Iranian Journal of Basic Medical Sciences ijbms.mums.ac.ir

Evaluation of the neuroprotective, anticonvulsant, and cognition- improvement effects of apigenin in temporal lobe epilepsy: Involvement of the mitochondrial apoptotic pathway Paria Hashemi 1, Javad Fahanik Babaei 2, Somaye Vazifekhah 1, Farnaz Nikbakht 3* 1 Department of Physiology, School of Medicine and Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran, Iran 2 Physiology Research Centre, Iran University of Medical Sciences, Tehran, Iran 3 Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O A B S T R A C T Article type: Objective(s): Original article the use of antiepileptic drugs includes their side effects on learning and memory. Herbal medicine Article history: Cognitive deficit is a common problem in epilepsy. A major concern emergent from Received: Aug 3, 2018 Accepted: Jan 22, 2019 iscognition considered in epilepsy. a complementary and alternative therapy in epilepsy. Apigenin is a safe flavone with Materialsantioxidant and properties. Methods: ForHowever, evaluating there the is anticonvulsant little information effect about of apigenin the beneficial in the kainite effect temporal of apigenin epilepsy on Keywords: model, apigenin was orally administered at 50 mg/kg for six days. Reference and working memory were examined via the Morris water maze and Y-maze task spontaneously. Results: P<0.01) and restored ApigeninCognition P AnticonvulsantCytochrome c number ofResults living showedneurons that in the apigenin hilus (P had<0.001). significant Immunohistochemical anticonvulsant activity analysis ( showed that apigenin Neuroprotection <0.01), suggesting an inhibitory role in the intrinsic apoptotic Temporal lobe epilepsy the memory-deficit induced by kainic acidP ( <0.05). Furthermore, apigenin significantly increased the pathway. Conclusion:reduced the Theserelease results of cytochrome suggest that c ( apigenin restores memory impairment via anticonvulsant and neuroprotective activity.

►Please cite this article as: Hashemi P, Fahanik Babaei J, Vazifekhah S, Nikbakht F. Evaluation of the neuroprotective, anticonvulsant, and cognition-improvement effects of apigenin in temporal lobe epilepsy: Involvement of the mitochondrial apoptotic pathway. Iran J Basic Med Sci 2019; 22:752-758. doi: 10.22038/ ijbms.2019.33892.8064 Introduction neurodegeneration, almost all available antiepileptic Epilepsy is one of the most common and chronic neurological diseases. It manifests itself by recurring, drugsbeen unsuccessful (AEDs) worsen in controlling memory and impairment reducing epileptic (9, 10). Recentseizures studies in drug-resistant have shown patients that AEDsbut have have also not led only to unprovokedepilepsy, is recognizedseizures that as havethe mostno clear frequent prognosis form (1). of Temporal lobe epilepsy (TLE), Partial a type evidence of partial indicates or focal need to replace these with other alternatives. Recently, that memory impairment is one of the main complaints undesirablescholars have side focused effects (9).on Therefore,medical herbs,there is especiallyan urgent acquired epilepsy in adults (2). of patients with TLE; it is caused by hippocampal flavonoids,many fruits onand neurodegenerative vegetables, with chamomile disorders, and including celery sclerosis (3). Hippocampal sclerosis is characterizedSome studies epilepsy (11). Apigenin, a natural flavone, is abundant in byhave the suggested loss of neurons, that there specifically is a clear ininteraction the CA1/CA3 between and particular interest because of its anxiolytic, sedative, thememory hilus loss areas and of theneurodegeneration hippocampus (4). in the temporal beingneuroprotective, the highest anticonvulsive, sources (12). Apigenin and antidepressant has received prolonged seizures lead to progressive hippocampal cortex,neuronal amygdala, death, whichand hippocampus is accompanied (5, 6). by Apparently, memory propertiescancer properties (13). Apigenin of apigenin can arealso due ameliorate to the induction memory dysfunction linked to Alzheimer’s disease (14). The anti- Mitochondrial dysfunction after prolonged seizure 17). However, in pathological conditions, apigenin acts isdysfunction the other (7). cause of neuronal cell death. One of the of apoptosis through different apoptotic pathways (15- mitochondrial dysfunction manifestations during suggesting the protective role of apigenin in cognition epilepsy is opening of the mitochondrial permeability asimprovement an anti-apoptotic through agent amelioration (18, 19). There of mitochondria are no data transition pore and release of cytochrome c into the cytosol. Cytochrome c is the key protein that can trigger to investigate the protective role of apigenin in cognition dysfunctionand memory in impairments TLE. Therefore, induced this studyby kainic was aciddesigned with emphasis on the possible involvement of mitochondrial mitochondrial cell death (8). Apart from seizures per se and vast hippocampal *Corresponding author: Farnaz Nikbakht. Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, Tehra, Iran. Tel: +98-21- 86704546; Email: [email protected] Protective effects of apigenin in temporal lobe epilepsy Hashemi et al.

Seizure intensity was measured according to a cell death pathway. Additionally, we examined the Stage 0: immobility; stage 1: rigid posture, eye Materialsanticonvulsant and effect Methods of apigenin in the rat model of TLE. modifiedblinking, Racine’sand /or scale mild values: facial clonus; stage 2: head Animals bobbing and multiple facial clonus; stage 3: myoclonic from the Experimental Studies Centre, Iran University forelimbs with rearing; and stage 5: generalized tonic- of MaleMedical Wistar Sciences, rats (200–250 Tehran, g)Iran. were The obtained animals (n=44) were jerksclonic in convulsions. the forelimbs; stage 4: clonic convulsions in the kept under the following standard conditions: 12 hr Training in the spatial version of the Morris water maze task light/dark50% with free cycle availability (lights on of from food 7:00and waterAM to throughout 7:00 PM), commonly accepted behavioral tests for assessing the environmental temperature 22±2 °C, and humidity 40– spatialMorris learning water and maze memory (MWM) capabilities is one of of rodents. the most In were compiled according to Guidelines for the Care and the study. All experimental protocols and procedures MWM was a circular black tank the present study, MWM was conducted five days after Useapproved of Laboratory by the Ethics Animals and as Research stipulated Committee by the National of Iran induction of TLE in rats. Institute of Health (NIH). In addition, this study was (100 cm in diameter and 75 cm in height). It was placed in a dim room with visual cues. Theconstantly tank was filledlocated to ExperimentalUniversity of Medical procedure Sciences (Tehran, Iran) in 2016. a1.5 depth cm beneathof 40 cm the with water water surface (21±1 °C).in one A hidden of the blackpool The rats were randomly divided into 4 groups as platformquadrants. (10 The cm task in consisted diameter) of 2 was stages: the acquisition follows: control-vehicle group, apigenin-treated sham phase for spatial learning assessment, and the probe group, kainic acid group, and apigenin-treated kainic test and retention retrieval of memory. The acquisition

in which the rats were given four trials per session acid group. For induction of TLE in rats, they were phaseper day was with performed the platform during in a constant five subsequent place. In days,each first anesthetized by the intraperitoneal (IP) injection session and between each trial, an interval of 20 sec was of ketamine (100 mg/kg) and xylazine (20 mg/kg). observed. If the platform was not found within 60 sec, Animalsincision inwere the scalp,then positioned the left lateral and ventriclefixed on wasa stereotaxic targeted the rat was forced into the platform and placed on it for apparatusat the anteroposterior (Stoelting, USA).position, After in aaccordance midline sagittalto the an additional 20 sec. During each trial session, the total platform was recorded using a video camera-based stereotaxic atlas, as follows: 1 mm; lateral; –1.5 mm from time (latency time) and movement to find the hidden bregma, and 3.5 mm ventral from the dura. An amount hours after the acquisition phase, the probe trial session of 1.8 μl of sterile normal saline solution containing 0.5 EthoVisionwas performed System in (Noldus,which the Netherlands). platform was Twenty-four removed μg of kainic acid was microinjected unilaterally into the from the pool and the rat was allowed to swim for 60 leftin lateraldistilled ventricle. water, Aftercontaining 5 min, the 5% needle sodium-carboxyl was removed sec to search for it. the target to minimize the drug withdrawn. Apigenin was dissolved quadrant, time spent in the target quadrant, and the total number of crossingsThe latency into theof first target entry quadrant to were methylcellulose (CMC-Na) and was administered orally recorded. at a dose of 50 mg/kg/day once a day five days before Behavioralsurgery till one testing day afterward (Figure 1). Y-maze spontaneous alternation performance Anticonvulsant activity of apigenin against kainite- This test was conducted 2 days after the MWM task. induced seizures To evaluate spatial working memory, we recorded In kainite-induced convulsion, the intensity of the spontaneous alternation behavior during a single- seizures, the onset of seizures, the onset of tonic-clonic session in a Y-maze on the 13th day of the experiment. general seizure, and the duration of tonic-clonic phase Testing occurred in a Y-shaped maze which consisted of were video monitored and recorded for 24 hr.

a gray-painted Plexiglas with three horizontal arms (40

Figure 1. was administrated orally at a dose of 50 mg/kg/day starting 5 days before surgery and continued 1-day post-surgery Experimental design of the study. For induction of TLE, 0.5 μg kainic acid was injected unilaterally into the left lateral ventricle. Apigenin

Iran J Basic Med Sci, Vol. 22, No. 7, Jul 2019 753 Hashemi et al. Protective effects of apigenin in temporal lobe epilepsy

Figure 2. P P P P<0.01; D) were recorded. The Anticonvulsant effect of apigenin (50 mg/kg/day). Following apigenin treatment, decrease in the onset of seizure ( <0.05; A), average seizure intensity ( <0.01; B), latency to the tonic-clonic stage ( <0.001; C), and duration time in tonic-clonic stage ( cmdata long,represented 30 cm as high, the mean±SEM, and 15 cmstatistically wide). significant The rat, differenceswithout comparedsolution with KAfor group about 30 min in darkness. The sections any prior experience of the maze, was put at the start were then air dried in the dark for 20 min. Finally, the arm and then allowed to move freely inside the maze dry slides were cleared in xylene and coverslipped with for a 10-min session. The arm entries were counted DPX, a non-aqueous mounting medium. For observation of neuronal degeneration, results were assessed using entries into all three arms on consecutive occasions andusing recorded the following visually. formula: Alternation rate was defined as Eight sections were used for cytochrome C theimmunoreactivity. FITC filter at 40X For magnification. this purpose, endogenous peroxidase activity was blocked in a dark room for 20 min in 3% H2O2/methanol solution after dehydration Alteration rate (%)=Number of alterations / (Number of Histologicaltotal arm entries–2) processing × 100 they were heated indirectly for 30 min in a bain in graded ethanol solutions. After washing with PBS, consisting of 3% goat serum and 0.3% Triton X-100 Five days after the KA injection, four rats in each mariefor 20 (70min. °C) The and sections then treated were withincubated blocking overnight serum groupthe left were cardiac anesthetized ventricle with with 4% ketamine paraformaldehyde (100 mg/kg) in and xylazine (20 mg/kg): they were then perfused via cytochrome c rabbit polyclonal antibody). Sections were withincubated 4 °C primaryfor 60 min antibodies at room at temperature 1:100 dilutions with (anti- goat 0.1four M microtome phosphate-buffered serial sections saline of (pHeach 7.4). brain The from brains the were removed and post-fixed overnight at 4 °C. Twenty Jade B, and cytochrome c immunoreactivity. Eight anti-rabbit IgG HRP conjugated as a secondary antibody. hippocampus area (5 μm) were used for Nissl, Fluoro- Chromogenic 3,3′-diaminobenzidine (DAB) solution used for each histological process. For Nissl staining, wascounterstaining. used for detecting cells, while Mayer’s hematoxylin sectionsthe slides with were three stained sections’ with interval 0.1% Cresylof each violet brain forwere 1 (Thermo Scientific Inc., Waltham, MA, USA) was used for min after dehydration in graded ethanol solutions: they Statistical analysis were then rehydrated and coverslipped. The number of The results were expressed as mean±SEM. Differences P<0.05. Statistical analyses were performed using SPSS v.16.0 Nissl-stainedThe second granule eight sections cells in wereCA3 and used the for hilar Fluoro-Jade areas of werefor windows. considered The data statistically related to significant MWM were at analyzed the hippocampus were counted using a 20X objective.

Bethanol staining for according3 min, followed to the by Schmued 2 min in (2000) 70% protocolethanol. by two way ANOVAs. MWMpost probe hoc testtest datato analyze and other the (20).Then, In the brief, sections the slides were were placed immersed in a solution in xylene: containing 100% studiesdifference. were analyzed using one-way ANOVA followed 1% sodium hydroxide in 80% alcohol. Subsequently, with subsequent Tukey’s they were rinsed in distilled water for 2 min before Results being transferred to a solution of permanganate for 10 Anticonvulsant activity of apigenin min. Following rinsing in distilled water, the sections The results show that apigenin has an anticonvulsant were immersed into a 0.004 Fluoro-Jade B working

effect on KA-induced seizure activity. Apigenin 754 Iran J Basic Med Sci, Vol. 22, No. 7, Jul 2019 Protective effects of apigenin in temporal lobe epilepsy Hashemi et al.

P statistical140): 19.41, analysis P<0.001]. revealed However, significant the interaction effect between on both factors: days [F (4,140): 30.04, <0.001] and groups [F (3, P daysrevealed and angroups increase failed in tothe reach total adistance statistical traveled significance in the [F (2, 140): 1.005, =0.44]. One-way ANOVA further P P P KA group as compared with the control group on day 1 ( <0.001), day 2 ( =0.01), and days 3 and 5 ( =0.05). Pre- treatmentP withP apigenin significantly decreasedP distance traveled as compared with the KA group on these days [1 (enhancement<0.001), 2 ( on=0.01), learning and as 3 comparedand 5 ( =0.05)]. with the Apigenin control treatmentrats, implying in the thatsham apigeningroup did notalone show cannot any significant improve

Effectmemory of inapigenin normal onrats retrieval (Figure 3B). phase of MWM Probe trial test was conducted to assess memory retrieval 24 hr after the last training session. Three

number of crossings into the target quadrant, and the parameterstotal time spent of firstin the entry target to region the target were analyzed quadrant, using the

one-way ANOVA. These results revealP a significantPost hoc effect test caused by treatment on latency to the first entry to the

target quadrant [F (3, 27): 6.30, =0.003]. Figure 3. confirmed that the KA group had higherP latencyP upon first entry to the target quadrant compared with control Effect of apigenin (50 mg/kg/day) on the acquisition phase and apigenin-treated sham groups ( =0.009, =0.003 of the Morris water maze (MWM) test. (A) Mean escape latency to reach the platform during the acquisition phase, (B) total distance respectively;(P Figure<0.05). 4A). The Apigenin number treatment of crossings significantly into the traveled to reach the platform. Apigenin treatment significantly reduced latency to the first entry as compared with the *ameliorated P<0.05, ** P learning-deficit<0.01, *** P induced by kainite KA group P<0.001, All data are expressed as mean±SEM Figuretarget region4B). Post in differenthoc groups revealed a significant <0.001 (KA vs control) showeddifference a betweenlower number groups of [F crossings (3, 27): 15.99;into the target analysis confirmed that KA rats P compared with the control and apigenin-treated groups significantly delayed the latent period to start seizure regionP (where the platform had been located before) as asseverity compared of the with seizure the KA recorded group ( by<0.05) the Racine(Figure scale 2A). AdministrationP of apigenin significantly decreased the ( <0.01). One-way ANOVA also showed that there Pwas a significant differencePost hoc betweenanalyses indicated all groups less for time the ( <0.01, Figure 2B)P in<0.001 comparison and P<0.01, with respectively).the KA group. total time spent in the target quadrant [F (3, 27): 7.69; Also, apigenin decreased the threshold and duration of P=0.001,<0.001): Figure this was 4C]. ameliorated by apigenin in the treated Effecttonic-clonic of oral stages administration ( of apigenin on learning spent in theP KA group as compared to the control group and memory deficits induced by KA in the Morris (the apigenin treated group and the control group, we water maze task KAconclude group that ( <0.05). the reference As there memory was no impairment difference between induced Effect of apigenin on acquisition phase of MWM by kainite was restored by apigenin. Figure 3 shows the results of learning in the acquisition phase of MWM. The latency to reach the Effect of oral administration of apigenin on spatial platform during the learning phase was analyzed by working memory in KA-induced rat model of TLE

P<0.001] experimental groups regarding the Y-maze task using two-way ANOVA. It revealed thatP<0.001], there was but a notsignificant for the Significant differences were detected amongst the influence of the factor days [F (4, 140): 56.05, P mean percent spontaneous alternation behavior for the andWhen groups this [Finteraction (3, 140): 13.76,was analyzed with one-way thecontrol-vehicle, one-way ANOVA apigenin-treated test. As shown sham, in kainic Figure acid, 5, and the interaction of the day group [F (12, 140): 1.19, =0.29]. apigenin-treated + kainic acid was 65±6.99, 69.47±5.53, longer latencies to reach the platform as compared with 37.53±7.58, and 61.99±4.4, respectively. Thus, a ANOVA, it was evident that KA ratsP<0.05; used P significantly<0.001; and P the control on days 1, 2, and 3 ( significant decrease in the percent alternation behaviorP<0.05). <0.05 respectively). Pre-treatment of the KAP group was observed in the KA group as compared with the with apigenin significantly decreased escape latency as control and apigenin-treated sham groups (P<0.05): compared with the KA group only on Day 2 ( =0.01). Treatmentthis indicates of the that KA groupapigenin with restores apigenin the significantly working ThereRegarding was no significantthe total distancedifference traveled, between groupsthe same on increased the percentage alternation behavior ( days 4 and 5 (Figure 3A). memory deficit induced by kainite. Iran J Basic Med Sci, Vol. 22, No. 7, Jul 2019 755 Hashemi et al. Protective effects of apigenin in temporal lobe epilepsy

Figure 4.

Effect of apigenin (50 mg/kg/day) on the retrieval phase of the Morris water maze (MWM) test. The statistical analysis of data from first entry to the target quadrant (A), theP <0.01,number *** of P crossings into the target regionP (B), andP the total time spent in the target region (c) revealed significant impairment in memory retrieval after status epilepticus. However, apigenin restored memory in apigenin-treated groups All data are expressed as mean±SEM. ** <0.001 (KA vs control) ; # <0.05, ## <0.01 (apigenin+kA vs KA)

Figure 5. memory shown by the percentage of alternative behavior in the Effect of apigenin (50 mg/kg/day) on spatial working P P Y-maze task. Apigenin treated rats restored the working memory deficit induced by KA. * <0.05 (vs control); # <0.05 (vs KA) Effect of oral administration of apigenin on neuronal loss and neurodegeneration in KA-induced rat model of TLE Figure 6. In the present study, the number of live neurons using hippocampus Nissl staining and neurodegeneration using Fluoro-Jabe Effect of apigenin (50 mg/kg/day) on neuronal loss in the B in the hilar area of the hippocampus of all experimental protection(A) Quantification after apigenin of Nissl-positive treatment cells was revealedobserved. significant neuronal damage in the hilus of the hippocampus. However, significant groups was counted and analyzed (Figures P6 & 7). Data ***(B)P Representative images showingP Nissl staining in the hilar area of shownshowed in thatFigure intracerebroventricular 6, in the hilar area compared microinjection with the the hippocampus. Scale bar = 100 μm controlof KA leads and toapigenin-sham a significant neuronaloperated loss,groups, <0.001. apigenin As <0.001 (KA vs control); ### <0.001 (apigenin+KA vs KA) P<0.001; treatment of the KA group at a dose of 50 mg/kg for 5 this reduction as compared to the KA group ( days before induction of epilepsy significantly prevented Figures 6 & 7). 756 Iran J Basic Med Sci, Vol. 22, No. 7, Jul 2019 Protective effects of apigenin in temporal lobe epilepsy Hashemi et al.

A

Figure 8. Effect of apigenin on Cytochrome C release in hilar area

B hilus of hippocampus. This effect was ameliorated after apigenin

treatment(A) Ka significantly increased the cytochrome c positive cells in the ***P P

hippocampus<0.001 (vs. from control); rat under ## apigenin<0.01 (apigenin+ treatments ka vs. ka) (B) Immunohistochemistry of caspase-3 positive cells in the

Scale Bar = 100 μm the apigenin group. To our knowledge, no study till now

hasrecurrent revealed seizure the and effect hippocampal of apigenin neuronal on memory loss are deficit the inducedtwo important by epilepsy. causes of Apart memory from impairment antiepileptic in epilepsy. drugs, Recurrent seizures have been found to be involved Figure 7. Effect of apigenin on neurodegeneration in the hippocampus.

(A) Quantitative analysis showed a significant decrease in Fluoro- in cognition and memory deficits in patients with BJade staining B positive in hilus cells region in apigenin-treated of hippocampus of compared ka and apigenin+ka to ka group groups. .### epilepsy (23). In fact, a close correlation between the P<0.001 by one-way ANOVA. (B) Representative sections of Fluoro-Jade degreeshowed of anti-seizure memory insufficiency activity whichand seizure may, frequencyto some hasextent, been be shownresponsible (24). for In improvement the present study,in the apigeninmemory Abundant Fluoro-Jade B positive neurons were seen in ka group, but to performance following apigenin pretreatment. This is a lesser degree in apigenin-treated rats. Scale Bar = 100 μm Han et al., who showed that apigenin has anticonvulsant activity in kainate- Effect of oral administration of apigenin on consistent with the findings of cytochrome c, mitochondrial cell death pathway in On the other hand, apigenin can interact with gamma- KA-induced rat model of TLE We further conducted cytochrome c induced seizure due to its anti-oxidative) receptors, properties suggesting (25). immunohistochemistry to evaluate whether apigenin A could protect hippocampal neurons against the aminobutyriccan also inhibit acid glutamate type A (GABA release in rat hippocampus deleterious effect of cytochrome c and proapoptotic an enhancement of the GABAergic system (26). Apigenin mitochondrial cell death pathway. In this regard, explain the anticonvulsant activity of apigenin is cytochrome c immunoreactivity in hilar neurons was (27).through Therefore, exerting a the balance other between probable the mechanisminhibitory and to excitatory neurotransmitters in the hippocampus. It has been documented that seizures result in P excessive neural loss and hyperactivity of the apoptotic significantly greater in KA than in the control group (P<0.001; Figure 8). Although apigenin treatment significantly reduced the release of cytochromeP<0.001). c ( <0.01 vs KA), there was still a significant difference pathways (28), especially in the hippocampus: this leads between control and apigenin treated groups ( to memory deficit in TLE patients. Neural cell loss in the Discussion CA3/CA1Both intrinsic and andhilar extrinsic regions ofapoptotic the hippocampus pathways initiatecan be In this study, anticonvulsant cognitive improvement directly correlated with memory loss in epilepsy (29). and neuroprotective effects of apigenin in a rat model afterfocus of seizures the intrinsic (28). pathway. Cytochrome In this c study, release hippocampal from the involvement of the mitochondrial apoptotic pathway mitochondrianeuronal loss andintermembrane cytochrome cspace release has were been found the major to be ofdue TLEto the have inhibitory been investigated,role of apigenin and on the cytochrome possible decreased in the apigenin-treated group: this suggests a c release has been assessed. The improvement of neuroprotective role for apigenin via attenuation of the learning and memory by apigenin has been shown in mitochondrial apoptotic pathway. In agreement with some neurodegenerative diseases, considering the anti- our results, data of a study has shown that apigenin oxidative and neuroprotective properties of apigenin can protect against the induced pluripotent stem cell by blocking caspase-3 asin our the study, main reasonno memory for memory improvement enhancement was observed (14, 21). in model of Alzheimer’s disease Apigenin can also stimulate neurogenesis (22): however, activity (30). Iran J Basic Med Sci, Vol. 22, No. 7, Jul 2019 757 Hashemi et al. Protective effects of apigenin in temporal lobe epilepsy

Our data not only shows the anti-apoptotic ability of apigenin but also reveals its protective role on Neuroprotective, anti-amyloidogenic and neurotrophic effects mitochondria. 14. Zhao L, Wang J-L, Liu R, Li X-X, Li J-F, Zhang L. 2013; 18:9949-9965. Conclusion of apigenin in an Alzheimer’s disease mouse model. Molecules We concluded that apigenin can exert a protective through p53-dependent pathway in human cervical carcinoma 15. Zheng P-W, Chiang L-C, Lin C-C. Apigenin induced apoptosis anticonvulsant and anti-apoptosis activity. Overall, 16. Shukla S, Gupta S. Molecular targets for apigenin-induced effectthis study on memory-deficitsuggests that apigenin induced offers by kainite great potential through cellcells. cycle Life arrest Sci 2005; and 76:1367-1379.apoptosis in prostate cancer cell xenograft. for further investigation as a therapeutic agent in the Mol Cancer Ther. 2006; 5:843-852.

through proteasomal degradation of HER2/neu in HER2/neu- 17. Way T-D, Kao M-C, Lin J-K. Apigenin induces apoptosis managementAcknowledgment of memory decline in TLE. overexpressing breast cancer cells via the phosphatidylinositol This work was supported by the Medical Research J Biol Chem 2004; 279:4479-4489. 3-kinase/Akt-dependent pathway. , et al. Protective Council [grant number 94-04-30-26998] of Iran isolated18. Hu J, rat Li Z,heart. Xu L-t, Cardiovasc Sun A-j, ToxicolFu X-y, Zhang2015; 15:241-249.L UniversityConflicts of MedicalInterest Sciences, Tehran, IRAN. effect of apigenin on ischemia/reperfusion injury of the of apigenin against acrylonitrile-induced subchronic sperm 19. Dang Y, Li Z,Food Luo Chem B, Pan Toxicol L, Wei 2017;Q, Zhang 109:517-525. Y. Protective effects ReferencesThere were no conflicts of interest. injury in rats. New evidence from animal models. Neuroscientist 2010; degeneration.20. Schmued LC,Brain Hopkins Res 2000; KJ. 874:123-130. Fluoro-Jade B: a high affinity 16:253-275.1. Giblin KA, Blumenfeld H. Is epilepsy a preventable disorder? fluorescent marker for the localization of neuronal Karl T, et al. neurogenesis and neural stem cells in temporal lobe epilepsy. 21. Liang H, Sonego S, Gyengesi E, Rangel A, Niedermayer G, Epilepsy2. Kuruba Behav. R, 2009; Hattiangady 14:65-73. B, Shetty AK. Hippocampal Anti-inflammatoryFree Radic Biol and Med neuroprotective 2017; 108:S10. effect 3. Pauli E, Hildebrandt M, Romstöck J, Stefan H, Blümcke I. of apigenin: studies in the GFAP-IL6 mouse model of chronic neurogenesis:neuroinflammation. Mars, Inc., the Salk Institute for Biological Studies: predicted by hippocampal granule cell loss. Neurology 2006; WO2008147483.22. Taupin P. Apigenin Expert and Opin related Ther compoundsPat 2009; 19:523-527. stimulate adult 67:1383-1389.Deficient memory acquisition in temporal lobe epilepsy is epilepsy. Neurosci Biobehav Rev 2013; 37:2887-2899. impairment23. Zhou JL, followingShatskikh recurrent TN, Liu X, seizures. Holmes Eur GL. JImpaired Neurosci single2007; 5.4. LévesqueHelmstaedter M, Avoli C, Elger M. The C. kainic Chronic acid temporal model of lobetemporal epilepsy: lobe 25:3667-3677.cell firing and long‐term potentiation parallels memory 24. Höller Y, Trinka E. What do temporal lobe epilepsy and Brain 2009; 132:2822-2830. 6.a neurodevelopmentalHöller Y, Trinka E. What or progressively do temporal dementing lobe epilepsy disease? and Front Syst Neurosci 2014; 8:58. progressive mild cognitive impairment have in common?, et al. Front Syst Neurosci 2014; 8. Protection of apigenin against kainate-induced excitotoxicity progressive mild cognitive impairment have in common? by25. anti-oxidative Han J-Y, Ahn S-Y, effects. Kim BiolC-S, PharmYoo S-K, Bull Kim 2012; S-K, 35:1440-1446.Kim H-C seizures induce progressive hippocampal neuron loss and 7. Kotloski R, LynchProg M, Brain Lauersdorf Res 2002; S, Sutula 135:95-110. T. Repeated brief , et al. 8. Cock H. The role of mitochondria in status epilepticus. 26. Gazola AC, Costa GM, Castellanos L, Ramos FA, Reginatto Epilepsiamemory deficits. 2007; 48:24-27. quadrangularisFH, Lima T pericarp. Involvement Revista of Brasileira GABAergic de pathwayFarmacognosia in the 2015;sedative 25:158-163. activity of apigenin, the main flavonoid from Passiflora challenges and solutions. Neuropsychiatr Dis Treat 2016; , et al. 12:2605-2616.9. Dalic L, Cook MJ. Managing drug-resistant epilepsy: 10. Meador KJ. Cognitive and memory effects of the new rat27. hippocampus.Chang CY, Lin TY, Eur Lu J PharmacolCW, Wang CC, 2015; Wang 762:72-81. YC, Chou SSP antiepileptic drugs. Epilepsy Res 2006; 68:63-67. 28.Apigenin, Henshall a natural DC, Simon flavonoid, RP. Epilepsy inhibits and glutamate apoptosis release pathways. in the J Cereb Blood Flow Metab 2005; 25:1557-1572.

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