molecules

Article Ameliorates Scopolamine-Induced Cognitive Dysfunction and Neuronal Damage in Mice

Yeojin Kim 1,2, Jihyun Kim 1 , Meitong He 1, Ahyoung Lee 3,* and Eunju Cho 1,*

1 Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University, Busan 46241, Korea; [email protected] (Y.K.); [email protected] (J.K.); [email protected] (M.H.) 2 Neural Circuit Research Group, Korea Brain Research Institute, Daegu 41062, Korea 3 Department of Food Science, Gyeongsang National University, Jinju 52725, Korea * Correspondence: [email protected] (A.L.); [email protected] (E.C.); Tel.: +82-55-772-3278 (A.L.); +82-51-510-2837 (E.C.)

Abstract: We investigated the protective effect and mechanisms of apigenin against cognitive im- pairments in a scopolamine-injected mouse model. Our results showed that intraperitoneal (i.p.) injection of scopolamine leads to learning and memory dysfunction, whereas the administration of apigenin (synthetic compound, 100 and 200 mg/kg/day) improved cognitive ability, which was confirmed by behavioral tests such as the T-maze test, novel objective recognition test, and Morris water maze test in mice. In addition, scopolamine-induced lipid peroxidation in the brain was attenuated by administration of apigenin. To further evaluate the protective mechanisms of apigenin on cognitive and memory function, Western blot analysis was carried out. Administration of apigenin decreased the B-cell lymphoma 2-associated X/B-cell lymphoma 2 (Bax/Bcl-2) ratio and suppressed

 caspase-3 and poly ADP ribose polymerase cleavage. Furthermore, apigenin down-regulated the  β-site amyloid precursor protein-cleaving enzyme, along with presenilin 1 (PS1) and PS2 protein

Citation: Kim, Y.; Kim, J.; He, M.; levels. Apigenin-administered mice showed lower protein levels of a receptor for advanced glycation Lee, A.; Cho, E. Apigenin end-products, whereas insulin-degrading enzyme, brain-derived neurotrophic factor (BDNF), and Ameliorates Scopolamine-Induced tropomyosin receptor kinase B (TrkB) expression were promoted by treatment with apigenin. There- Cognitive Dysfunction and Neuronal fore, this study demonstrated that apigenin is an active substance that can improve cognitive and Damage in Mice. Molecules 2021, 26, memory functions by regulating apoptosis, amyloidogenesis, and BDNF/TrkB signaling pathways. 5192. https://doi.org/10.3390/ molecules26175192 Keywords: apigenin; cognitive ability; oxidative stress; neuronal damage; scopolamine

Academic Editor: Raffaele Capasso

Received: 8 July 2021 1. Introduction Accepted: 24 August 2021 Published: 27 August 2021 The brain, a central organ of the human body, is responsible for memory and cognitive function [1]. Behavioral abnormalities and cognitive decline in the central nervous system

Publisher’s Note: MDPI stays neutral (CNS) are linked to oxidative stress [2]. This means that the overproduction of reactive with regard to jurisdictional claims in oxygen species (ROS) leads to neuron damage in the hippocampus, causing the impairment published maps and institutional affil- of cognitive and memory functions [3]. Moreover, memory loss and cognitive impairment iations. are associated with neurological problems in brain function related to neurodegenerative diseases such as Alzheimer’s disease (AD) [4]. Mainly, the pathogenesis of AD includes amyloid beta (Aβ) production, phosphorylated tau protein, the accumulation of nerve fiber tangles, and oxidative stress [5,6]. Among them, the main characteristic of AD is the accumulation of Aβ plaque and phosphorylated tau protein in neurons of the brain. Aβ Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. plaque is produced from amyloid precursor protein (APP), which is sequentially cleaved β γ This article is an open access article by -secretase (BACE) and -secretase (presenilin 1 and 2) [7]. The principal component of distributed under the terms and senile plaques is Aβ peptides that act as neurotoxins and interfere with synaptic communi- conditions of the Creative Commons cation [8]. Furthermore, the toxicity of Aβ is related to increased oxidative stress and lipid Attribution (CC BY) license (https:// peroxidation in neurons [9]. Oxidative stress caused by overproduction of ROS and reactive – creativecommons.org/licenses/by/ species (RNS), such as superoxide anion radical (O2 ), hydroxyl radical (·OH), – 4.0/). (NO), and peroxynitrite (ONOO ), can cause oxidation of lipids, proteins, cell

Molecules 2021, 26, 5192. https://doi.org/10.3390/molecules26175192 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 5192 2 of 16

membranes, and DNA, eventually leading to cellular aging and deformation [10,11]. In particular, the brain requires more oxygen to perform hippocampal synaptic functions, though the brain is vulnerable to oxidative stress [12]. Hence, the overproduction of ROS contributes to AD progression, which causes oxidative damage by a mechanism of neurotoxicity and induces memory deficit [13]. Scopolamine is a widely used in vivo model for inducing damage to the nerve sys- tem, resulting in memory loss and cognitive impairment mimicking those observed in AD [14,15]. According to previous studies, injection of scopolamine induced oxidative stress, along with memory and cognitive deficits, in the hippocampus of a Wistar rat and Institute of Cancer Research (ICR) mouse model [16–19]. Furthermore, a recent study con- firmed that scopolamine-injected mice showed a decrease in brain-derived neurotrophic (BDNF)/tropomyosin receptor kinase B (TrkB) expressions, resulting in long-term and working memory deficits [20]. Therefore, we used the scopolamine-injected mouse model to assess potential agents for the treatment of AD. The current drugs clearly cannot treat the progression of AD, but there are some drugs (donepezil, rivastigmine, and galantamine) used for temporary delay of AD progress [21]. However, these drugs have been reported to show side effects such as vomiting, loss of appetite, and insomnia [22]. Therefore, researchers have focused on examining the prevention of AD with natural products with beneficial effects deriving from their daily consumption [20]. Apigenin (40,5,7-trihydroxyflavone), a less-toxic and non-mutagenic compound, is an edible, plant-derived flavonoid abundant in various vegetables and fruits, such as parsley, celery, onions, and oranges. According to the USDA (United States Department of Agriculture) database, parsley has a significant amount of apigenin [23,24]. Apigenin has been reported to exhibit anti-inflammation [25], anti-apoptosis [26], and free radical-scavenging activities [27] in vitro and in vivo. Previous study [28] indicated that oral administration of apigenin protects from Aβ25–35–induced oxidative damage of the neurovascular system by regulation of BDNF/TrkB levels in amnesic mice. In addition, apigenin reduced Aβ deposition in the APP/PS1 transgenic AD mouse model [29]. Despite the therapeutic effect of apigenin on neuronal function, the protective role of apigenin against scopolamine-induced cognitive impairment and potential mechanisms has not yet been studied. In the present study, we investigated the protective effect of apigenin, which was obtained from Sigma-Aldrich Co., Ltd. (St Louis, MO, USA), against memory and cogni- tive deficits via behavioral tests (T-maze, novel objective recognition, and Morris water maze tests) in mice injected with scopolamine. In addition, we studied whether apigenin attenuates oxidative stress by measuring the malondialdehyde levels, and protective mech- anisms of apigenin on the apoptosis, amyloidogenic, and BDNF/TrkB pathways were also analyzed in the brains of mice.

2. Results and Discussion 2.1. Effect of Apigenin on the Body Weight Change and Organs Weight in Scopolamine-Injected Mice Decline in the body weight and internal organ weight is a simple and sensitive index with which to determine toxicity after injection of scopolamine or treatment of samples. The changes in body weight and organ weights were measured in scopolamine-injected mice (Table1). Before oral administration of apigenin, the initial body weights among the five groups did not show significant differences, and we found no statistical differences in the body weight gain for each group during the experiment. After dissection, there were no significant differences in organ (brain, kidney, and liver) weights among all groups, indicating that mice did not have any problems, such as inflammation or edema. Moreover, these results demonstrated that scopolamine, apigenin, and donepezil pose no detectable abnormalities to the brain, liver, and kidney. Molecules 2021, 26, x FOR PEER REVIEW 3 of 16

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weights among all groups, indicating that mice did not have any problems, such as in- Table 1. Effectsflammation of apigenin or edema. on body Moreover, and organs these weight result in scopolamine-injecteds demonstrated that mice.scopolamine, apigenin, and donepezil pose no detectable abnormalities to the brain, liver, and kidney. Normal Control AP10 AP20 DO Table 1. Effects of apigenin on body and organs weight in scopolamine-injected mice. Initial body weight 34.1 ± 1.4 33.3 ± 1.1 32.9 ± 0.8 33.7 ± 0.9 33.1 ± 0.7 NS (g) Normal Control AP10 AP20 DO Final body weight Initial body weight (g)35.4 ± 2.0 34.1 ± 1.4 33.6 ± 1.4 33.3 ± 1.1 34.6 32.9± 1.5 ± 0.8 34.1 33.7± ±0.6 0.9 33.134.2 ± ±0.70.9 NS NS (g) Final body weight (g) 35.4 ± 2.0 33.6 ± 1.4 34.6 ± 1.5 34.1 ± 0.6 34.2 ± 0.9 NS Body weight gain NS 1.3 ± 1.5 0.3 ± 2.0 1.7 ± 2.2 0.4 ± 1.2 1.1 ± 1.5NS Body(g) weight gain (g) 1.3 ± 1.5 0.3 ± 2.0 1.7 ± 2.2 0.4 ± 1.2 1.1 ± 1.5 Brain (g)Brain (g) 0.50 ± 0.01 0.50 ± 0.01 0.49 ± 0.02 0.49 ± 0.02 0.51 0.±510.01 ± 0.01 0.50 0.50± ±0.01 0.01 0.500.50 ± ±0.010.01 NS NS Liver (g)Liver (g) 2.47 ± 0.51 2.47 ± 0.51 2.45 ± 0.21 2.45 ± 0.21 2.25 2.25± 0.23 ± 0.23 2.71 2.71± ±0.39 0.39 2.252.25 ± ±0.580.58 NS NS KidneyKidney (g) (g) 0.53 ± 0.04 0.53 ± 0.04 0.53 ± 0.06 0.53 ± 0.06 0.56 0.56± 0.07 ± 0.07 0.59 0.59± ±0.07 0.07 0.560.56 ± ±0.080.08 NS NS ValuesValues are are the the mean mean± SD.± SD. NS: NS: non-significance. non-significance. Normal: Normal: oral oral administration administration of of drinking drinking water water + + 0.9% 0.9% NaCl NaCl i.p.; i.p.; Control: Control: oral administrationoral administration of drinking of waterdrinking + scopolamine water + scopolamine 1.5 mg/kg i.p.; 1.5 AP10: mg/kg oral i.p.; administration AP10: oral of administration apigenin 10 mg/kg of apigenin + scopolamine 10 mg/kg 1.5 mg/kg + i.p.;scopolamine AP20: oral administration 1.5 mg/kg i.p.; of AP20: apigenin oral 20 administration mg/kg + scopolamine of apigenin 1.5 mg/kg 20 mg/kg i.p.; and + scopolamine DO: oral administration 1.5 mg/kg ofi.p.; donepezil and DO: 5 mg/kgoral + scopolamineadministration 1.5 mg/kg of donepezil i.p. 5 mg/kg + scopolamine 1.5 mg/kg i.p.

2.2.2.2. EffectEffect ofof Apigenin on on the the T-Maze T-Maze Test Test in in Scopolamine-Injected Scopolamine-Injected Mice Mice TheThe T-mazeT-maze test was performed performed to to evaluate evaluate the the spatial spatial memory memory function function of mice. of mice. ExploringExploring aa new new route route rather rather than than a familiara familiar route route is consideredis considered to demonstrateto demonstrate improved im- spaceproved perception space perception ability [ability30]. As [30]. presented As presented in Figure in Figure1, the scopolamine-injected1, the scopolamine-injected control groupcontrol did group not showdid not a significantshow a significant difference difference in spatial in explorationspatial exploration for the for old the route old and newroute route. and new In contrast,route. In thecontrast, non-scopolamine the non-scopolamine treated normaltreated groupnormal demonstratedgroup demon- sig- nificantlystrated significantly increased explorationincreased exploration in the new in routethe new (60.4%) route compared(60.4%) compared with the with old the route (39.6%),old route suggesting (39.6%), suggesting that injection that injection of scopolamine of scopolamine leads to leads impairment to impairment of cognition of cogni- and a memorytion and deficita memory in mice. deficit The in AP10 mice. and The AP20 AP10 groups and AP20 significantly groups significantly increased the increased new route explorationthe new route from exploration 44.5% to from 55.5% 44.5% and fromto 55.5% 45.5% and to from 54.6%, 45.5% respectively. to 54.6%, respectively. The DO group showedThe DO angroup increase showed in explorationan increase in between exploration the oldbetween route the and old new route route and from new 46.5%route to 53.5%.from 46.5% These to results 53.5%. showed These results that apigenin-administered showed that apigenin-administered mice improved mice spatial improved cognitive abilityspatial against cognitive scopolamine. ability against scopolamine.

Figure 1. Effect of apigenin on spatial alternation in the T-maze test. Values are the mean ± SD. a–c Figure 1. Effect of apigenin on spatial alternation in the T-maze test. Values are the mean ± SD. a–cThe different letters among groups represent significant differences (p < 0.05) by Duncan’s multi- pleThe range different test. * The letters space among perceptive groups abilities represent for old significant and new routes differences are significantly (p < 0.05) different by Duncan’s as multipledetermined range by test.Student’s * The spacet-test ( perceptivep < 0.05). Normal: abilities oral forold administrati and newon routes of drinking are significantly water + different0.9% asNaCl determined i.p.; Control: by Student’s oral administrationt-test (p < 0.05).of drinking Normal: water oral + scopolamine administration 1.5 ofmg/kg drinking i.p.; AP10: water oral + 0.9% NaCladministration i.p.; Control: of apigenin oral administration 10 mg/kg + ofscopolamine drinking water 1.5 mg/kg + scopolamine i.p.; AP20:1.5 oral mg/kg administration i.p.; AP10: oforal administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral administration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

Molecules 2021, 26, 5192 apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil4 of 16 5 mg/kg + scopolamine 1.5 mg/kg i.p.

2.3. Effect of Apigenin on Novel Object Recognition Test in Scopolamine-Injected Mice 2.3. Effect of Apigenin on Novel Object Recognition Test in Scopolamine-Injected Mice A novel object recognition test was conducted to confirm the memory retention, A novel object recognition test was conducted to confirm the memory retention, based based on evidence that rodents prefer to explore novel objects rather than familiar ob- on evidence that rodents prefer to explore novel objects rather than familiar objects [31]. To jects [31]. To elucidate the effect of apigenin on object recognition ability, a novel object elucidate the effect of apigenin on object recognition ability, a novel object recognition test wasrecognition carried out test (Figure was 2carried). The scopolamine-treated out (Figure 2). The control scopolamine-treated group showed no control significance group inshowed exploration no significance between familiar in exploration and novel betw objects,een familiar indicating and that novel scopolamine objects, indicating induced thethat impairment scopolamine of theirinduced object the recognition impairment ability. of their However, object recognition the untreated-normal ability. However, group significantlythe untreated-normal increased the group number significantly of touches increased for new objectthe number (56.5%) of compared touches for to oldnew object object (47.7%).(56.5%) Moreover,compared to AP10 old andobject AP20 (47.7%). groups Moreover, significantly AP10 increasedand AP20 in groups their explorationsignificantly ofincreased the novel inobject, their exploration from 47.9% of to the 56.2% novel and object, from from 48.2% 47.9% to 55.2%, to 56.2% respectively. and from 48.2% These to results55.2%, suggest respectively. that the These administration results suggest of apigenin that the improved administration scopolamine-induced of apigenin improved object recognitionscopolamine-induced deficit. object recognition deficit.

FigureFigure 2. 2.Effect Effect ofof apigeninapigenin onon recognition memory in the novel novel object object recognition recognition test. test. a–ba–b TheThe dif- differentferent letters letters among among groups groups repr representesent significant significant differences ((pp< < 0.05)0.05) bybyDuncan’s Duncan’s multiple multiple range range test.test. * The* The space space perceptive perceptive abilities abilities for oldfor andold and new new routes routes are significantly are significantly different different as determined as deter- mined by Student’s t-test (p < 0.05). Normal: oral administration of drinking water + 0.9% NaCl by Student’s t-test (p < 0.05). Normal: oral administration of drinking water + 0.9% NaCl i.p.; Control: i.p.; Control: oral administration of drinking water + scopolamine 1.5 mg/kg i.p.; AP10: oral ad- oral administration of drinking water + scopolamine 1.5 mg/kg i.p.; AP10: oral administration of ministration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral administration of apigeninapigenin 10 20 mg/kg mg/kg + scopolamine+ scopolamine 1.5 1.5 mg/kg mg/kg i.p.; i.p.; AP20: and oral DO: administration oral administration of apigenin of donepezil 20 mg/kg 5 +mg/kg scopolamine + scopolamine 1.5 mg/kg 1.5 i.p.;mg/kg and i.p. DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. 2.4. Effect of Apigenin on Morris Water Maze Test in Scopolamine-Injected Mice 2.4. EffectThe ofMorris Apigenin water on Morris maze Watertest was Maze conducted Test in Scopolamine-Injected to analyze the effect Mice of apigenin on long-termThe Morris and waterspatial maze memory test was ability conducted in mice to analyze[32]. A theswimming-tracking effect of apigenin path on long- was termmeasured and spatial for four memory days using ability a in SMART mice [32 video]. A swimming-trackingtracking program (Figure path was 3A,B). measured On the forfirst four day, days the using swimming-tracking a SMART video paths tracking of all program mice showed (Figure a3 A,B).long-complicated On the first day, route, the as swimming-trackingall groups were untrained. paths of allAs mice the showedexperiment a long-complicated of the learn-training route, session as all groups progressed, were untrained.however, Asthe the normal experiment group ofshowed the learn-training a shorter swim session trace progressed, to reach however,the hidden the platform normal groupduring showed the four a shorter days. swimIn contrast, trace to the reach scop theolamine-injected hidden platform control during mice the four indicated days. Inno contrast,significant the differences scopolamine-injected in the length control and miceshape indicated of the swim no significant trace for differencesthe Morris in water the lengthmaze andtest shapeduring of the the four swim days. trace However, for the Morris the AP10, water AP20, maze testand during DO groups the four revealed days. However,progressively the AP10, shorter AP20, swim and traces, DO indicating groups revealed that treatments progressively of apigenin shorter and swim donepezil traces, indicatingeffectively that protected treatments spatial of apigenin learning andand donepezilmemory function effectively against protected scopolamine spatial learning in mice. and memory function against scopolamine in mice. In addition, these results suggested that the longer swim trace led to a longer latency to search for the hidden platform in the scopolamine-injected control group compared with the untreated normal group during Molecules 2021, 26, x FOR PEER REVIEW 5 of 16

Molecules 2021, 26, 5192 In addition, these results suggested that the longer swim trace led to a longer latency5 of 16to search for the hidden platform in the scopolamine-injected control group compared with the untreated normal group during the training days. However, the normal group theshowed training that days. the latency However, to find the normalthe hidden group platform showed was that gradually the latency shortened to find the compared hidden platformwith the control was gradually group over shortened four days. compared In addition, with thethe controlAP10, AP20, group and over DO four groups days. sig- In addition,nificantly thedecreased AP10, AP20, the latency and DO to groupsreach th significantlye hidden platform, decreased compared the latency with to the reach control the hiddengroup. On platform, the last compared day of the with experiment, the control the group. spent On time the lastin the day target of the quadrant experiment, with the a spentvisual timesign inwas the recorded target quadrant after the with eliminat a visualion sign of the was hidden recorded platform after the (Figure elimination 3C). The of thecontrol hidden group platform spent the (Figure shortest3C). Thetimecontrol in the target group quadrant spent the (20.8%) shortest compared time in the with target the quadrantnormal group (20.8%) (25.0%). compared In contrast, with the AP10, normal AP20, group and (25.0%). the donepezil-administered In contrast, AP10, AP20, group and thespent donepezil-administered a significantly longer group time spentin the a significantlytarget quadrant, longer showing time in the23.9%, target 23.0%, quadrant, and showing24.7%, respectively. 23.9%, 23.0%, As andshown 24.7%, in Figure respectively. 3D, the As time shown to reach in Figure the exposed3D, the timeplatform to reach was thenot exposedsignificantly platform different was notamong significantly all the groups, different suggesting among all that the the groups, effect suggesting of apigenin that on thecognitive effect ofimprovement apigenin on was cognitive not related improvement to visual or was swimming not related abilities, to visual but or learning swimming and abilities,memory butabilities. learning Similarly, and memory previous abilities. studies Similarly, also demonstrated previous studies that apigenin also demonstrated improved thatspatial apigenin working improved memory spatial function working in behavioral memory function tasks such in behavioral as the Y-maze tasks such and as Morris the Y-

mazewater andmaze Morris tests waterin Aβ maze25–35- or tests -induced in Aβ25–35- or isoflurane-induced in vivo model [33,34].in vivo Thesemodel findings [33,34]. Thesedemonstrated findings demonstratedthat apigenin thatcould apigenin ameliorate could amelioratecognitive dysfunction cognitive dysfunction induced inducedby sco- bypolamine. scopolamine.

FigureFigure 3. EffectsEffects of of apigenin apigenin on on latency latency to to reach reach the the hidden hidden plat platformform during during four four days days in the in theMorris Morris water water maze maze test test(A), (representativeA), representative swimming-tracking swimming-tracking path pathss of the of theMorris Morris water water maze maze test test(B), ( Boccupancy), occupancy time time to stay to stay in target in target quadrant quadrant on onthe the final final day day in the in the Morris Morris water water maze maze test test (C), ( Clatency), latency to reach to reach the thehidden hidden and and expo exposedsed platforms platforms on the on thefinal final day day in the in Morris water maze test (D). Values are the mean ± SD. a–b The different letters among groups represent significant dif- the Morris water maze test (D). Values are the mean ± SD. a–b The different letters among groups represent significant ferences (p < 0.05) by Duncan’s multiple range test. NS: non-significance. Normal: oral administration of drinking water p differences+ 0.9% NaCl ( i.p.;< 0.05) Control: by Duncan’s oral administration multiple range of test.drinking NS: non-significance. water + scopolamine Normal: 1.5 mg/kg oral administration i.p.; AP10: oral of drinkingadministration water +of 0.9% apigenin NaCl 10 i.p.; mg/kg Control: + scopolamine oral administration 1.5 mg/kg of drinkingi.p.; AP20: water oral +administration scopolamine 1.5 of mg/kgapigenin i.p.; 20AP10: mg/kg oral + scopolamine administration 1.5 ofmg/kg apigenin i.p.; and 10 mg/kgDO: oral + administration scopolamine 1.5 of mg/kgdonepezil i.p.; 5 AP20:mg/kg oral+ scopolamine administration 1.5 mg/kg of apigenin i.p. 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p.

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2.5.2.5. Effect Effect of of Apigenin Apigenin on on Lipid Lipid Peroxidation Peroxidation in in the Brain of Scopolamine-Injected Mice ItIt has has been been well established that that oxidative stress stress is implicated in the progression of AD [35]. [35]. Abnormal Abnormal proteolytic proteolytic processing processing of of APP APP and and secretase enzymes is involved in Aβ deposition, leadingleading toto oxidative oxidative stress, stress, neuronal neuronal cell cell death, death, and and cognitive cognitive impairment. impair- ment.Aβ-mediated Aβ-mediated oxidative oxidative stress stress can cause can cause metabolic metabolic alteration alteration in the in brain, the brain, such assuch lipid as lipidperoxidation peroxidation and ROS and production ROS production [36]. Previous [36]. Previous studies studies have shown have that shown injection that injection of scopo- oflamine scopolamine caused oxidativecaused oxidative stress by stress decreasing by decreasing antioxidant antioxidant enzymes’ enzymes’ activity (superoxideactivity (su- peroxidedismutase dismutase or glutathione or glutathione peroxidase) peroxidase and increasing) and lipidincreasing peroxidation lipid peroxidation in the brain [37in ,the38]. brainMalondialdehyde [37,38]. Malondialdehyde (MDA), an end-product (MDA), an of en lipidd-product peroxidation, of lipid is peroxidation, considered to is be consid- one of eredthe markers to be one of radicalof the generation.markers of Toradical investigate generation. whether To apigenininvestigate protects whether scopolamine- apigenin protectsinduced scopolamine-induced oxidative stress, we measured oxidative the stress levels, we of MDAmeasured in the the brains levels of mice.of MDA As shownin the brainsin Figure of 4mice., the miceAs shown injected in Figure with scopolamine 4, the mice showedinjected anwith increase scopolamine in the MDAshowed contents an in- creasewhen comparedin the MDA to non-injectedcontents when mice. compared On the to other non-injected hand, supplementation mice. On the other of apigenin hand, supplementation(10 and 20 mg/kg) of showed apigenin a significant(10 and 20 decreasemg/kg) showed in MDA a levels, significant compared decrease to the in control MDA levels,group. compared These findings to the control are consistent group. withThese previous findings studiesare consistent showing with that previous treatment stud- of iesapigenin showing provides that treatment protection of against apigenin oxidative provides stress protection through reduction against oxidative in MDA concen- stress throughtrations inreduction a diabetes-associated in MDA concentrations cognitive decline in a mousediabetes-associated model [39]. In addition,cognitive thedecline ROS mouseand MDA model levels [39]. were In addition, significantly the ROS decreased and MDA by apigenin, levels were resulting significantly in the decreased protection by of apigenin,neuronal apoptosisresulting [in40 ].the Therefore, protection our of results neuronal suggest apoptosis that apigenin [40]. Therefore, may contribute our results to the suggestimprovement that apigenin of scopolamine-induced may contribute learning to the andimprovement memory impairment of scopolamine-induced by attenuating learningoxidative and stress. memory impairment by attenuating oxidative stress.

FigureFigure 4. EffectEffect of of apigenin apigenin on levels of MDA induced by scopolamine in the brain of mice. Values areare the mean ± SD.SD. a–ca–c TheThe different different letters letters among among groups groups represent represent significant significant differences differences ( p < 0.05) 0.05) byby Duncan’s multiple range test. NS: non-significance.non-significance. Normal: oral oral administration of drinking water + 0.9% NaCl i.p.; Control: oral administrationadministration of drinkingdrinking waterwater ++ scopolaminescopolamine 1.51.5 mg/kgmg/kg i.p.; AP10: oral administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral ad- i.p.; AP10: oral administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral ministration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of administration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. 2.6. Effect of Apigenin on Apoptosis-Related Protein Expressions in the Brains of Scopolamine-Injected2.6. Effect of Apigenin Mice on Apoptosis-Related Protein Expressions in the Brains of Scopolamine-InjectedOxidative stress Mice induced by scopolamine can cause inflammation and apoptosis in the brain,Oxidative leading stress to inducedthe death by of scopolamine hippocampal can neurons cause inflammation[41,42]. The B-cell and apoptosis lymphoma in (Bcl)-familythe brain, leading is known to theas an death important of hippocampal mediator of neurons apoptosis [41 ,in42 ].the The nervous B-cell system lymphoma [43]. The(Bcl)-family Bcl-2 protein is known acts as as anti-apoptosis an important mediatorfactor via ofsuppression apoptosis inof thecytochrome nervous C system release [43 in]. mitochondria,The Bcl-2 protein whereas acts as the anti-apoptosis Bcl-2-associated factor X via(Bax) suppression protein is ofrelated cytochrome to induction C release of

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apoptosis [44]. These proteins regulate cytochrome C release or mitochondrial mem- inbrane mitochondria, depolarization, whereas thereby the Bcl-2-associated deciding cell death X (Bax) [45]. protein Moreover, is related cytochrome to induction C can of apoptosiscleave and [44 activate]. These proteinscaspase-3, regulate which cytochromedirectly degrades C release poly or mitochondrial ADP ribose polymerase membrane depolarization,(PARP), a key therebyprotein decidingfactor forcell apoptosis death [ 45[46–48].]. Moreover, Therefore, cytochrome caspase-3 C canand cleave PARP and are activatebeing studied caspase-3, as the which most directly important degrades executor polys of ADP apoptosis. ribose polymeraseA previous (PARP),study showed a key proteinthat scopolamine factor for apoptosisincreased [levels46–48 ].of Therefore, Bax and caspase-3, caspase-3 and and reduced PARP are levels being of studied Bcl-2 pro- as theteins most in the important brains of executors mice [49]. of In apoptosis. addition, A scopolamine previous study increased showed expression that scopolamine of PARP increasedand caspase-3 levels in of the Bax brain and caspase-3,tissue of the and dementia reduced animal levels of model, Bcl-2 proteinsresulting in in the neuronal brains ofapoptosis mice [49 [50].]. In addition, scopolamine increased expression of PARP and caspase-3 in the brainTo tissue confirm of the the dementia protective animal mechanisms model, resulting of apigenin in neuronal against apoptosis scopolamine-induced [50]. neuronalTo confirm damage, the the protective levels of mechanisms apoptosis-related of apigenin protein against expr scopolamine-inducedession were analyzed neu- in a ronalmouse damage, brain (Figure the levels 5). ofThe apoptosis-related ratio of Bax/Bcl-2 protein was expressionsignificantly were increased analyzed in inthe a mousecontrol braingroup (Figure treated5). with The scopolamine, ratio of Bax/Bcl-2 compared was significantly with the normal increased grou inp. theHowever, control supple- group treatedmentation with with scopolamine, apigenin significantly compared with decreased the normal the Bax/Bcl-2 group. However, expression supplementation levels. In addi- withtion, apigeninthe levels significantly of cleaved decreased caspase-3 the and Bax/Bcl-2 cleaved expression PARP protein levels. expression In addition, were the levelsup-regulated of cleaved in caspase-3the control and group, cleaved compar PARPed protein with expressionthe scopolamine-untreated were up-regulated normal in the controlgroup. group,In contrast, compared the AP10 with theand scopolamine-untreated AP20 groups showed normallower levels group. of In these contrast, apopto- the AP10sis-related and AP20 protein groups expressions. showed lower Several levels studie of theses also apoptosis-related reported the protective protein expressions. activity of Severalapigenin studies from alsoapoptosis. reported Apigenin the protective reduced activity caspase-3 of apigenin and Bax from levels apoptosis. in apoptotic Apigenin neu- reducedrons in caspase-3early brain and injury Bax levelsafter subarachnoid in apoptotic neurons hemorrhage in early [40]. brain Moreover, injury after treatment subarach- of noidapigenin hemorrhage suppressed [40]. neuronal Moreover, cell treatment death by of inactivation apigenin suppressed of caspases neuronal (-8, -9, and cell -3) death and bycleavage inactivation of PARP of caspases [51]. Thus, (-8, these -9, and results -3) and indi cleavagecate that of apigenin PARP [51 had]. Thus, a protective these results effect indicateon neuronal that apigenin damage had through a protective inhibition effect onof neuronalthe apoptosis damage signaling through pathway inhibition in of theAD apoptosismouse models signaling induced pathway by scopolamine. in AD mouse models induced by scopolamine.

FigureFigure 5. 5.Effect Effect of of apigenin apigenin on on levels levels of of Bax/Bcl-2, Bax/Bcl-2, cleaved cleaved caspase-3, caspase-3, and and PARP PARP protein protein expression expression induced induced by scopolamine by scopola- inmine the in brain the ofbrain mice. of mice.a–c The a–c The different different letters letters among among groups groups represent represent significant significant differences differences (p (

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sion levels of BACE and γ-secretase are expected to play an important role in producing Aβ. ToTo elucidate thethe effecteffect ofof apigenin apigenin on on the the amyloidogenic amyloidogenic pathway, pathway, Aβ Ageneration-β genera- tion-relatedrelated protein protein expression expression was evaluated was evaluate in a scopolamine-injectedd in a scopolamine-injected mouse brain.mouse Figure brain.6 Figureshowed 6 thatshowed the proteinthat the levels protein of BACE,levels presenilinof BACE, 1presenilin (PS1), and 1 presenilin(PS1), and 2presenilin (PS2) in the 2 (PS2)scopolamine-injected in the scopolamine-injected control group control were higher group than were in thehigher non-injected than in the group. non-injected However, group.the AP10, However, AP20, andthe DOAP10, groups AP20, exhibited and DO a grou decreaseps exhibited in the levels a decrease of BACE, in PS1,the levels and PS2 of BACE,protein PS1, expression. and PS2 protein expression.

FigureFigure 6. 6. EffectEffect of of apigenin apigenin on on levels levels of ofBACE, BACE, PS1, PS1, and and PS2 PS2 (A), ( AIDE), IDE and and RAGE RAGE (B) protein (B) protein expression expression induced induced by sco- by polaminescopolamine in the in thebrain brain of mice. of mice. Values Values are arethe themean mean ± SD.± SD.a–d Thea–d differentThe different letters letters among among groups groups represent represent significant significant dif- ferencesdifferences (p < ( p0.05)< 0.05) by Duncan by Duncan′s multiple0s multiple range range test. test.Results Results of expression of expression were were calculated calculated using using β-actinβ-actin as the as standard. the stan- Normal:dard. Normal: oral administration oral administration of drinking of drinking water water+ 0.9% + NaCl 0.9% NaCli.p.; Control: i.p.; Control: oral administration oral administration of drinking of drinking water water + sco- + polamine 1.5 mg/kg i.p.; AP10: oral administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral ad- scopolamine 1.5 mg/kg i.p.; AP10: oral administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral ministration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + administration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. scopolamine 1.5 mg/kg i.p.

TheThe receptor receptor for for advanced glycosylation end products (RAGE) isis knownknown asas aa multi-mul- ti-ligandligand receptor receptor that that binds binds to Atoβ ,A andβ, and plasma-derived plasma-derived Aβ isAβ transported is transported by RAGE by RAGE from fromthe blood the blood to the to brain the viabrain the via blood–brain the blood–bra barrierin [barrier58]. RAGE [58]. blocking RAGE blocking protects neuronalprotects neuronalcells from cells Aβ-induced from Aβ oxidative-induced stressoxidative [59]. stress The insulin [59]. The degrading insulin enzymedegrading (IDE) enzyme is one (IDE)of the is extracellular one of the extracellular proteolytic enzymesproteolytic involved enzymes in involved Aβ removal in A [β60 removal]. A previous [60]. A study pre- viousdemonstrated study demonstrated that low levels that oflow IDE levels were of observedIDE were inobserved the brain in the of ADbrain patients of AD [pa-61]. tientsTherefore, [61]. Therefore, increased RAGEincreased expression RAGE expr or decreasedession or IDEdecreased expression IDE expression could contribute could contributeto Aβ deposition. to Aβ deposition. Similar to Similar a previous to a previous study [62 st],udy the [62], protein the levelprotein of RAGElevel of was RAGE up- wasregulated up-regulated by scopolamine by scopolamine in the control in the group. control In group. contrast, In the contrast, AP10, AP20,the AP10, and DOAP20, groups and DOcould groups significantly could significantly down-regulate down-regulate the levels ofthe RAGE levels proteinof RAGE expression. protein expression. In addition, In addition,extremely extremely low expression low expression of IDE protein, of IDE one pr ofotein, the principalone of the proteases principal involved proteases in thein- degradation of Aβ, was obtained in mice injected with scopolamine. However, the AP10-

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volved in the degradation of Aβ, was obtained in mice injected with scopolamine. However, the AP10- and AP20-administered groups reversed the reduction in IDE. and AP20-administered groups reversed the reduction in IDE. From these results, apigenin Fromcould these suppress results, the cognitiveapigenin impairmentcould suppress and neuronalthe cognitive death inducedimpairment by scopolamineand neuronal via deathregulation induced of the by amyloidogenicscopolamine via pathway regulati andon Aofβ thedegradation amyloidogenic protein pathway expressions and in A theβ degradationmouse brain. protein expressions in the mouse brain.

2.8.2.8. Effect Effect of of Apigenin Apigenin on on TrkB/BDNF TrkB/BDNF Pa Pathwaythway Protein Protein Expressions Expressions in in the the Brains Brains of of Scopolamine-InjectedScopolamine-Injected Mice Mice BDNF/TrkBBDNF/TrkB signaling signaling plays plays a fundamentalfundamental rolerole in in learning learning and and memory memory abilities abilities [63]. [63].The BDNF,The BDNF, a member a member of the neurotrophin of the neurotrophin family, is extensively family, is expressed extensively throughout expressed the throughoutbrain [64]. BDNFthe brain has [64]. a regulatory BDNF has function a regulato of synapticry function interactions of synaptic that affect interactions learning that and affectmemory learning formation and memory [65,66]. Information addition, [65,66]. BDNF In stimulates addition, the BDNF activation stimulates of TrkB, the whichactiva- is tionassociated of TrkB, with which neuroprotection is associated andwith cognitive neuroprotection improvement and cognitive [67]. According improvement to a previous [67]. Accordingstudy, scopolamine to a previous exposure study, inhibited scopolamine BDNF exposure and TrkB inhibited expression, BDNF leading and toTrkB impairment expres- sion,of cognitive leading to and impairment memory abilities of cognitive in mouse and modelsmemory [20 abilities]. in mouse models [20]. InIn the the present present study, study, decreased decreased levels levels of of BDNF BDNF and and TrkB TrkB protein protein expression expression were were observedobserved in in a a scopolamine-injected scopolamine-injected mouse brain,brain, comparedcompared toto the the normal normal group group(Figure (Figure7) . 7).However, However, protein protein expression expression of of BDNF BDNF was wa significantlys significantly enhanced enhanced in in the the AP10 AP10 group group in incomparison comparison with with the the control control group, group, which which is is consistent consistent with with previous previous reports reports showing show- ingthe the requirement requirement for BDNFfor BDNF in cognitive in cognitive improvement improvement by treatment by treatment with apigenin with apigenin [68]. In [68].addition, In addition, TrkB protein TrkB expressionprotein expression was also significantlywas also significantly up-regulated up-regulated in the AP20 in group. the AP20These group. results These suggest results that suggest the protective that the effect prot ofective apigenin effect against of apigenin scopolamine-induced against scopol- amine-inducedlearning and memory learning impairment and memory might be impairment associated with might up-regulation be associated of BDNF/TrkB with up-regulationexpression in of the BDNF/TrkB brain. expression in the brain.

FigureFigure 7. 7. EffectEffect of of apigenin apigenin on on levels levels of of BDNF BDNF and and TrkB TrkB protein protein expression expression induced induced by by scopolamine scopolamine in in the the brain brain of of mice. mice. ValuesValues are are the the mean mean ±± SD.SD. a–ca–c TheThe different different letters letters among among groups groups represent represent significant significant differences differences (p (p << 0.05) 0.05) by by Duncan’s Duncan’s multiplemultiple range range test. test. Results Results of of expression expression were were calculated calculated using using ββ-actin-actin as as the the standard. standard. Normal: Normal: oral oral administration administration of of drinking water + 0.9% NaCl i.p.; Control: oral administration of drinking water + scopolamine 1.5 mg/kg i.p.; AP10: oral drinking water + 0.9% NaCl i.p.; Control: oral administration of drinking water + scopolamine 1.5 mg/kg i.p.; AP10: oral administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral administration of apigenin 20 mg/kg + administration of apigenin 10 mg/kg + scopolamine 1.5 mg/kg i.p.; AP20: oral administration of apigenin 20 mg/kg + scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. scopolamine 1.5 mg/kg i.p.; and DO: oral administration of donepezil 5 mg/kg + scopolamine 1.5 mg/kg i.p. Apigenin is a bioactive present in edible vegetables and fruits, such as Apigenin is a bioactive flavonoid present in edible vegetables and fruits, such as parsley,parsley, celery, celery, chamomile, guava,guava, oranges,oranges, etc. etc. Furthermore, Furthermore, apigenin apigenin has has been been considered consid- ereda non-toxic a non-toxic flavonoid, flavonoid, even even at high at high doses do (upses to (up 500 to mg/kg) 500 mg/kg) [69]. Although[69]. Although quantification quanti- ficationof the dietaryof the dietary intake ofintake apigenin of apigenin is subject is subject to large to variations large variations depending depending on age, sex,on age, and sex,country, and country, the doses the of doses apigenin of apigenin used in used this in study this study were equivalentwere equivalent to the to average the average daily dailyconsumption consumption of flavonoids of in humans in humans (0.13–4.9 (0.13–4.9 mg/day) mg/day) [70] based [70] onbased the equivalenton the equiva- body lentsurface body area surface index. area Liu index. et al. [Liu28] showedet al. [28] that showed treatment that withtreatment apigenin with (20 apigenin mg/kg/day (20 mg/kg/day for eight days) attenuated learning and memory impairment in Aβ25– for eight days) attenuated learning and memory impairment in Aβ25–35-injected mice. In 35addition,-injected mice. oral doses In addition, of apigenin oral atdoses 10and of apigenin 20 mg/kg at exerted10 and neuroprotective20 mg/kg exerted effects neuro- by inhibiting inflammation, oxidative stress, and inflammation in rats [71]. Based on this

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Molecules 2021, 26, 5192 protective effects by inhibiting inflammation, oxidative stress, and inflammation in10 ofrats 16 [71]. Based on this evidence, we decided to choose apigenin at concentrations of 10 and 20 mg/kg/day, and this aspect suggests a possibility of apigenin as a therapeutic agent for the treatment of neurodegenerative diseases. evidence, we decided to choose apigenin at concentrations of 10 and 20 mg/kg/day, and Flavonoids, including apigenin, are able to directly scavenge ROS by donating hy- this aspect suggests a possibility of apigenin as a therapeutic agent for the treatment of neurodegenerativedrogen. However, ingested diseases. flavonoids are metabolized in the intestine and liver, result- ing inFlavonoids, forms that including are very different apigenin, from are able those to directlyfound in scavenge foods. There ROS byare donating low circulating hydro- gen.concentrations However, ingestedof flavonoids flavonoids in the arebrain, metabolized thereby it in isthe most intestine likely andthat liver,apigenin resulting inhibits in formsoxidative that stress are very through different modulation from those of foundan intracellular in foods. Theresignaling are lowpathway circulating such as concen- amy- trationsloidogenic of flavonoidspathway or in BDNF/TrkB the brain, thereby pathway it is [7 most2]. A likely previous that apigeninstudy suggested inhibits oxidativethat mice stressfed with through celery-based modulation apigenin-rich of an intracellular diets (aglycone signaling form) pathway showed such improvement as amyloidogenic in the pathwayabsorption or of BDNF/TrkB apigenin and pathway possessed [72]. anti-inflammatory A previous study activities suggested [73]. that Although mice fed there with celery-basedis little known apigenin-rich about the oral diets bioavailability (aglycone form) of apigenin showedimprovement in humans, one inthe of absorptionthe studies ofdemonstrated apigenin and that possessed apigenin anti-inflammatory was found in ur activitiesine samples [73]. after Although apigenin-rich there is little parsley known in- abouttake. Another the oral bioavailabilitystudy on the bioavailability of apigenin in of humans, apigenin one in ofhumans the studies indicated demonstrated that apigenin that apigeninconcentration was foundwas detected in urine in samples the plasma after following apigenin-rich parsley parsley consumption intake. Another [74]. In studyaddi- ontion, the apigenin bioavailability can cross of the apigenin blood–brain in humans barrier indicated [75], suggesting that apigenin that apigenin concentration may have was detectedachievable in thebioavailable plasma following levels by parsley oral administration consumption [74and]. Inexert addition, a neuroprotective apigenin can effect cross theas a blood–brainmetabolic modulator. barrier [75 ], suggesting that apigenin may have achievable bioavailable levels by oral administration and exert a neuroprotective effect as a metabolic modulator. 3. Materials and Methods 3.3.1. Materials Chemicals and and Methods Reagents 3.1. Chemicals and Reagents Apigenin (40 ′,5,7-Trihydoxyflavon, ≥95.0% purity by HPLC) (Figure 8A), scopola- mine,Apigenin and donepezil (4 ,5,7-Trihydoxyflavon, hydrochloride were≥95.0% obta purityined byfrom HPLC) Sigma-Aldrich (Figure8A), scopolamine,Co., Ltd. (St andLouis, donepezil MO, USA). hydrochloride Apigenin and were donepezil obtained we fromre dissolved Sigma-Aldrich in water Co., and Ltd. orally (St Louis, adminis- MO, USA).tered in Apigenin doses of and 10 mg/kg/day, donepezil were 20 mg/kg/ dissolvedday in(apigenin), water and and orally 5 mg/kg/day administered (donepezil). in doses ofScopolamine 10 mg/kg/day, was 20dissolved mg/kg/day in 0.9% (apigenin), sodium chloride and 5 mg/kg/day (NaCl) and (donepezil). administered Scopolamine by i.p. in- was dissolved in 0.9% sodium chloride (NaCl) and administered by i.p. injection to mice. jection to mice. NaCl was purchased from LPS Solution (Seoul, Korea). Radioimmuno- NaCl was purchased from LPS Solution (Seoul, Korea). Radioimmunoprecipitation assay precipitation assay (RIPA) buffer was purchased from Elpis Biotech (Daejeon, Korea). (RIPA) buffer was purchased from Elpis Biotech (Daejeon, Korea). The polyvinylidene The polyvinylidene fluoride (PVDF) membrane was obtained from Millipore Co. (Biller- fluoride (PVDF) membrane was obtained from Millipore Co. (Billerica, MA, USA). ica, MA, USA).

FigureFigure 8.8. StructureStructure ofof apigenin apigenin ( A(A)) and and illustrative illustrative diagram diagram course course of of behavioral behavioral tests, tests, including including the the treatment treatment of of scopo- sco- laminepolamine and and apigenin apigenin (B). (B).

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3.2. Animals and Experimental Protocols Male ICR mice (5 weeks old, 25–30 g) were purchased from Orient Inc. (Seongnam, Korea). The mice were housed in plastic cages with a maintained 12 h light/dark cycle and controlled temperature (20 ± 2 ◦C) and humidity (50 ± 10%). The mice were free to access to water and food (5L79, Orient Inc., Seongnam, Korea). The animal protocols were confirmed by the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC; approval number: PNU-2019-2480). The mice were acclimated for a week before the experiments. They were randomly divided into five groups (n = 10 per group): normal, control, AP10, AP20, and DO. The mice in the normal and control groups were orally administered 100 µL of drinking water via oral gavage. The mice in the AP10 and AP20 groups were orally administered 100 µL of apigenin at doses of 10 and 20 mg/kg/day, respectively. In addition, the DO group mice (positive control group) were orally given 100 µL of donepezil at dose of a 5 mg/kg/day. Apigenin and donepezil were dissolved in the drinking water prior to oral administration, and all samples were treated daily for 14 days. The normal group mice were i.p. injected with 0.9% NaCl and the other four groups (control, AP10, AP20, and DO) were injected scopolamine (1.5 mg/kg) 30 min before every behavioral experiment, in the same way (Figure8B). Daily water intake was observed in terms of the water remaining in the water bottle every day, and there were no significant differences in water consumption among all the groups (data not shown).

3.3. T-Maze Test The T-maze test was performed according to the procedure established by Mont- gomery [76]. To investigate the spatial memory ability, the T-maze test was carried out. The T-shaped maze consisted of a start box, a left arm, and a right arm. On the first day, the right arm was blocked using a movable black acrylic wall, and the mice were placed at start area then left to explore the maze for 10 min. After 24 h, the black acrylic wall was removed and the mice were placed in the same apparatus, allowing them to move to the left (old route) or right (new route) arm for 10 min. The numbers entering the left and right arms were recorded.

3.4. Novel Object Recognition Test The novel object recognition test was carried out using the procedure of Bevins and Besheer [77] in a black plastic square box (40 × 40 × 40 cm). On the first day, during the test, two of the same objects (A and A’) were placed at both sides of the center of the box. The mice started at the center of the objects and the numbers of touches were counted for 10 min. After 24 h, one of the objects was replaced with a new object (B), which was totally different from the others. The mice were allowed to touch both of the objects (A and B) for 10 min. The numbers of contacts with each object were recorded.

3.5. Morris Water Maze Test To confirm the long-term memory, the Morris water maze test was performed [78]. The test proceeded in a circle water tank (diameter 150 cm, height 60 cm), which was filled with water and mixed with non-toxic soluble opaque black liquid gel. The water temperature was maintained at 22 ± 2 ◦C. The water tank was randomly divided into four quadrants and each quadrant was marked with a different sign as a space perception cue. The hidden platform was placed at approximately 1 cm below the water surface in the center of one of the quadrants. In all experiments, SMART video tracking software 3.0 (Panlab, Barcelona, Spain) was used with a camera above the circle water tank to analyze mouse movements. The program visually analyzes mouse movements to identify significant differences for each group. Training sessions were performed over three days; the intention each day was for the mice to swim and find the escape platform in 60 s using the visual clues. If the mice could not find the platform, we guided the mice carefully to the hidden platform and allow them stay there for 15 s. On the fourth day, three trials were conducted. The first trial was the same procedure as the past three days. In the second trial, we removed the hidden Molecules 2021, 26, 5192 12 of 16

platform and allowed the mice to swim and find the quadrant where the platform used to be located in 60 s. In the final trial, the black water was replaced with clean water and a visible platform was provided for the mice; we recorded the time they then took to reach the platform.

3.6. Measurement of MDA Levels MDA levels were measured by the method described by Ohkawa et al. [79]. After completion of the behavioral tests, dissected brain tissue was homogenized with saline solution, and mixed with 1% phosphoric acid and 0.67% thiobarbituric acid solution. After boiling at 95 ◦C for 45 min, the solution mixture was cooled in an ice bath, and 2 mL of 1-butanol was added followed by centrifugation at 3000 rpm for 10 min. The absorbance values of the supernatant were measured at 535 and 520 nm. The level of lipid peroxidation was calculated using an MDA standard curve.

3.7. Western Blot Analysis The brain tissues were homogenized with a lysis buffer containing a protease inhibitor cocktail. The homogenates were centrifuged at 12,000 rpm for 30 min at 4 ◦C to obtain the supernatant. After, the total concentration of protein in the supernatant was determined with bovine serum albumin (BSA) as the standard, and samples were prepared with an equal amount of protein. Equal amounts of proteins were separated by gel electrophoresis with 10% or 13% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGEs), then trans- ferred to the PVDF membranes. The proteins were blocked by 5% skim milk for 60 min and washed with PBS. The membranes with primary antibodies were incubated over night at 4 ◦C (each antibody at a dilution of 1:200–1:1000; Bax, Bcl-2, cleaved caspase-3, cleaved PARP, BACE, PS1, PS2, RAGE, IDE, BDNF, TrkB, and β-actin). Then, the membranes were incubated with appropriate HRP-conjugated secondary antibodies for 60 min. Protein expressions were visualized using a chemiluminescent imaging system (Davinci Chemi, Seoul, Korea).

3.8. Statistical Analysis All data in this experiment were presented as the mean ± standard deviation (SD). Statistical differences were calculated by analysis of variance (ANOVA) and Duncan’s multiple range test using the statistical program Statistical Package for the Social Sciences (version 25 SPSS Inc., Chicago, IL, USA). In the T-maze test and novel object recognition test, the perceptive ability between the training and test sessions was compared by Student’s t-test. p value of < 0.05 was considered significant.

4. Conclusions In conclusion, oral administration of apigenin alleviated the cognitive and mem- ory deficits in an AD mouse model induced by scopolamine. Apigenin also attenuated scopolamine-induced lipid peroxidation in the brains. In addition, apigenin decreased expressions of apoptosis factors, such as Bax/Bcl-2, cleaved capase-3, and cleaved PARP. Apigenin inhibited BACE, PS1, PS2, RAGE, and elevated IDE protein expression, suggest- ing that apigenin regulated the amyloidogenic pathway and promoted degradation of Aβ. Up-regulation of BDNF and TrkB expression by apigenin also contributed to an improve- ment effect on learning and memory abilities. Therefore, apigenin treatment inhibited scopolamine-induced cognitive impairment by regulating the apoptosis, amyloidogenic and BDNF/TrkB pathways. The present study suggests that apigenin could be a promising agent with the effect of improving cognitive and memory impairment in neurodegenerative diseases, such as AD.

Author Contributions: Conceptualization, A.L. and E.C.; methodology, Y.K., J.K. and M.H.; inves- tigation, Y.K., J.K. and M.H.; writing—original draft, Y.K.; writing—review and editing, A.L. and E.C.; and supervision, A.L. and E.C. All authors have read and agreed to the published version of the manuscript. Molecules 2021, 26, 5192 13 of 16

Funding: This research received no external funding. Institutional Review Board Statement: The animal protocols were confirmed by the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC; approval number: PNU- 2019-2480). Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are available from the authors.

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