy to protect vascular endothelial cell survival from oxidative stress damage. Autophagy 8, 812-825.

He, J., Yu, J.J., Xu, Q., Wang, L., Zheng, J.Z., Liu, L.Z., Jiang, B.H., 2015. Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin- induced apoptosis by inhibiting cyto-protective autophagy. Autophagy 11, 373-384. Jiang, T.F., Zhang, Y.J., Zhou, H.Y., Wang, H.M., Tian, L.P., Liu, J., Ding, J.Q., Chen, S.D., 2013. Curcumin ameliorates the neurodegenerative pathology in A53T alpha-synuclein cell model of Parkinson's disease through the downregulation of mTOR/p70S6K signaling and the recovery of macroautophagy. Journal of neuroimmune pharmacology : the official journal of 24

Journal Pre-proof

the Society on NeuroImmune Pharmacology 8, 356-369. Lei, Z.N., Liu, F., Zhang, L.M., Huang, Y.L., Sun, F.Y., 2012. Bcl-2 increases stroke- induced striatal neurogenesis in adult brains by inhibiting BMP-4 function via activation of beta-catenin signaling. Neurochem Int 61, 34-42. Li, Y., Li, X., Zhao, J., Li, L., Wang, Y., Zhang, Y., Chen, Y., Liu, W., Gao, L., 2018.

f

Midazolam Attenuates Autophagy and Apoptosis Caused by Ketamine by Decreasing

oo

Reactive Oxygen Species in the Hippocampus of Fetal Rats. Neuroscience 388, 460-471.

pr

Lin, W.M., Li, Z.G., 2015. Blockage of cisplatin- induced autophagy sensitizes cervical cancer

e-

cells to cisplatin. Genet Mol Res 14, 16905-16912.

Pr

Lindqvist, L.M., Heinlein, M., Huang, D.C., Vaux, D.L., 2014. Prosurvival Bcl-2 family members affect autophagy only indirectly, by inhibiting Bax and Bak. Proc Natl Acad Sci U S

al

A 111, 8512-8517.

rn

Manohar, S., Jamesdaniel, S., Salvi, R., 2014. Cisplatin inhibits hippocampal cell proliferation

Jo u

and alters the expression of apoptotic genes. Neurotox Res 25, 369-380. Mythri, R.B., Bharath, M.M., 2012. Curcumin: a potential neuroprotective agent in Parkinson's disease. Current pharmaceutical design 18, 91-99. Ng, T.P., Chiam, P.C., Lee, T., Chua, H.C., Lim, L., Kua, E.H., 2006. Curry consumption and cognitive function in the elderly. American journal of epidemiology 164, 898-906. Nikoletopoulou, V., Sidiropoulou, K., Kallergi, E., Dalezios, Y., Tavernarakis, N., 2017. Modulation of Autophagy by BDNF Underlies Synaptic Plasticity. Cell Metab 26, 230-242 e235. O'Farrell, E., MacKenzie, J., Collins, B., 2013. Clearing the air: a review of our current 25

Journal Pre-proof

understanding of "chemo fog". Current oncology reports 15, 260-269. Piccirillo, J.F., Hardin, F.M., Nicklaus, J., Kallogjeri, D., Wilson, M., Ma, C.X., Coalson, R.S., Shimony, J., Schlaggar, B.L., 2015. Cognitive impairment after chemotherapy related to atypical network architecture for executive control. Oncology 88, 360-368. Pisanu, M.E., Noto, A., De Vitis, C., Morrone, S., Scognamiglio, G., Botti, G., Venuta, F.,

f

Diso, D., Jakopin, Z., Padula, F., Ricci, A., Mariotta, S., Giovagnoli, M.R., Giarnieri, E.,

oo

Amelio, I., Agostini, M., Melino, G., Ciliberto, G., Mancini, R., 2017. Blockade of

pr

Stearoyl-CoA-desaturase 1 activity reverts resistance to cisplatin in lung cancer stem cells.

e-

Cancer letters 406, 93-104.

Pr

Selamat, M.H., Loh, S.Y., Mackenzie, L., Vardy, J., 2014. Chemobrain experienced by breast cancer survivors: a meta-ethnography study investigating research and care implications. PloS

al

one 9, e108002.

rn

Sousa, N., Lukoyanov, N.V., Madeira, M.D., Almeida, O.F., Paula-Barbosa, M.M., 2000.

Jo u

Reorganization of the morphology of hippocampal neurites and synapses after stress- induced damage correlates with behavioral improvement. Neuroscience 97, 253-266. Sun, Q., Fan, J., Billiar, T.R., Scott, M.J., 2017. Inflammasome and autophagy regulation - a two-way street. Mol Med 23. Vardy, J., Tannock, I., 2007. Cognitive function after chemotherapy in adults with solid tumours. Critical reviews in oncology/hematology 63, 183-202. Vichaya, E.G., Molkentine, J.M., Vermeer, D.W., Walker, A.K., Feng, R., Holder, G., Luu, K., Mason, R.M., Saligan, L., Heijnen, C.J., Kavelaars, A., Mason, K.A., Lee, J.H., Dantzer, R., 2016. Sickness behavior induced by cisplatin chemotherapy and radiotherapy in a murine 26

Journal Pre-proof

head and neck cancer model is associated with altered mitochondrial gene expression. Behav Brain Res 297, 241-250. Wang, Y., Liu, J., Tao, Z., Wu, P., Cheng, W., Du, Y., Zhou, N., Ge, Y., Yang, Z., 2016. Exogenous HGF Prevents Cardiomyocytes from Apoptosis after Hypoxia via Up-Regulating Cell Autophagy. Cell Physiol Biochem 38, 2401-2413.

f

Waseem, M., Parvez, S., 2013. Mitochondrial dysfunction mediated cisplatin induced toxicity:

oo

modulatory role of curcumin. Food Chem Toxicol 53, 334-342.

pr

Wu, X., Chen, H., Huang, C., Gu, X., Wang, J., Xu, D., Yu, X., Shuai, C., Chen, L., Li, S., Xu,

e-

Y., Gao, T., Ye, M., Su, W., Liu, H., Zhang, J., Wang, C., Che n, J., Wang, Q., Cui, W., 2017.

Pr

Curcumin attenuates surgery-induced cognitive dysfunction in aged mice. Metabolic brain disease 32, 789-798.

al

Wu, X., Fleming, A., Ricketts, T., Pavel, M., Virgin, H., Menzies, F.M., Rubinsztein, D.C.,

rn

2016. Autophagy regulates Notch degradation and modulates stem cell development and

Jo u

neurogenesis. Nature communications 7, 10533. Xiong, Y., Lu, D., Qu, C., Goussev, A., Schallert, T., Mahmood, A., Chopp, M., 2008. Effects of erythropoietin on reducing brain damage and improving functional outcome after traumatic brain injury in mice. Journal of neurosurgery 109, 510-521. Yang, Y., Ma, F., Liu, Z., Su, Q., Liu, Y., Liu, Z., Li, Y., 2018. The ER- localized Ca2+-binding protein

calreticulin

couples

ER

stress

to

autophagy

by

associating

with

microtubule-associated protein 1A/1B light chain 3. The Journal of biological chemistry. Yazdankhah, M., Farioli- Vecchioli, S., Tonchev, A.B., Stoykova, A., Cecconi, F., 2014. The autophagy regulators Ambra1 and Beclin 1 are required for adult neurogenesis in the brain 27

Journal Pre-proof

subventricular zone. Cell death & disease 5, e1403. Zhang, R., Xue, Y.Y., Lu, S.D., Wang, Y., Zhang, L.M., Huang, Y.L., Signore, A.P., Chen, J., Sun, F.Y., 2006. Bcl-2 enhances neurogenesis and inhibits apoptosis of newborn neurons in adult rat brain following a transient middle cerebral artery occlusion. Neurobiol Dis 24, 345-356.

f

Zhang, X.Y., Zhang, M., Cong, Q., Zhang, M.X., Zhang, M.Y., Lu, Y.Y., Xu, C.J., 2017.

oo

Hexokinase 2 confers resistance to cisplatin in ovarian cancer cells by enhancing

pr

cisplatin- induced autophagy. Int J Biochem Cell Biol 95, 9-16.

e-

Zhou, W., Kavelaars, A., Heijnen, C.J., 2016. Metformin Prevents Cisplatin-Induced

Pr

Cognitive Impairment and Brain Damage in Mice. PloS one 11, e0151890.

rn

al

Figure legends

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Fig. 1. The effects of curcumin on crossing number (A) and rearing number in the open field test (B), recognition index in the novel object recognition test (C), and mouse escape latency (D) and crossing number (E) in the Morris water maze test (n=15). # P < 0.05 and

##

P < 0.01

versus the control-vehicle group. *P < 0.05 and **P < 0.01 versus the cisplatin- vehicle group.

Fig. 2. The effects of curcumin on hippocampal neurogenesis and synaptogenesis in mice (n=3). (A) Immature neurons, DCX (green), Iba1 (red). (B) Dendritic spine density. 0.01 versus the control- vehicle group. * P < 0.05 and group. 28

**

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P<

P < 0.01 versus the cisplatin- vehicle

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Fig. 3. The effects of curcumin on the levels of apoptosis-related proteins (n=4). Bars are relative to control=1.0.

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P < 0.01 versus the control-vehicle group. * P < 0.05 versus the

cisplatin- vehicle group.

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Fig. 4. The effects of curcumin on hippocampal neuron autophagy in mice (n=3-4). (A)

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Representative photomicrograph of autophagosomes in hippocampal neurons from animals of

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each group. (B) The protein ratio of LC3-II/ LC3-I and (C) the levels of p62. Bars are relative

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to control=1.0. (D) Immunofluorescent image of the autophagy marker LC3 (green). (E) Immunofluorescent image of the autophagy marker p62 (red). # P < 0.05 and

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P < 0.01

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versus the control-vehicle group. * P < 0.05 versus the cisplatin- vehicle group.

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Fig. 5. The effects of curcumin on hippocampal Akt, mTOR, AMPK, JNK and ATF4 in mice

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(n=4). (A) Western blot image. (B) The levels of p-Akt/Akt. (C) The levels of p-mTOR/mTOR. (D) The levels of p-AMPK/AMPK. (D) The levels of p-JNK/JNK. (D) The levels of ATF4. Bars are relative to control=1.0. control-vehicle group. * P < 0.05 and

**

#

P < 0.05 and

##

P < 0.01 versus the

P < 0.01 versus the cisplatin- vehicle group.

Fig. 6. Pretreatment with 3-MA abolished the effects of curcumin on crossing number (A) and rearing number (B) in the open field test, recognition index in the novel object recognition test (C), and mouse escape latency (D) and crossing number (E) in the Morris water maze test (n=15). # P < 0.05 and

##

P < 0.01 versus the control- vehicle group. * P < 0.05 and 29

**

P < 0.01

Journal Pre-proof versus the cisplatin-vehicle group. +P < 0.05 versus the cisplatin-curcumin group.

Fig. 7. Pretreatment with 3-MA abolished the effects of curcumin on hippocampal neurogenesis and synaptogenesis in mice receiving chemotherapy (n=3). (A) Immature neurons, DCX (green). (B) Dendritic spine density. # P < 0.05 and

##

P < 0.01 versus the ++

P < 0.01

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control- vehicle group. * P < 0.05 versus the cisplatin- vehicle group. +P < 0.05 and

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versus the cisplatin-curcumin group.

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Fig. 8. Pretreatment with 3-MA abolished the effects of curcumin on apoptosis-related proteins (n=4). The levels of Bcl-2, Bax and Bim. Bars are relative to control=1.0. **

P < 0.01

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versus the control- vehicle group. * P < 0.05 and

##

+

P < 0.01 versus the cisplatin- vehicle group.

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P < 0.05 versus the cisplatin-curcumin group.

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Fig. 9. Pretreatment with 3-MA abolished the effects of curcumin on hippocampal neuron autophagy in mice (n=3-4). (A) Representative photomicrograph of autophagosomes in hippocampal neurons from animals of each group. (B) The protein ratio of LC3-II/ LC3-I. (C) The levels of p62 protein. (D) Immunofluorescent image of the autophagy marker LC3 (green). € Immunofluorescent image of the autophagy marker p62 (red). # P < 0.05 and 0.01 versus the control- vehicle group. * P < 0.05 and group.

**

P<

P < 0.01 versus the cisplatin- vehicle

++

P < 0.001 versus the cisplatin-curcumin group.

Fig. 10. Regulation of curcumin- induced autophagy in mice receiving chemotherapy. 30

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Li-Tao Yi: Conceptualization, Investigation, Writing-Original draft preparation/ Reviewing and Editing, Supervision. Shu-Qi Dong.: Investigation, Writing-Original draft preparation/ Reviewing and Editing. Shuang-Shuang Wang: Investigation. Min Chen: Investigation.

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Cheng-Fu Li: Formal analysis, Validation.

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Di Geng: Formal analysis, Validation.

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Ji-Xiao Zhu: Formal analysis.

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Qing Liu: Methodology.

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Jie Cheng: Methodology.

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Journal Pre-proof Curcumin attenuates the impaired cognition induced by cisplatin.

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Curcumin promotes neurogenesis and synaptogenesis in the hippocampus..

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Curcumin enhances autophagy in the hippocampus.

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The autophagy enhanced by curcumin is mediated by AMPK-JNK signaling.

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3-MA blocks the effects of curcumin on cognitive improvement in chemotherapy.

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