molecules

Article Astaxanthin Protects PC12 Cells against Homocysteine- and Glutamate-Induced Neurotoxicity

1, 2,3,4, 1 4, Chi-Huang Chang †, Kuan-Chou Chen †, Kuo-Chun Liaw , Chiung-Chi Peng * and Robert Y. Peng 1,*

1 Department of Biotechnology, College of Medical and Health Care, Hungkuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu District, Taichung City 43302, Taiwan; [email protected] (C.-H.C.); [email protected] (K.-C.L.) 2 Department of Urology, School of Medicine, College of Medicine, Taipei Medical University, 250 Wu-Shing St., Taipei 11031, Taiwan; [email protected] 3 Department of Urology, Taipei Medical University Shuang-Ho Hospital, 291, Zhong-Zheng Rd., Zhong-He, Taipei 23561, Taiwan 4 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan * Correspondence: [email protected] (C.-C.P.); [email protected] (R.Y.P.) Two authors contributed equally. †


Received: 30 September 2019; Accepted: 11 December 2019; Published: 5 January 2020


Abstract: Memory impairment has been shown to be associated with glutamate (Glu) excitotoxicity, homocysteine (Hcy) accumulation, and oxidative stress. We hypothesize that Glu and Hcy could damage neuronal cells, while astaxanthin (ATX) could be beneficial to alleviate the adverse effects. Using PC12 cell model, we showed that Glu and Hcy provoked a huge amount of reactive oxygen species (ROS) production, causing mitochondrial damage at EC50 20 and 10 mm, respectively. The mechanisms of action include: (1) increasing calcium influx; (2) producing ROS; (3) initiating lipid peroxidation; (4) causing imbalance of the Bcl-2/Bax homeostasis; and (5) activating cascade of caspases involving caspases 12 and 3. Conclusively, the damages caused by Glu and Hcy to PC12 cells can be alleviated by the potent antioxidant ATX.

Keywords: PC12 cells; glutamate (Glu); homocysteine (Hcy); intrinsic apoptotic pathways; neuroprotective; astaxanthin (ATX)

1. Introduction Glutamate is the most well-known excitatory neurotransmitter, occurring in over 50% of nervous tissues [1]; it is involved in the process of learning, cognition, and neurodegeneration [2]. Two major classes of glutamate receptors are recognized, the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs) [3]. All the iGluRs function as nonselective cation channels, allowing the passage of Na+,K+, and small amounts of Ca2+ in some cases [2]. The mGluRs modify neuronal and glial excitability through G protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and cAMP [2]. Excess extracellular glutamate level could induce brain lesions, neuronal death, and other pathological changes in several organs associated with endocrine function; this pathway is called excitotoxicity, which was coined by Olney (1969) [4]. During the development process for Alzheimer’s disease (AD), excessive activated extracellular glutamate receptors prompt changes in ion channels and signaling systems [5–7]. The intracellular calcium produces an avalanche of reactive oxygen species (ROS), leading to mitochondria-mediated apoptotic process, in particular, during such a pathological condition [5–7]. ROS and the oxidative-stress-induced

Molecules 2020, 25, 214; doi:10.3390/molecules25010214 www.mdpi.com/journal/molecules Molecules 2020, 25, 214 2 of 17 injuries lead to nuclear degradation in both neuronal and vascular systems, causing early loss of cellular membrane asymmetry that mitigates inflammation and vascular occlusion [8]. The mechanism has been reported to involve the Wnt pathway and the serine-threonine kinase Akt as well as the downstream substrates like GSK-3β, Bad, and Bcl-xL [8]. Homocysteine (Hcy), a neurotoxic amino acid, accumulates in several neurodegenerative disorders, including AD [9,10]. In AD patients, the blood plasma Hcy level has been found to rise above 20 µM[2]. Hcy acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor [11]. The PC12 cell line, originally isolated from a pheochromocytoma in the rat adrenal medulla, has become an in vitro model for studying numerous problems in neurobiology, neurochemistry [12,13], and Alzheimer’s and Parkinson’s diseases [14]. Like glutamate, Hcy induced calcium influx, depolarization of mitochondrial membrane potential, and changes of Bcl-2 and Bcl-xL in the PC12 cell model [15]. Accumulating studies of animal and cell culture models have clearly shown that the ability of neurons to regulate cellular Ca2+ levels and dynamics properly is compromised by both oxidative stress and impaired cellular energy metabolism [16], which features the common molecular pathological mechanisms raised by glutamate and homocysteine. Abnormalities in calcium regulation in astrocytes, oligodendrocytes, and microglia have been documented in studies of experimental models of AD [17]. Human studies suggest that Hcy plays a role in brain damage and in cognitive and memory decline. Numerous studies in recent years have implicated the role of Hcy as a cause of brain damage [18]. Astaxanthin (ATX) is a red-orange xanthophyll carotenoid, exhibiting a diversity of bioactivities, including antioxidant and peroxyl radical scavenging, anti-inflammation, cardiovascular health protection, anticancer, and antioxidant bioactivity [19]. ATX, with unique cell membrane actions and diverse biological activities, acts as a potential neuroprotective agent for neurological diseases [20]. We hypothesize that Glu and Hcy may cooperatively attack the glutamate receptor; whether ATX can effectively issue its protective effect against the neurotoxicity induced by cotreatment with these two ligands is still unclear. To verify this, we carried out this present experiment.

2. Results and Discussions

2.1. Cell Viability Affected by Astaxanthin, Homocysteine, and Glutamate ATX (Figure1a) did not show any cytotoxicity until up to a dose of 100 µM. Within the doses from 10 µM to 100 µM, it is evidently seen that the cell viability was inhibited in a dose-dependent manner (Figure1b). The 50% inhibition only occurred at 100 µM after incubation for 72 h (Figure1b), rather consistent with the observation of Ye et al. (2013) [21]. Both Hcy and Glu were revealed to be highly cytotoxic. The cytotoxicity also occurred in a dose-dependent manner. The IC50 was found to occur at 10 mM Hcy and 20 mM Glu at 24 h (Figure1c,d). PC12 cells were found to be more susceptible to Hcy than Glu, with susceptibilities of 3.4%/mM and 1.3%/mM, respectively, at 48 h of incubation (Figure1c,d). On the other hand, Hcy or Glu have been reported to induce apoptosis in neuronal cells by upregulating GRP78 [22–24]. Molecules 2020, 25, 214 3 of 17 Molecules 2020, 25, x 3 of 17

(a) (b)

(c) (d)

FigureFigure 1.1.Chemical Chemical structure structure of of astaxanthin astaxanthin and and the etheffect effect of each of compoundeach compound of interest of interest on the viability on the ofviability PC12 cellof PC12 line culturedcell line cultured for 24, 48, for and 24, 7248, h. and 24 72 h: h. empty 24 h: bars;empty 48 bars; h: gray 48 h: bars; gray 72 bars; h: dark72 h: netdark bars. net PC12bars. cellsPC12 were cells seededwere seeded onto a 24-wellonto a 24-well plate at plate 5 10 at4 cells5 × /10mL4 cells/mL and cultured and incultured serum-free in serum-free medium × overnight,medium overnight, then treated then with treated astaxanthin with astaxanthin (ATX), homocysteine (ATX), homocysteine (Hcy), or glutamate (Hcy), or glutamate (Glu). (a) Chemical(Glu). (a) structureChemical of structure ATX (depicted of ATX from (depicted PubChem, from https:PubChem,//pubchem.ncbi.nlm.nih.gov https://pubchem.ncbi.nlm.nih.gov/)./). (b) ATX (0–100 (b) µATXM). ((0–100c) Hcy μ (0–40M). ( mM).c) Hcy (d (0–40) Glu (0–20mM). mM).(d) Glu Data (0–20 are mM). expressed Data as are means expressedSD (nas= means3). * p ±< SD0.05; (n ** = p3).< 0.01;* p < ± ***0.05;p < **0.005 p < 0.01; vs. control*** p < 0.005 at the vs. same control time. at the same time.

AA concentrationconcentration ofof HcyHcy upup toto 1010 µμMM hashas beenbeen measuredmeasured inin brainbrain [[25].25]. ElevatedElevated concentrationconcentration ofof totaltotal HcyHcy in in plasma plasma ( >(>1212 µ μmolmol/L)/L) is is a riska risk factor factor for for several several diseases diseases of the of centralthe central nervous nervous system system [10]. However,[10]. However, more more modest modest levels levels (15–50 (15–50 mM) mM) are foundare found very very commonly commonly in the in the general general population population (a condition(a condition known known as as hyper-homocysteinemia) hyper-homocysteinemia) [26 [26,27].,27]. The The cell cell viability viability could could be be a affectedffected byby severalseveral factorsfactors includingincluding thethe kindkind ofof cellcell lineline andand mediummedium forfor incubationincubation [[28].28]. FroissardFroissard andand DuvalDuval (1994)(1994) reportedreported thatthat glutamate glutamate (1–10 (1–10 mM) mM) led led to to a dose-dependenta dose-dependent cell cell damage damage (70% (70% of cell of lysiscell lysis at 10 at mM) 10 mM) [29]. In[29]. contrast, In contrast, according according to Wang to etWang al. (2016), et al. 50%(2016), of severe 50% of suppression severe suppression of Y79 cell of viability Y79 cell by viability glutamate by occurredglutamate at occurred a dose ofat a 20 dose mM of [ 2030 ].mM Literature [30]. Literatu elsewherere elsewhere also often also usedoften 25used mM 25 ofmM glutamate of glutamate for conductingfor conducting similar similar studies studies [5, 31[5,31–33],–33], consequently, consequently, a dose a dose of of 10 10 mM mM Hcy Hcy and and 20 20 mM mM Glu Glu has has beenbeen adoptedadopted inin thisthis article.article.

2.2. Potentiation of Glutamate on the Cytotoxicity of Homocysteine 2.2. Potentiation of Glutamate on the Cytotoxicity of Homocysteine BothBoth GluGlu andand HcyHcy areare cytotoxic,cytotoxic, andand wewe questionedquestioned whetherwhether thesethese twotwo compoundscompounds maymay exertexert anyany additiveadditive oror synergisticsynergistic eeffectffect onon thethe cellcell viability.viability. GluGlu atat 55 mMmM seemedseemed toto bebe totallytotally ineineffectiveffective toto potentiate the cytotoxicity of 10 mM Hcy. At a dose >10 mM (up to 20 mM), Glu significantly potentiated the Hcy cytotoxicity in a dose- and time-dependent manner (Figure 2a). Much of

Molecules 2020, 25, 214 4 of 17

potentiate the cytotoxicity of 10 mM Hcy. At a dose >10 mM (up to 20 mM), Glu significantly potentiated the Hcy cytotoxicity in a dose- and time-dependent manner (Figure2a). Much of literature has evidenced Molecules 2020, 25, x 4 of 17 that Hcy not only induces direct neurotoxicity, but also potentiates both amyloid-β and glutamate neurotoxicityliterature [34 has]. Similarly,evidenced itthat has Hcy been not revealed only induces that activationdirect neurotoxicity, of group IIIbut metabotropic also potentiates glutamate both receptorsamyloid- stimulatesβ and theglutamate excitotoxic neurotoxicity action of[34]. Hcy Similarly, and homocysteic it has been revealed acid [2]. that Previously, activation Lecleric of group et al. evidencedIII metabotropic the occurrence glutamate of NMDA receptors receptor stimulates inPC12 the excitotoxic cells and action concluded of Hcy and that homocysteic PC12 cells acid express predominantly[2]. Previously, the splice Lecleric variant et al. NMDAR1-4a evidenced the and occurrence smaller amountsof NMDA of receptor NMDAR1-1a, in PC12 NMDAR1-2a, cells and and NMDAR1-3aconcluded that [35]. PC12 More cells recently, express Sibarov predominantly et al. implicated the splice that variant GluN2A NMDAR1-4a subunit-containing and smaller NMDA amounts of NMDAR1-1a, NMDAR1-2a, and NMDAR1-3a [35]. More recently, Sibarov et al. receptors as the preferential neural targets of Hcy [36]. Moreover, Hcy has been confirmed not only implicated2+ that GluN2A subunit-containing NMDA receptors2+ as the preferential neural targets of to be CaHcy- [36]. and Moreover, NMDA receptor-dependent,Hcy has been confirmed but not also only Ca to be-independent, Ca2+- and NMDA mainly receptor-dependent, mediated by the “synapticbut type”also Ca GluN12+-independent,/2 NMDAR mainly [36]. Suggestively,mediated by the degree“synaptic of bindingtype” GluN1/2 by diff erentNMDAR ligands [36]. like Glu andSuggestively, Hcy for these the receptordegree of subunits binding by and different the outcome ligands responseslike Glu and may Hcy be for synergistically these receptor subunits additive on one hand,and butthe outcome competitively responses expelling may be onsynergistically the other hand. additive on one hand, but competitively expelling on the other hand. 2.3. Protective Effect of Astaxanthin against The Insult Exerted by Homocysteine and Glutamate 2.3. Protective Effect of Astaxanthin against The Insult Exerted by Homocysteine and Glutamate ATX at 2–5 µM significantly alleviated the cell viability from the insult caused by Hcy (10 mM) μ (Figure2b), GluATX (20at 2–5 mM) M (Figure significantly2c), and alleviated the combined the cell viability Hcy (10 from mM) the plus insult Glu caused (20 mM) by Hcy (Figure (10 mM)2d) at (Figure 2b), Glu (20 mM) (Figure 2c), and the combined Hcy (10 mM) plus Glu (20 mM) (Figure 2d) 24 and 48 h. As seen, ATX at 2 and 5 µM secured the cell viability for about 12%–14% and 21%–22%, at 24 and 48 h. As seen, ATX at 2 and 5 μM secured the cell viability for about 12%–14% and 21%– respectively, (Figure2b) when insulted by Hcy. ATX at 2 and 5 µM secured the cell viability for about 22%, respectively, (Figure 2b) when insulted by Hcy. ATX at 2 and 5 μM secured the cell viability for and 14%–19%about and and 14%–19% 19%–22% and (Figure 19%–22%2c) when(Figure insulted 2c) when by in Glusulted for by 24–48 Glu for h, respectively.24–48 h, respectively. The alleviation The againstalleviation the combined against insults the combined was seen insults as rather was seen comparable as rather comparable at 2 µM of at ATX 2 μM (11% of ATX and (11% 13% and for 13% 24 and 48 h, respectively)for 24 and 48 buth, respectively) was much but better was at much 5 µM better ATX at (26%) 5 μM atATX 48 (26%) h (Figure at 482 hd). (Figure 2d).

(a) (b)

(c) (d)

Figure 2. Combined effect of target compounds on the viability of PC12 cell line. PC12 cells were

Molecules 2020, 25, 214 5 of 17 MoleculesMolecules 2020 2020, ,25 25, ,x x 55 ofof 1717

FigureFigure 2.2. CombinedCombined effecteffect ofof targettarget compoundscompounds onon thethe viabilityviability ofof PC12PC12 cellcell line.line. PC12PC12 cellscells werewere 4 seededseeded ontoontoseeded 24-well24-well onto platesplates 24-well atat 55 plates×× 10104 cells/mLcells/mL at 5 10 and4andcells culturedcultured/mL and inin cultured serum-freeserum-free in serum-free mediummedium overnight,overnight, medium overnight,thenthen then × treatedtreated with withtreated different different with combinations combinations different combinations of of ATX, ATX, Hcy Hcy of ATX,an and/ord/or Hcy Glu Glu and separately. separately./or Glu separately. In In all all panels, panels, In all empty empty panels, bars: bars: empty 24 24 bars: 24 h. h.h. In In Figure FigureIn 2a, 2a, Figure gray gray2 bars: a,bars: gray 48 48 bars: h; h; dark dark 48 h;net net dark bars bars net: :72 72 bars: h. h. In In 72 Figure Figure h. In Figure2b–d, 2b–d, 2net netb–d, dark dark net bars: darkbars: bars:48 48 h. h. 48( (aa) )h.Glu Glu ( a(5–20 )(5–20 Glu (5–20 mM) μ μ mM)mM) plus plus Hcy plusHcy (10 Hcy(10 mM). mM). (10 mM).( (bb)) ATX ATX (b) ATX(1–10 (1–10 (1–10 μ M)M) µplus plusM) plusHcy Hcy Hcy(10 (10 mM). (10mM). mM). ( (cc)) ATX ATX (c) ATX (1–10 (1–10 (1–10 μ M)M)µ plusM) plus plus Glu Glu Glu (20 (20 (20mM). mM). mM). (d) ATX μ ((dd)) ATX ATX (1–10 (1–10(1–10 μ M)M)µM) plus plus plus Hcy Hcy Hcy (10 (10 (10 mM) mM) mM) plus plus plus Glu Glu Glu (20 (20 (20 mM). mM). mM). Data Data are are expressed expressed as asas means means ± ± SD SDSD ( n(n = = 3). 3).3). *: *: compared ± comparedcompared to toto the thethe control; control;control; 〒〒:: :vs. vs. Hcy Hcy Hcy 10 10 10 mM mM mM at at at the the the same same same time; time; time;  : :vs. vs. vs. 24 24 24 h h h at at at the the the same same same dose; dose; dose; ⊗ ⊗: :vs. vs. vs. Glu Glu Glu 20 mM at ⊗ 2020 mMmM atat thethe same samesame time; time;time; #: #:#: vs. vs. HcyHcy 1010 mM mM ++ GluGlu 2020 mM mM at at the the same samesame time. time.time. The The significance significancesignificance of of the thethe difference 〒 ## 〒 ⊗⊗   differencedifference was was judged judged by by confidence confidence levels levels of of * * p p < < 0.05; 0.05; # p p < < 0.05; 0.05; pp < < 0.05; 0.05; pp < < 0.05; 0.05; pp < < 0.05; 0.05; ** ** p p was judged⊗⊗ by confidence levels of * p < 0.05; p < 0.05; p < 0.05; ⊗ p < 0.05; p < 0.05; ** p < 0.01; << 0.01; 0.01; ## ## p p < < 0.01; 0.01; ⊗⊗ p p < < 0.01; 0.01;   p p < < 0.01; 0.01; *** *** p p < < 0.005. 0.005. ## p < 0.01; ⊗⊗ p < 0.01; p < 0.01; *** p < 0.005. Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating Literature Literaturehas implicated has implicated that ATX inhibits that ATX homocysteine-induced inhibits homocysteine-induced neurotoxicity neurotoxicity via alleviating via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac mitochondrialmitochondrial dysfunction dysfunction and signal and crosstalk signal [37]. crosstalk A similar [37]. result A similar was resultfound was in in found vitro incardiacin vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple cell culturescell [38]. cultures Previously, [38]. Previously, Zhang et al. Zhang found et th al.at foundATX exerted that ATX neuroprotective exerted neuroprotective effect via multiple effect via multiple signaling pathways including NFκκB and MAPK pathways, and implicated ATX to be used as a signaling signalingpathways pathwaysincluding includingNF B and NF MAPKκB and pathways, MAPK pathways,and implicated and implicated ATX to be ATX used to as be a used as a prophylactic or remediation agent against neuronal disorder [39]. prophylacticprophylactic or remediation or remediation agent against agent neuronal against disorder neuronal [39]. disorder [39].

2.4.2.4. Effect Effect of 2.4.of Astaxanthin Astaxanthin Effect of Astaxanthin on on the the Intracellular Intracellular on the Intracellular Calc Calciumium Ion Ion Calcium Level Level Affected Affected Ion Level by by A Homocysteine Homocysteineffected by Homocysteine and and Glutamate Glutamate and Glutamate CalciumCalcium ion ionCalcium dysregulation dysregulation ion dysregulation is is relevant relevant is to to relevant the the initiation initiation to the of initiationof Alzheimer's Alzheimer's of Alzheimer’s disease disease (AD), (AD), disease the the PC12 (AD),PC12 cell thecell PC12 cell modelmodel inin realitymodelreality has inhas reality pertinentlypertinently has pertinently reflectedreflected such reflectedsuch aa possibilitypossibility such a possibility [40].[40]. TheThe calcium [calcium40]. The ionion calcium influxinflux ion waswas influx highlyhighly was highly raisedraised by by the raisedthe insult insult by of theof 20 20 insult mM mM Glu ofGlu 20 (144%), (144%), mM Glu and/or and/or (144%), 10 10 mM andmM/ orHcy Hcy 10 (153%), (153%), mM Hcy and/or and/or (153%), the theand combined combined/or the combinedtreatment treatment treatment μμ (176%)(176%) (Figure(Figure(176%) 3a).3a). (Figure ATXATX 3 atata). 55 ATXMM fullyfully at 5 alleviatedµalleviatedM fully alleviated thethe calciumcalcium the influxinflux calcium raisedraised influx byby raised2020 mMmM by Glu,Glu, 20 butmMbut waswas Glu, but was slightlyslightly less lessslightly effective effective less against against effective that that against by by Hcy Hcy that (10 (10 by mM), mM), Hcy and (10and mM),was was much much and wasless less mucheffective effective less against against effective the the against combined combined the combined therapytherapy (133%)(133%)therapy (Figure(Figure (133%) 3a),3a), (Figure implicatingimplicating3a), implicating insufficientinsufficient insu mole ffimolecient numbernumber mole number forfor thethe for interactionsinteractions the interactions betweenbetween between ATXATX ATX and andand (Glu (Glu + (Glu+ Hcy); Hcy);+ Hcy);suggestively, suggestively, suggestively, a a higher higher a higher dose dose of doseof ATX ATX of may ATXmay be maybe required required be required for for such such for suchaa combined combined a combined insult. insult. insult. InIn addition,addition,In addition, HcyHcy hashas Hcy beenbeen has implicatedimplicated been implicated toto indirectlyindirectly to indirectly increaseincrease increase intracellularintracellular intracellular calciumcalcium calcium levelslevels byby activating activatingactivating ionotropicionotropic ionotropic andand and metabotropicmetabotropic metabotropic receptors receptors receptors [ 41[41], [41],], which, which, which, compared compared compared with with with glutamate, glutamate, glutamate, may may may create create create the reasons thethe reasonsreasonsto to elicitto elicitelicit diff differenterentdifferent outcomes outcomesoutcomes by such byby suchsuch a combination aa combinationcombination of pharmacological ofof pharmacologicalpharmacological actions actionsactions of Hcy ofof [ 2 Hcy,Hcy36,41 ]. [2,36,41].[2,36,41]. Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are TwoTwo involvedsubcellularsubcellular in organelles, theorganelles, cellular namelynamely pathogenesis thethe mitochondriamitochondria of AD; an increased andand endoplasmicendoplasmic oxidative reticulumreticulum stress and (ER),(ER), dysregulation areare of involvedinvolved incalciumin thethe cellularcellular homeostasis pathogenesispathogenesis also have ofof AD;AD; been anan reported increasedincreased [42 oxidativeoxidative]. Owing stressstress to the andand important dysregulationdysregulation role of ofof ER in the calciumcalcium homeostasis homeostasisregulation also ofalso Ca have have2+-signaling, been been reported reported ER-mitochondrial [42]. [42]. Owing Owing to to distancethe the important important in the role role neurons of of ER ER isin in tightlythe the regulation regulation controlled in the 2+ ofof Ca Ca2+-signaling,-signaling,physiological ER-mitochondrial ER-mitochondrial conditions. When distance distance the distancein in the the neurons neurons is decreased, is is tightly tightly Ca 2controlled +controlled-overload in in occurs the the physiological physiological both in the cytosol and 2+ conditions.conditions.mitochondria WhenWhen thethe [40distancedistance]. The reduction isis decreased,decreased, in the distanceCaCa2+-overload-overload between occursoccurs ER and bothboth mitochondria inin thethe cytosolcytosol may be andand implicated in mitochondriamitochondriaAlzheimer’s [40]. [40]. The The diseasereduction reduction (AD) in in pathologythe the distance distance by be be enhancedtweentween ER ER Ca and and2+-signaling mitochondria mitochondria [40]. may Linmay etbe be al.’s implicated implicated study indicated in in that 2+ Alzheimer’sAlzheimer’sATX disease disease attenuated (AD) (AD) pathology glutamate-inducedpathology by by enhanced enhanced elevation Ca Ca2+-signaling-signaling of CHOP [40]. [40]. and Lin ERLin et chaperoneet al.’s al.’s study study glucose-regulated indicated indicated that that protein ATXATX attenuated attenuated(GRP78), glutamate-induced glutamate-induced inhibiting glutamate-induced elevation elevation of of CH CH apoptosisOPOP and and throughERER chaperone chaperone rescuing glucose-regulated glucose-regulated the redox balance protein protein and inhibiting (GRP78),(GRP78), inhibiting glutamate-inducedinhibiting glutamate-induced glutamate-induced calcium influx apoptosis apoptosis [24]. th throughrough rescuing rescuing the the redox redox balance balance and and inhibiting inhibiting glutamate-inducedglutamate-induced calcium calcium influx influx [24]. [24]. 2.5. Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate 2.5.2.5. Effect Effect of of Astaxanthin Astaxanthin on on the the ROS ROS Produc Productiontion Caused Caused by by Homocysteine Homocysteine and and Glutamate Glutamate The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), TheThe levellevelHcy (10ofof reactivereactive mM), and oxygenoxygen the combined speciesspecies (ROS)(ROS) treatment waswas highly forhighly 24 h inducedinduced (Figure 3 bybyb). treatingtreating The ROS withwith produced GluGlu (20(20 were mM),mM), e ffectively HcyHcy (10 (10 mM), mM),suppressed and and the the bycombined combined ATX at 5 treatment treatmentµM (Figure for for3b). 24 24 h h (Figure (Figure 3b). 3b). The The ROS ROS produced produced were were effectively effectively suppressedsuppressed by by TheATX ATX mitochondrial at at 5 5 μ μMM (Figure (Figure Ca 3b). 3b).2+-overload can lead to increased generation of reactive oxygen species, TheThe mitochondrialinducingmitochondrial the opening CaCa2+2+-overload-overload of the mitochondrial cancan leadlead toto increased permeabilityincreased genegene transitionrationration ofof pore reactivereactive and ultimately oxygenoxygen species,species, causing neuronal inducinginducing apoptoticthethe openingopening and ofof necrotic thethe mitochondrialmitochondrial cell death [40 ].permeabilitypermeability In addition, transition thetransition NO pathway porepore andand relating ultimatelyultimately with hyperexcitability causingcausing is neuronalneuronal inducedapoptoticapoptotic by and homocysteineand necroticnecrotic thiolactonecellcell deathdeath (HcyT)[40].[40]. InIn [43 addition,addition,]. ATX is athethe nutrient NONO withpathwaypathway unique relatingrelating cell membrane withwith actions hyperexcitabilityhyperexcitabilityand diverse is is induced induced clinical by by benefits homocysteine homocysteine [44]. ATX thiola thiola isctone thectone most (HcyT) (HcyT) effective [43]. [43]. ATX antioxidant.ATX is is a a nutrient nutrient This with with molecule unique unique neutralizes cellcell membrane membranefree radicals actions actions orandand other diverse diverse oxidants clinical clinical by benefits benefits either accepting[44]. [44]. ATX ATX oris is the donatingthe most most effective electrons,effective antioxidant. antioxidant. without being This This destroyed moleculemolecule neutralizes orneutralizes becoming free free a radicals radicals pro-oxidant or or other other in oxidants theoxidants process by by either [either44]. ATXaccepting accepting blocks or or oxidativedonating donating electrons, electrons, DNA damage without without and lowers beingbeing destroyed destroyed or or becoming becoming a a pro-oxidant pro-oxidant in in the the process process [44]. [44]. ATX ATX blocks blocks oxidative oxidative DNA DNA damage damage andand lowerslowers C-reactiveC-reactive proteinprotein (CRP)(CRP) andand otherother inflammationinflammation biomarkersbiomarkers [44].[44]. ATX-mediatedATX-mediated

Molecules 2020, 25, 214 6 of 17

C-reactiveMolecules protein 2020, 25, (CRP)x and other inflammation biomarkers [44]. ATX-mediated neuroprotection6 of 17 in experimental models of neurological disorders involves antioxidant, anti-inflammatory, and neuroprotection in experimental models of neurological disorders involves antioxidant, anti- antiapoptotic mechanisms [45–47]. inflammatory, and antiapoptotic mechanisms [45–47]. 2.6. Effect of Astaxanthin on the MDA Production Resulting from Insult of Homocysteine and Glutamate 2.6. Effect of Astaxanthin on the MDA Production Resulting from Insult of Homocysteine and Glutamate MDAMDA was highlywas highly stimulated stimulated when when treated treated with with GluGlu (20 mM), mM), Hcy Hcy (10 (10 mM), mM), and and the thecombined combined therapytherapy to levels to levels of 1.48, of 1.48, 1.80, 1.80, and and 2.20 2.20µ molμmol//µμgg of of protein,protein, respectively, respectively, which which were were suppressed suppressed by by 2+ treatmenttreatment with with 5 µM 5 ATXμM ATX (Figure (Figure3c), 3c), similar similar to to the the outcomes outcomes forfor CaCa2+ influxinflux (Figure (Figure 3a)3 anda) and ROS ROS inductioninduction (Figure (Figure3b). ATX3b). ATX was was evidenced evidenced to to be be a a strong strong peroxylperoxyl radical scavenger, scavenger, exerting exerting a strong a strong protectiveprotective effect effect on the on humanthe human brain brain [19 [19,48].,48].

(a)

(b)

(c)

Figure 3. Protective effect of astaxanthin against the calcium ion influx and oxidative stress induced Molecules 2020, 25, x 5 of 17

Figure 2. Combined effect of target compounds on the viability of PC12 cell line. PC12 cells were Molecules 2020, 25, 214 7 of 17 seeded onto 24-well plates at 5 × 104 cells/mL and cultured in serum-free medium overnight, then treated with different combinations of ATX, Hcy and/or Glu separately. In all panels, empty bars: 24 by target compounds. PC12 cells were cultured in serum-freeh. In Figure medium 2a, overnight,gray bars: then48 h; treateddark net with bars: 72 h. In Figure 2b–d, net dark bars: 48 h. (a) Glu (5–20 different combinations of ATX (5 µM), Hcy (10 mM), and/mM)or Glu plus (20 Hcy mM). (10 (a )mM). Intracellular (b) ATX calcium (1–10 μM) ion plus Hcy (10 mM). (c) ATX (1–10 μM) plus Glu (20 mM). level. (b) Reactive oxygen species (ROS) production. (c) Malondialdehyde(d) ATX (1–10 μM) (MDA) plus production.Hcy (10 mM) Data plus are Glu (20 mM). Data are expressed as means ± SD (n = 3). *: expressed as means SD (n = 3). *: compared to the control;compared: vs. Glu to 20the mM control; only; 〒: vs. vs. Hcy Hcy 10 10 mM at the same time; : vs. 24 h at the same dose; ⊗: vs. Glu ± ⊗ only; #: vs. Hcy 10 mM + Glu 20 mM. The significance20 of themM di atff erencethe same was time; judged #: vs. by Hcy confidence 10 mM + Glu 20 mM at the same time. The significance of the 〒 ⊗  # difference was judged## by confidence levels of * p < 0.05; # p < 0.05; p < 0.05; p < 0.05; p < 0.05; ** p levels of * p < 0.05; ⊗ p < 0.05; p < 0.05; p < 0.05; ** p < 0.01; ⊗⊗ p < 0.01; p < 0.01; p < 0.01; ## ⊗⊗  *** p < 0.005; ### p < 0.005. < 0.01; p < 0.01; p < 0.01; p < 0.01; *** p < 0.005.

2.7. Western Blot Analysis Indicated Astaxanthin Restored BaxLiterature and Bcl-2 has Homeostasis implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating Bcl-2 family members either promote or repressmitochondrial programmed dysfunction cell death. and Bax, signal a proapoptotic crosstalk [37]. A similar result was found in in vitro cardiac member of the Bcl-2 family of proteins, is a pore-forming,cell cultures mitochondria-associated [38]. Previously, Zhang protein et [al.49 ]found as well that ATX exerted neuroprotective effect via multiple κ as an activator for the mitochondrial permeability transitionsignaling porepathways (mPTP) including [50]. NF B and MAPK pathways, and implicated ATX to be used as a Western analysis revealed that the Bax (proapoptoticprophylactic protein) or remediation level was highly agent upregulatedagainst neuronal to disorder [39]. 128%, 134%, and 170% by Glu (20 mM), Hcy (10 mM), and the combined treatments, respectively (Figure4a). Interestingly, results clearly implicated2.4. theEffect synergistic of Astaxanthin e ffect on the of GluIntracellular plus Hcy, Calc i.e.,ium Ion Level Affected by Homocysteine and Glutamate 70% > (28% + 34%). ATX at 5 µM completely attenuatedCalcium the suppression ion dysregulation caused is by relevant glutamate to the or initiation of Alzheimer's disease (AD), the PC12 cell homocysteine alone and also ameliorated significantlymodel that in reality caused has by thepertinently combined reflected treatments such ofa possibility [40]. The calcium ion influx was highly glutamate with homocysteine (Figure4a). Conversely,raised Bcl-2 by the was insult severely of 20 down-regulatedmM Glu (144%), and/or by the 10 mM Hcy (153%), and/or the combined treatment insult of Glu (20 mM), Hcy (10 mM), and the combined(176%) treatment (Figure to 3a). 78%, ATX 76%, at and5 μM 56%, fully respectively alleviated the calcium influx raised by 20 mM Glu, but was (Figure4b). Hcy induced neurotoxicity via causing mitochondrialslightly less effective dysfunction against to regulatethat by Hcy Bcl-2 (10 family mM), and was much less effective against the combined and opening of mitochondrial permeability transitiontherapy pores (133%) [37]. Our (Figure results 3a), have implicating revealed thatinsufficient ATX mole number for the interactions between ATX was unable to have completely attenuated such inhibitions.and (Glu Although + Hcy); suggestively, ATX at 5 µM a alonehigher was dose able of toATX may be required for such a combined insult. increase the Bcl-2 level (antiapoptotic protein) to 116%, comparedIn addition, to 100%Hcy has of the been control implicated (Figure 4tob), indirectly increase intracellular calcium levels by the % recovery only attained 83% and 84% by theactivating single insult ionotropic exerted and by Glumetabotropic and homocysteine, receptors [41], which, compared with glutamate, may create respectively, and 70% by the combined insults. As thethe counterbalancereasons to elicit of different Bcl-2 and outcomes Bax is responsible by such a combination of pharmacological actions of Hcy for the homeostasis of the intrinsic pathway, we further[2,36,41]. examined the variation of Bcl-2/Bax ratio (Figure4c). More apparent results were seen when insultedTwo by subcellular Glu, Hcy andorganelles, the combined namely therapy; the mitochondria and endoplasmic reticulum (ER), are the Bcl-2/Bax ratios reached 60%, 58%, and 34%, respectively,involved in comparedthe cellular with pathogenesis 100% of the of controlAD; an increased oxidative stress and dysregulation of (Figure4c), for which ATX at 5 µM was found to havecalcium attenuated homeostasis the levels also to onlyhave 75%,been 72%,reported and 52%,[42]. Owing to the important role of ER in the regulation respectively, only. ATX (5 µM) alone increased the ratioof Ca to2+-signaling, 108% (Figure ER-mitochondrial4c). In contrast, Wangdistance et al.’sin the neurons is tightly controlled in the physiological study showed the complete reversal of Hcy (8 mM)-inducedconditions. neurotoxicity When the afterdistance treatment is decreased, with ATX Ca2+-overload occurs both in the cytosol and (5 µM) [37]; suggestively, such a deviation could be duemitochondria to the diff erence[40]. The in thereduction cell model in the and distance dose of between ER and mitochondria may be implicated in Hcy used. Alzheimer’s disease (AD) pathology by enhanced Ca2+-signaling [40]. Lin et al.’s study indicated that In cultured cells, astaxanthin protected the mitochondriaATX attenuated against glutamate-induced endogenous oxygen elevation radicals, of CHOP and ER chaperone glucose-regulated protein conserved their redox (antioxidant) capacity, and enhanced(GRP78), inhibiting their energy glutamate-induced production efficiency apoptosis [44]. through rescuing the redox balance and inhibiting Two distinct mechanisms leading to cytochrome C releaseglutamate-induced were described calcium by Eskes influx et [24]. al. (1998): one being stimulated by calcium and inhibited by cyclosporine A; the other being Bax-dependent, Mg2+ sensitive, but cyclosporine insensitive [49]. Apparently,2.5. Effect the of treatment Astaxanthin of PC12on the cellsROS withProduc Glu,tion Hcy Caused by Homocysteine and Glutamate and/or the combined therapy could suppress the cell viability (Figure1) by up-regulating apoptosis The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), (Figure4)[51]. Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b). The mitochondrial Ca2+-overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated

Molecules 2020, 25, 214 8 of 17 Molecules 2020, 25, x 8 of 17

(a)

(b)

Molecules 2020, 25, x 5 of 17

Figure 2. Combined effect of target compounds on the viability of PC12 cell line. PC12 cells were seeded onto 24-well plates at 5 × 104 cells/mL and cultured in serum-free medium overnight, then treated with different combinations of ATX, Hcy and/or Glu separately. In all panels, empty bars: 24 (c) h. In Figure 2a, gray bars: 48 h; dark net bars: 72 h. In Figure 2b–d, net dark bars: 48 h. (a) Glu (5–20 FigureFigure 4. Western 4. Western blot blot and mM)and quantified quantified plus Hcy protein protein (10 mM). expression expre (bssion) ATX affected aff (1–10ected by μ byM) different di plusfferent Hcy treatments. treatments. (10 mM). (a) Bax;( (ca)) ATX ( Bax;b) (1–10 (b) μM) plus Glu (20 mM). Bcl-2; andBcl-2; (c and) Bcl-2 (c)/ Bax.Bcl-2/Bax. Western(d )Western ATX blot (1–10 analysisblot μanalysisM) quantifiedplus quantified Hcy (10 into mM)into bar bar diagram.plus diagram. Glu Data(20 Data mM). are are expressed Data expressed are asexpressed as means as means ± SD (n = 3). *:

SD (n = 3). *: vs. control;compared: vs. Glu to 20 the mM control; only; 〒: vs. vs. Hcy Hcy 10 10 mM mM at only; the #:same vs. time; Hcy 10: mM vs. 24+ Gluh at the same dose; ⊗: vs. Glu ± ⊗ 20 mM. The significance of20 themM di atfference the same was time; judged #: vs. by Hcy confidence 10 mM levels+ Glu of20* mMp < 0.05;at the⊗ samep < 0.05; time. The significance of the # 〒 ⊗  p < 0.05; # p < 0.05; ** pdifference< 0.01; *** wasp < 0.005. judged by confidence levels of * p < 0.05; p < 0.05; p < 0.05; p < 0.05; p < 0.05; ** p < 0.01; ## p < 0.01; ⊗⊗ p < 0.01;  p < 0.01; *** p < 0.005.

Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

2.4. Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult. In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41]. Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca2+-signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca2+-overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer’s disease (AD) pathology by enhanced Ca2+-signaling [40]. Lin et al.’s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

2.5. Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b). The mitochondrial Ca2+-overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated

Molecules 2020, 25, x 9 of 17

means ± SD (n = 3). *: vs. control; ⊗: vs. Glu 20 mM only; 〒: vs. Hcy 10 mM only; #: vs. Hcy 10 mM + Glu 20 mM. The significance of the difference was judged by confidence levels of * p < 0.05; ⊗ p < 0.05; 〒 p < 0.05; # p < 0.05; ** p < 0.01; *** p < 0.005. Molecules 2020, 25, 214 9 of 17 2.8. Effect of Astaxanthin on the Expression of Caspase-12 and Caspase-3 in PC12 Cells Treated with 2.8.Homocysteine Effect of Astaxanthin and Glutamate on the Expression of Caspase-12 and Caspase-3 in PC12 Cells Treated with HomocysteineResults andfrom Glutamate Western blot analysis showed the levels of caspase-12 (42 kDa) and caspase-3 (17 kDa)Results were fromhighly Western upregulated blot analysis due to showed the insult the levelsof Glu of (20 caspase-12 mM), Hcy (42 kDa)(10 mM), and caspase-3and the combined (17 kDa) weretreatment highly (Figures upregulated 5 and due 6). to This the insultagain ofconfirms Glu (20 that mM), Glu, Hcy Hcy (10 mM),and/or and the the combined combined insult treatment could (Figuressuppress5 and the6 ).cell This viability again confirms(Figure 1), that up-regulating Glu, Hcy and apoptosis/or the combined (Figure 4) insult and the could activities suppress of caspase- the cell viability12 (Figure (Figure 5) and1), up-regulatingcaspase-3 (Figure apoptosis 6) and (Figure indicating4) and the the neurotoxic activities ofrole caspase-12 of glutamate (Figure and5) Hcy and to caspase-3PC12 cells (Figure [51]. 6) and indicating the neurotoxic role of glutamate and Hcy to PC12 cells [51].

Molecules 2020, 25, x 5 of 17

Figure 2. Combined effect of target compounds on the viability of PC12 cell line. PC12 cells were seeded onto 24-well plates at 5 × 104 cells/mL and cultured in serum-free medium overnight, then treated with different combinations of ATX, Hcy and/or Glu separately. In all panels, empty bars: 24 h. In Figure 2a, gray bars: 48 h; dark net bars: 72 h. In Figure 2b–d, net dark bars: 48 h. (a) Glu (5–20 mM) plus Hcy (10 mM). (b) ATX (1–10 μM) plus Hcy (10 mM). (c) ATX (1–10 μM) plus Glu (20 mM). (d) ATX (1–10 μM) plus Hcy (10 mM) plus Glu (20 mM). Data are expressed as means ± SD (n = 3). *: FigureFigure 5. 5.Caspase-12 Caspase-12 expression expression affected affected by di ffbyerent different treatments. treatments. (a) Western (a) blotWestern analysis; blot (b analysis;) quantified (b) ⊗ bar diagram. Data are expressed as means SD (n = 3). *: vs. control;compared: vs. Glu to 20 the⊗ mM control; only; 〒: vs. vs. Hcy 10 mM at the same time; : vs. 24 h at the same dose; : vs. Glu quantified bar diagram. Data are expressed± as means ± SD (n = 3). *:⊗ vs. control; : vs. Glu 20 mM 20 mM at the same time; #: vs. Hcy 10 mM + Glu 20 mM at the same time. The significance of the Hcyonly; 10 〒 mM: vs. only; Hcy#: 10 vs. mM Hcy10 only; mM#: vs.+ Hcy10Glu 20 mM mM. + TheGlu 20 significance mM. The significance of the difference of the was difference judged was by # 〒 ⊗  ⊗ 〒# difference was judged by confidence levels of * p < 0.05; p < 0.05; p < 0.05; p < 0.05; p < 0.05; ** p confidencejudged bylevels confidence of * p

Literature has implicated that ATX inhibits homocysteine-induced neurotoxicity via alleviating mitochondrial dysfunction and signal crosstalk [37]. A similar result was found in in vitro cardiac cell cultures [38]. Previously, Zhang et al. found that ATX exerted neuroprotective effect via multiple signaling pathways including NFκB and MAPK pathways, and implicated ATX to be used as a prophylactic or remediation agent against neuronal disorder [39].

2.4. Effect of Astaxanthin on the Intracellular Calcium Ion Level Affected by Homocysteine and Glutamate Calcium ion dysregulation is relevant to the initiation of Alzheimer's disease (AD), the PC12 cell model in reality has pertinently reflected such a possibility [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%), and/or 10 mM Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully alleviated the calcium influx raised by 20 mM Glu, but was slightly less effective against that by Hcy (10 mM), and was much less effective against the combined therapy (133%) (Figure 3a), implicating insufficient mole number for the interactions between ATX and (Glu + Hcy); suggestively, a higher dose of ATX may be required for such a combined insult. In addition, Hcy has been implicated to indirectly increase intracellular calcium levels by activating ionotropic and metabotropic receptors [41], which, compared with glutamate, may create the reasons to elicit different outcomes by such a combination of pharmacological actions of Hcy [2,36,41]. Two subcellular organelles, namely the mitochondria and endoplasmic reticulum (ER), are involved in the cellular pathogenesis of AD; an increased oxidative stress and dysregulation of calcium homeostasis also have been reported [42]. Owing to the important role of ER in the regulation of Ca2+-signaling, ER-mitochondrial distance in the neurons is tightly controlled in the physiological conditions. When the distance is decreased, Ca2+-overload occurs both in the cytosol and mitochondria [40]. The reduction in the distance between ER and mitochondria may be implicated in Alzheimer’s disease (AD) pathology by enhanced Ca2+-signaling [40]. Lin et al.’s study indicated that ATX attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced apoptosis through rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].

2.5. Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b). The mitochondrial Ca2+-overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated

Molecules 2020, 25, 214 10 of 17 Molecules 2020, 25, x 10 of 17

Molecules 2020, 25, x 5 of 17

Figure 2. Combined effect of target compounds on the viability of PC12 cell line. PC12 cells were seeded onto 24-well plates at 5 × 104 cells/mL and cultured in serum-free medium overnight, then treated with different combinations of ATX, Hcy and/or Glu separately. In all panels, empty bars: 24 h. In Figure 2a, gray bars: 48 h; dark net bars: 72 h. In Figure 2b–d, net dark bars: 48 h. (a) Glu (5–20 mM) plus Hcy (10 mM). (b ) ATX (1–10 μM) plus Hcy (10 mM). (c) ATX (1–10 μM) plus Glu (20 mM). (d) ATX (1–10 μM) plus Hcy (10 mM) plus Glu (20 mM). Data are expressed as means ± SD (n = 3). *: FigureFigure 6. 6.Caspase-3 Caspase-3 expression expression affected affected by di ffbyerent different treatments. treatments. (a) Western (a) blotWestern analysis; blot (b )analysis; quantified (b) ⊗ bar diagram. Data are expressed as means SD (n = 3). *: vs. control;compared: vs Glu to⊗ 20 the mM control; only; 〒: vs. vs. Hcy 10 mM at the same time; : vs. 24 h at the same dose; : vs. Glu quantified bar diagram. Data are expressed± as means ± SD (n = 3). *: vs.⊗ control; : vs Glu 20 mM only; Hcy〒: 10vs. mMHcy only;10 mM #: only; vs. Hcy10 #: vs. Hcy10 mM + mMGlu + 20 Glu mM, 20 ThemM, significance The significance of20 themM of di theatfference thedifference same was time; judgedwas judged#: vs. by Hcy 10 mM + Glu 20 mM at the same time. The significance of the # 〒 ⊗  ⊗ 〒 # difference was judged by confidence levels of * p < 0.05; p < 0.05; p < 0.05; p < 0.05; p < 0.05; ** p confidenceby confidence levels levels of * p of< *0.05; p < 0.05;⊗ p < 0.05;p < 0.05;p < p0.05; < 0.05;p #

2.5. Effect of Astaxanthin on the ROS Production Caused by Homocysteine and Glutamate The level of reactive oxygen species (ROS) was highly induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatment for 24 h (Figure 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figure 3b). The mitochondrial Ca2+-overload can lead to increased generation of reactive oxygen species, inducing the opening of the mitochondrial permeability transition pore and ultimately causing neuronal apoptotic and necrotic cell death [40]. In addition, the NO pathway relating with hyperexcitability is induced by homocysteine thiolactone (HcyT) [43]. ATX is a nutrient with unique cell membrane actions and diverse clinical benefits [44]. ATX is the most effective antioxidant. This molecule neutralizes free radicals or other oxidants by either accepting or donating electrons, without being destroyed or becoming a pro-oxidant in the process [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP) and other inflammation biomarkers [44]. ATX-mediated

Molecules 2020, 25, 214 11 of 17 release of cytochrome c, which in turn upregulated caspase-3 and provoked cell apoptosis. The role of ATX was seen to have dose-dependently alleviated all these adverse effects. Literature elsewhere has indicated that Aβ induced GRP78/Bip expression and activated caspases 4 and 12 [56], implicating the possible alternate target of ATX.

3. Materials and Methods

3.1. Chemicals Astaxanthin (ATX) and other chemicals used in this experiment were purchased from Sigma Aldrich Co. (St. Louis, MO, USA) unless otherwise stated. RPMI-1640 medium, trypsin, Fungizone (C47H73NO17), L-glutamine, penicillin and streptomycin were purchased from Invitrogen (Thermo-Fischer Scientific, Waltham, MA, USA). Purified NGF was purchased from Sigma-Aldrich (Sydney, Australia).

3.2. Source of Cell Line The cell line PC12 cells (BCRC 6008), originating from the pheochromocytoma on the rat adrenal gland, was purchased from the Bioresource Collection and Research Center (Hsin-Chu, Taiwan).

3.3. Cultivation of PC12 Cell Line The method for cultivation of PC12 cell line was conducted as previously reported [14].

3.4. Preparation of Complete Medium RPMI 1640 medium was prepared according to the supplier’s protocol in reverse osmosis/double distilled water (https://biocyclopedia.com/index/cellbiology_methods/cultured_for_neuronal_pc12_ cells.php)[57].

3.5. Preparation of Differentiating Medium

Serum-free RPMI 1640 medium stored at 4 ◦C was used. The PC12 cells were dislodged from stock culture dishes and triturated well using a glass Pasteur pipette to break up cell clumps. The cells were seeded onto 150-mm, 100-mm and 35-mm-dishes at densities of 5 106, (1–2) 106, and (2–5) 106 × × × cells, respectively, on poly-l-lysine-coated dishes and cultured in medium supplemented with a final concentration of NGF at 50 ng/mL. The cells were incubated at 37 ◦C under saturated water vapor and 7.5% CO2 atmosphere. The medium was changed three times per week. The culture was observed for 72 h. According to the instruction, by 7–10 days of treatment, at least 90% of the cells can generate neurites. (https://biocyclopedia.com/index/cell_biology_methods/cultured_for_neuroanal_ pc12_cells.php)[57]. The differentiated cells were roughly counted by a hematocytometer.

3.6. Preparation of Astaxanthin Solution The work of Bolin et al. (2010) was followed for preparation of astaxanthin DMSO stock solution, which was stored at 20 C in a brown-colored Eppendorf tube. Appropriate dilution was performed − ◦ before use [58].

3.7. Preparation of Homocysteine and Glutamate Solutions The work of Zhou et al. (2005) was followed to prepare homocysteine DMSO solution [59], while the method of Kawakami et al. (2009) was conducted to prepare glutamic acid solution (pH 7.0) (denoted as Glu) [60]. With a slight modification, an appropriate amount of Hcy was weighed, dissolved in DMSO, and stored at 20 C in a brown-colored Eppendorf tube. Appropriate dilution − ◦ was performed before use. Molecules 2020, 25, 214 12 of 17

3.8. MTT Assay Cell Viability Test The cell viability test with MTT assay was carried out as previous reported [14]. The doses of Glu and Hcy were applied as indicated. The absorbance was read with ELISA Reader (ClarioStar, BMG Labtech Japan Ltd., Saitama, Japan) at 570 nm. Cell viability was calculated according to Equation (1):

Cell viability = (As/Ac) 100%, (1) × where As is the absorbance of the sample and Ac is the absorbance of the control.

3.9. The Cytotoxicity of Astaxanthin Test for ATX cytotoxicity was conducted as previously reported [45]. The dose of ATX was applied to 24-well culture plates as indicated. The cell viability was calculated according to Equation (1).

3.10. Protective Effect of Astaxanthin against the Cytotoxicity of Homocysteine, Glutamate, or Homocysteine Plus Glutamate The PC12 cells were seeded at a density of 7 104 cells/well and incubated for 24 h until adhesion. × The medium was changed to the differentiating media containing ATX at 0.0, 1.0, 2.0, 5.0, 10.0, and 100.0 µM and incubated for 24 h The medium was replaced fresh with differentiating medium. Hcy (10 mM), Glu (20 mM), or Hcy (10 mM) plus Glu (20 mM) were added and the incubation was continued for 24 and 48 h. MTT assay was carried out as mentioned above. The cell viability was calculated according to Equation (1).

3.11. Determination of Intracellular Calcium Ion Concentration The work of Sul et al. (2009) was followed to determine the intracellular calcium ion concentration [61]. In brief, PC12 cells were seeded onto 24-well plates at a density 7 104 cells/well and × incubated for 24 h until entirely adhered. The following protocol was carried out as cited. The doses of ATX, Hcy, and Glu were 5 µM, 10 mM, and 20 mM respectively. The final supernatant was discarded, and the pellets were rinsed with PBS. Then, 1 trypsin was added and incubated at 37 C for 5 min. × ◦ The cell density was counted with the hematocytometer, the cells were seeded onto 96-well plates at a density 2 104 cells/well, and the medium was replaced fresh with the incomplete medium × containing 1 µM Fluo-3/AM (C51H50Cl2N2O23) and incubated at 37 ◦C for 30 min avoiding direct sunlight. The fluorescence produced after the reaction of Fluo-3/AM with calcium ions was measured at excitation wavelength Ex = 488 nm, and emitted wavelength Em = 532 nm. The untreated sample was used as the blank and set at 100% to estimate the change of intracellular calcium ion concentration within different groups.

3.12. Determination of Intracellular Reactive Oxygen Species (ROS) According to Zhang et al. (2008), the intracellular ROS level was determined [62]. The cultivation of PC12 cells and the doses of ATX, Hcy, and Glu were similarly conducted as mentioned in the above section. The final culture was centrifuged at 12,500 rpm for 20 min. The supernatant was discarded, × and the pellets were rinsed with PBS. Then, 1 trypsin was added and incubated at 37 C for 5 min. × ◦ The cell density was counted with the hematocytometer, the cells were seeded onto 96-well plates at a density 2 104 cells/well, and the medium was replaced fresh with the incomplete medium × containing 10 µM 20,70-dichlorodihydrofluorescein diacetate (H2DCFDA) and incubated at 37◦C for 30 min avoiding direct sunlight. The fluorescence of the product dichlorofluorescein (DCF) produced after the reaction of H2DCFDA with ROS was measured at excitation wavelength Eexcitation = 488 nm, and emitted wavelength Eemission = 532 nm using a fluorescence ELISA Reader (Bio-Rad, Hercules, CA, USA). The untreated sample was used as the blank and set at 100% to calculate the amount of ROS produced within different groups. Molecules 2020, 25, 214 13 of 17

3.13. Protein Extraction Method of protein extraction was carried out as previously cited [63]. The cultivation of PC12 cells and the doses of ATX (5 µM), Hcy (10 mM), and Glu (20 mM) were similarly conducted as mentioned in the above section. The protein content of the final supernatant was determined using the authentic bovine serum protein to establish the calibration curve, and the results were expressed in µg/µL. The remaining supernatant was transferred to a new 5-mL Eppendorf tube and stored at –80 ◦C in a freezer to keep the integrity of the cells for further use.

3.14. Assay for the Malondialdehyde (MDA) Concentration The determination of MDA was carried out using the protocol reported by Chang et al. (2011) [64]. The final OD was read at 532 nm using an ELISA Reader (Bio-Rad, CA, USA). Authentic 1,1,3,3-tetramethoxy propane (TMP) was used to establish the calibration curve, from which the concentration of MDA in samples was calculated and expressed in µM/µg protein.

3.15. Western Blot Analysis The SDS PAGE (7.5%–12%) electrophoresis was conducted at 100 V as instructed by the manufacturer (Life Science, CA, USA). The 100 µg of sample protein was loaded in each well. The primary antibodies of monoclonal anti-βactin, polyclonal anti-Bax, polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-cleaved caspase-12, and polyclonal anti-BcL-2 (BioVision Inc., Milpitas, CA, USA) were applied as 1/1000 of dilution and left in a 4 ◦C ice box overnight. The PVDF membranes were rinsed with TBST buffer once for 30 min. The secondary antibodies Gt Ms IgG(H + L) HRP and goat anti-rabbit IgG pAb HRP (Stressgen × Biotechnologies Corporation Corporate, San Diego, CA, USA) were applied as 1/5000 of dilution and left in 4 ◦C ice box for 30 min. while shaken. The membranes were rinsed with TBST buffer twice, each time for 30 min. Enhanced chemiluminescence (ECL) was added and mixed well for 5 min to facilitate Molecules 2020, 25, x Molecules 2020, 25, x 5 of 17 5 of 17 the reaction. The emitted chemiluminescence was taken by the Hansor LIS02 photo system, and the intensityFigure was 2. quantified Combined witheffect Image of target J (NIH compoundsFigure Image, 2. Combined on Bethesda, the viability effect MD, ofof USA). PC12target cell compounds line. PC12 on cells the wereviability of PC12 cell line. PC12 cells were seeded onto 24-well plates at 5 × 104 cells/mLseeded ontoand 24-wellcultured plates in serum-free at 5 × 10 4medium cells/mL overnight, and cultured then in serum-free medium overnight, then 3.16. Statistical Analysis treated with different combinations of ATX,treated Hcy with an differentd/or Glu separately.combinations In allof ATX,panels, Hcy empty and/or bars: Glu 24 separately. In all panels, empty bars: 24 Datah. In obtainedFigure 2a, ingray the bars: same 48 grouph; dark wereneth. barsIn analyzed Figure: 72 h. 2a,Inusing Figuregray bars: the 2b–d, SPSS48 neth; dark 10.0dark net statisticalbars: bars 48: h.72 software(h.a) InGlu Figure (5–20 (SPSS, 2b–d, net dark bars: 48 h. (a) Glu (5–20 Chicago,mM) IL, plus USA). Hcy Analysis(10 mM). of(b)variance ATX (1–10 (ANOVA) mM)μM) plusplus Hcy andHcy (10 Tukey’s(10 mM).mM). test ((cb)) ATX ATX were (1–10 (1–10 used μ μM) toM) analyzeplus plus Glu Hcy the(20 (10 mM). variance. mM). (c) ATX (1–10 μM) plus Glu (20 mM). Data are(d) ATX expressed (1–10 μM) as meansplus Hcy (10SD mM) from plus(d triplicate) ATX Glu (20(1–10 mM). experiments. μM) Data plus are Hcy expressed Data(10 mM) with asplus means significant Glu (20± SD mM). ( din =ff Data3).erences *: are expressed as means ± SD (n = 3). *: ± ⊗ compared to the control; 〒: vs. Hcy 10compared mM at the to same the control; time;, ,#,  ,: 〒vs.: vs.24 hHcy at the10**, mMsame,##, at dose; the ,same : vs. time; Glu : vs. 24 h at the same dose; ⊗: vs. Glu between groups were identified with statistical level of * ⊗ p < 0.05; ⊗⊗ p < 0.01; 20 mM at the same time; #: vs. Hcy 1020 mM mM + atGlu the 20 same mM time;at the #: same vs. Hcytime. 10 The mM significance + Glu 20 mM of theat the same time. The significance of the , ,###, , 〒 ⊗ 〒 *** ⊗⊗⊗difference was judgedp < by0.005. confidence levelsdifference of * p neuron-mimicium and Ion Glutamate Level PC12 Affected by Homocysteine and Glutamate cell viabilityCalcium via ion alleviating dysregulation these adverseis relevantCalcium eff ects.to the Suggestively, ioninitiation dysregulation of Alzheimer's drugs is that relevant exhibit disease to potent the (AD), initiation antioxidative the PC12 of Alzheimer's cell disease (AD), the PC12 cell capabilitymodel in canreality be consideredhas pertinently beneficial reflectedmodel for treatment insuch reality a possibility ofhas certain pertinently [40]. neuro-excitatory The reflected calcium such diseases.ion influxa possibility was highly [40]. The calcium ion influx was highly raised by the insult of 20 mM Glu (144%),raised byand/or the insult 10 mM of Hcy 20 mM (153%), Glu (144%),and/or theand/or combined 10 mM treatment Hcy (153%), and/or the combined treatment (176%) (Figure 3a). ATX at 5 μM fully(176%) alleviated (Figure the3a). calcium ATX at influx5 μM fullyraised alleviated by 20 mM the Glu, calcium but was influx raised by 20 mM Glu, but was slightly less effective against that byslightly Hcy (10 less mM), effective and was against much that less by effective Hcy (10 against mM), and the wascombined much less effective against the combined therapy (133%) (Figure 3a), implicatingtherapy insufficient (133%) (Figure mole number 3a), implicating for the interactions insufficient between mole number ATX for the interactions between ATX and (Glu + Hcy); suggestively, a higherand (Gludose +of Hcy); ATX suggestively,may be required a higher for such dose a ofcombined ATX may insult. be required for such a combined insult. In addition, Hcy has been implicatedIn addition, to indirectly Hcy hasincrease been intracellularimplicated to calcium indirectly levels increase by intracellular calcium levels by activating ionotropic and metabotropicactivating receptors ionotropic [41], which, and metabotropic compared with receptors glutamate, [41], maywhich, create compared with glutamate, may create the reasons to elicit different outcomesthe reasons by such to aelicit combination different ofoutcomes pharmacological by such aactions combination of Hcy of pharmacological actions of Hcy [2,36,41]. [2,36,41]. Two subcellular organelles, namelyTwo the subcellular mitochondria organelles, and endoplasmic namely the reticulum mitochondria (ER), andare endoplasmic reticulum (ER), are involved in the cellular pathogenesisinvolved of AD; in anthe increased cellular pathogenesis oxidative stress of AD;and andysregulation increased oxidative of stress and dysregulation of calcium homeostasis also have beencalcium reported homeostasis [42]. Owing also to the have important been reported role of [42].ER in Owing the regulation to the important role of ER in the regulation of Ca2+-signaling, ER-mitochondrialof distance Ca2+-signaling, in the neurons ER-mitochondrial is tightly controlled distance inin thethe neuronsphysiological is tightly controlled in the physiological conditions. When the distance isconditions. decreased, When Ca2+ -overloadthe distance occurs is decreased,both in the Ca cytosol2+-overload and occurs both in the cytosol and mitochondria [40]. The reduction inmitochondria the distance be [40].tween The ER reduction and mitochondria in the distance may be between implicated ER and in mitochondria may be implicated in Alzheimer’s disease (AD) pathologyAlzheimer’s by enhanced disease Ca2+-signaling (AD) pathology [40]. Lin by et enhanced al.’s study Ca indicated2+-signaling that [40]. Lin et al.’s study indicated that ATX attenuated glutamate-inducedATX elevation attenuated of CH glutamate-inducedOP and ER chaperone elevation glucose-regulated of CHOP and protein ER chaperone glucose-regulated protein (GRP78), inhibiting glutamate-induced(GRP78), apoptosis inhibiting through glutamate-induced rescuing the redox apoptosis balance th andrough inhibiting rescuing the redox balance and inhibiting glutamate-induced calcium influx [24].glutamate-induced calcium influx [24].

2.5. Effect of Astaxanthin on the ROS 2.5.Produc Effecttion of Caused Astaxanthin by Homocysteine on the ROS and Produc Glutamatetion Caused by Homocysteine and Glutamate The level of reactive oxygen speciesThe (ROS) level was of reactive highly inducedoxygen speciesby treating (ROS) with was Glu highly (20 mM), induced by treating with Glu (20 mM), Hcy (10 mM), and the combined treatmentHcy (10 mM),for 24 andh (Figure the combined 3b). The treatmentROS produced for 24 were h (Figure effectively 3b). The ROS produced were effectively suppressed by ATX at 5 μM (Figuresuppressed 3b). by ATX at 5 μM (Figure 3b). The mitochondrial Ca2+-overload canThe lead mitochondrial to increased Ca gene2+-overloadration of can reactive lead to oxygen increased species, gene ration of reactive oxygen species, inducing the opening of the mitochondrialinducing thepermeability opening of transition the mitochondrial pore and permeabilityultimately causing transition pore and ultimately causing neuronal apoptotic and necrotic neuronalcell death apoptotic [40]. In andaddition, necrotic the cellNO deathpathway [40]. relating In addition, with the NO pathway relating with hyperexcitability is induced by homocysteinehyperexcitability thiolactone is induced (HcyT) by [43]. homocysteine ATX is a nutrient thiolactone with (HcyT)unique [43]. ATX is a nutrient with unique cell membrane actions and diversecell clinical membrane benefits actions [44]. ATX and isdiverse the most clinical effective benefits antioxidant. [44]. ATX This is the most effective antioxidant. This molecule neutralizes free radicals ormolecule other oxidants neutralizes by either free acceptingradicals or or other donating oxidants electrons, by either without accepting or donating electrons, without being destroyed or becoming a pro-oxidantbeing destroyed in the process or becoming [44]. ATX a pro-oxidant blocks oxidative in the processDNA damage [44]. ATX blocks oxidative DNA damage and lowers C-reactive protein (CRP)and lowersand other C-reactive inflammation protein biomarkers(CRP) and [44].other ATX-mediated inflammation biomarkers [44]. ATX-mediated

Molecules 2020, 25, 214 14 of 17 Molecules 2020, 25, x 14 of 17

Figure 7. Summary of the apoptotic pathways induced by glutamate and homocysteine. This figure figure shows how glutamate and homocysteine induce mitochondrial damages, while astaxanthin alleviates the upregulation upregulation of of Ca Ca2+2 +influx,influx, ROS, ROS, Bax, Bax, caspase-12, caspase-12, and and caspase-3 caspase-3 and and the downregulation the downregulation of Bcl- of Ψ 2.Bcl-2. ER: ER:endoplasmic endoplasmic reticulum; reticulum; mito: mito: mitochondria; mitochondria; ROS: ROS: reactive reactive oxygen oxygen species; species; MMP MMP and and Ψm:: mitochondrial membranemembrane potential. potential. Symbols Symbols expressed expressed inlarger in larger characters characters are the are experimental the experimental results, whileresults, symbols while symbols in smaller in characters smaller characters are pathway-linking are pathway-linking items. PARP: items. Poly(ADP-ribose) PARP: Poly(ADP-ribose) polymerase; Apaf-1:polymerase; (apoptotic Apaf-1: protease (apoptotic activating protease factor activating 1). factor 1).

Chi-Huang Chang 1,†, Kuan-Chou Chen 2,3,4,†, Kuo-Chun Liaw 1, Chiung-Chi Peng 4,* and Author Contributions: Conceptualization, C.-H.C. and R.Y.P.; Methodology, C.-H.C. and K.-C.L.; Validation: 1, RobertK.-C.C. andY. Peng C.-C.P.; * Analysis: K.-C.L. and C.-C.P.; Project administration: K.-C.C. and C.-H.C.; Funding acquisition: K.-C.C., C.-H.C. and C.-C.P. Writing manuscript—C.-C.P. and R.Y.P.; Revising the manuscript: C.-C.P., K.-C.C. and Author Contributions: Conceptualization, C.-H.C. and R.Y.P.; Methodology, C.-H.C. and K.-C.L.; Validation: R.Y.P. All authors have read and agreed to the published version of the manuscript. K.-C.C. and C.-C.P.; Analysis: K.-C.L. and C.-C.P.; Project administration: K.-C.C. and C.-H.C.; Funding acquisition:Funding: The K.-C.C., authors C.-H.C. acknowledge and C.-C.P. Writing the funding manuscript—C.-C.P. offered by Ministryand R.Y.P.; of Revising Science the and manuscript: Technology C.- (MOST103-2313-B-038-002-MY3, MOST105-2320-B-038-034-MY3, MOST106-2320-B-038-032 & MOST108-2320-B-038-051) C.P., K.-C.C. and R.Y.P. and by Taipei Medical University-Shuang Ho Hospital (Grant No. 107 TMU-SHH-11). Funding:Conflicts ofThe Interest: authorsThe acknowledge authors declare the funding no conflict offered of interest.by Ministry of Science and Technology (MOST103-2313- B-038-002-MY3, MOST105-2320-B-038-034-MY3, MOST106-2320-B-038-032 & MOST108-2320-B-038-051) and by Taipei Medical University-Shuang Ho Hospital (Grant No. 107 TMU-SHH-11). References Conflicts of Interest: The authors declare no conflict of interest. 1. Meldrum, B.S. Glutamate as a neurotransmitter in the brain: Review of physiology and pathology. J. Nutr. 2000, 130 (Suppl. S4), 1007S–1015S. [CrossRef] References 2. Boldyrev, A.A.; Johnson, P. Homocysteine and its derivatives as possible modulators of neuronal and 1. Meldrum,non-neuronal B.S. cell Glutamate glutamate as receptorsa neurotransmitter in Alzheimer’s in the disease. brain: ReviewJ. Alzheimers of physiology Dis. 2007, and11, 219–228. pathology. [CrossRef J. Nutr.] 2000[PubMed, 130 ](Suppl. S4), 1007S–1015S, doi:10.1093/jn/130.4.1007S. 2.3. Boldyrev,Traynelis, A.A.; S.F.; Johnson, Wollmuth, P. Homocysteine L.P.; McBain, and C.J.; its Menniti, derivatives F.S.; as Vance, possible K.M.; modulators Ogden, of K.K.; neuronal Hansen, and non- K.B.; neuronalYuan, H.; cell Myers, glutamate S.J.; Dingledine, receptors in R. Alzheimer’s Glutamate receptor disease. ionJ. Alzheimers channels: Dis. Structure, 2007, 11 regulation,, 219–228. and function. 3. Traynelis,Pharmacol. S.F.; Rev. Wollmuth,2010, 62, 405–496. L.P.; McBain, [CrossRef C.J.;][ Menniti,PubMed ]F.S.; Vance, K.M.; Ogden, K.K.; Hansen, K.B.; Yuan, 4. H.;Olney, Myers, J.W. BrainS.J.; Dingledine, lesions, obesity, R. Glutamate and other receptor disturbances ion channels: in mice treatedStructure, with regulation, monosodium and glutamate. function. Pharmacol.Science 1969 Rev., 164 2010, 719–721., 62, 405–496, [CrossRef doi:10.1124/pr.109.002451.][PubMed] 4.5. Olney,Zhang, J.W. J.; An, Brain S.; lesions, Hu, W.; obesity, Teng, M.; and Wang, other X.; distur Qu,bances Y.; Liu, in Y.; mice Yuan, treated Y.; Wang, with monosodium D. The Neuroprotective glutamate. ScienceProperties 1969 of, 164 Hericium, 719–721, erinaceus doi:10.1126/science.164.3880.719. in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer’s 5. Zhang,Disease J.; Mouse An, S.; Model. Hu, W.;Int. Teng, J. Mol. M.; Sci. Wang,2016, 17X.;, 1810.Qu, Y.; [CrossRef Liu, Y.;][ Yuan,PubMed Y.; ]Wang, D. The Neuroprotective Properties of Hericium erinaceus in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer’s Disease Mouse Model. Int. J. Mol. Sci. 2016, 17, doi:10.3390/ijms17111810. 6. Murphy, E.; Steenbergen, C. Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol. Rev. 2008, 88, 581–609, doi:10.1152/physrev.00024.2007.

Molecules 2020, 25, 214 15 of 17

6. Murphy, E.; Steenbergen, C. Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol. Rev. 2008, 88, 581–609. [CrossRef][PubMed] 7. Cheriyan, J.; Balsara, R.D.; Hansen, K.B.; Castellino, F.J. Pharmacology of triheteromeric N-Methyl-D-Aspartate Receptors. Neurosci. Lett. 2016, 617, 240–246. [CrossRef] 8. Chong, Z.Z.; Li, F.; Maiese, K. Stress in the brain: Novel cellular mechanisms of injury linked to Alzheimer’s disease. Brain Res. Brain Res. Rev. 2005, 49, 1–21. [CrossRef] 9. Ho, P.I.; Ortiz, D.; Rogers, E.; Shea, T.B. Multiple aspects of homocysteine neurotoxicity: Glutamate excitotoxicity, kinase hyperactivation and DNA damage. J. Neurosci. Res. 2002, 70, 694–702. [CrossRef] 10. Herrmann, W.; Lorenzl, S.; Obeid, R. [Review of the role of hyperhomocysteinemia and B-vitamin deficiency in neurological and psychiatric disorders–current evidence and preliminary recommendations]. Fortschr. Neurol. Psychiatr. 2007, 75, 515–527. [CrossRef] 11. Lipton, S.A.; Rosenberg, P.A. Excitatory amino acids as a final common pathway for neurologic disorders. N. Engl. J. Med. 1994, 330, 613–622. [CrossRef][PubMed] 12. Greene, L.A.; Tischler, A.S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 1976, 73, 2424–2428. [CrossRef] [PubMed] 13. Westerink, R.H.; Ewing, A.G. The PC12 cell as model for neurosecretion. Acta Physiol. (Oxf.) 2008, 192, 273–285. [CrossRef][PubMed] 14. Chang, C.H.; Chen, H.X.; Yu, G.; Peng, C.C.; Peng, R.Y. Curcumin-Protected PC12 Cells Against Glutamate-Induced Oxidative Toxicity. Food Technol. Biotechnol. 2014, 52, 468–478. [CrossRef] 15. Wang, D.; Hu, S.; Zhang, J.; Li, Q.; Liu, X.; Li, Y. Investigation of the neuroprotective effects of a novel synthetic compound via the mitochondrial pathway. Mol. Med. Rep. 2017, 16, 1133–1138. [CrossRef] 16. Gleichmann, M.; Mattson, M.P. Neuronal calcium homeostasis and dysregulation. Antioxid. Redox Signal. 2011, 14, 1261–1273. [CrossRef] 17. Mattson, M.P.; Chan, S.L. Neuronal and glial calcium signaling in Alzheimer’s disease. Cell Calcium 2003, 34, 385–397. [CrossRef] 18. Obeid, R.; Herrmann, W. Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia. FEBS Lett. 2006, 580, 2994–3005. [CrossRef] 19. Naguib, Y.M. Antioxidant activities of astaxanthin and related carotenoids. J. Agric. Food Chem. 2000, 48, 1150–1154. [CrossRef] 20. Wu, H.; Niu, H.; Shao, A.; Wu, C.; Dixon, B.J.; Zhang, J.; Yang, S.; Wang, Y. Astaxanthin as a Potential Neuroprotective Agent for Neurological Diseases. Mar. Drugs 2015, 13, 5750–5766. [CrossRef] 21. Ye, Q.; Zhang, X.; Huang, B.; Zhu, Y.; Chen, X. Astaxanthin suppresses MPP(+)-induced oxidative damage in PC12 cells through a Sp1/NR1 signaling pathway. Mar. Drugs 2013, 11, 1019–1034. [CrossRef][PubMed] 22. Wang, C.Y.; Zou, W.; Liang, X.Y.; Jiang, Z.S.; Li, X.; Wei, H.J.; Tang, Y.Y.; Zhang, P.; Tang, X.Q. Hydrogen sulfide prevents homocysteineinduced endoplasmic reticulum stress in PC12 cells by upregulating SIRT1. Mol. Med. Rep. 2017, 16, 3587–3593. [CrossRef][PubMed] 23. Olatunji, O.J.; Feng, Y.; Olatunji, O.O.; Tang, J.; Ouyang, Z.; Su, Z.; Wang, D.; Yu, X. Neuroprotective effects of adenosine isolated from Cordyceps cicadae against oxidative and ER stress damages induced by glutamate in PC12 cells. Environ. Toxicol. Pharmacol. 2016, 44, 53–61. [CrossRef][PubMed] 24. Lin, X.; Zhao, Y.; Li, S. Astaxanthin attenuates glutamate-induced apoptosis via inhibition of calcium influx and endoplasmic reticulum stress. Eur. J. Pharmacol. 2017, 806, 43–51. [CrossRef][PubMed] 25. Broch, O.J.; Ueland, P.M. Regional distribution of homocysteine in the mammalian brain. J. Neurochem. 1984, 43, 1755–1757. [CrossRef][PubMed] 26. Lindgren, A.; Brattstrom, L.; Norrving, B.; Hultberg, B.; Andersson, A.; Johansson, B.B. Plasma homocysteine in the acute and convalescent phases after stroke. Stroke 1995, 26, 795–800. [CrossRef][PubMed] 27. Perry, I.J.; Refsum, H.; Morris, R.W.; Ebrahim, S.B.; Ueland, P.M.; Shaper, A.G. Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 1995, 346, 1395–1398. [CrossRef] 28. Kritis, A.A.; Stamoula, E.G.; Paniskaki, K.A.; Vavilis, T.D. Researching glutamate—Induced cytotoxicity in different cell lines: A comparative/collective analysis/study. Front. Cell Neurosci. 2015, 9, 91. [CrossRef] Molecules 2020, 25, 214 16 of 17

29. Froissard, P.; Duval, D. Cytotoxic effects of glutamic acid on PC12 cells. Neurochem. Int. 1994, 24, 485–493. [CrossRef] 30. Wang, K.; Zhu, X.; Zhang, K.; Wu, Z.; Sun, S.; Zhou, F.; Zhu, L. Neuroprotective Effect of Puerarin on Glutamate-Induced Cytotoxicity in Differentiated Y-79 Cells via Inhibition of ROS Generation and Ca(2+) Influx. Int. J. Mol. Sci. 2016, 17, 1109. [CrossRef] 31. Wang, D.; Tan, Q.R.; Zhang, Z.J. Neuroprotective effects of paeoniflorin, but not the isomer albiflorin, are associated with the suppression of intracellular calcium and calcium/calmodulin protein kinase II in PC12 cells. J. Mol. Neurosci. 2013, 51, 581–590. [CrossRef][PubMed] 32. Li, W.; Cheong, Y.K.; Wang, H.; Ren, G.; Yang, Z. Neuroprotective Effects of Etidronate and 2,3,3-Trisphosphonate Against Glutamate-Induced Toxicity in PC12 Cells. Neurochem. Res. 2016, 41, 844–854. [CrossRef][PubMed] 33. Hu, S.; Wang, D.; Zhang, J.; Du, M.; Cheng, Y.; Liu, Y.; Zhang, N.; Wang, D.; Wu, Y. Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells. Int. J. Mol. Sci. 2016, 17, 133. [CrossRef][PubMed] 34. Shea, T.B.; Lyons-Weiler, J.; Rogers, E. Homocysteine, folate deprivation and Alzheimer neuropathology. J. Alzheimers Dis. 2002, 4, 261–267. [CrossRef][PubMed] 35. Leclerc, C.L.; Chi, C.L.; Awobuluyi, M.; Sucher, N.J. Expression of N-methyl-D-aspartate receptor subunit mRNAs in the rat pheochromocytoma cell line PC12. Neurosci. Lett. 1995, 201, 103–106. [CrossRef] 36. Sibarov, D.A.; Abushik, P.A.; Giniatullin, R.; Antonov, S.M. GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine. Front. Cell Neurosci. 2016, 10, 246. [CrossRef] 37. Wang, X.J.; Chen, W.; Fu, X.T.; Ma, J.K.; Wang, M.H.; Hou, Y.J.; Tian, D.C.; Fu, X.Y.; Fan, C.D. Correction to: Reversal of homocysteine-induced neurotoxicity in rat hippocampal neurons by astaxanthin: Evidences for mitochondrial dysfunction and signaling crosstalk. Cell Death Discov. 2019, 5, 70. [CrossRef] 38. Fan, C.D.; Sun, J.Y.; Fu, X.T.; Hou, Y.J.; Li, Y.; Yang, M.F.; Fu, X.Y.; Sun, B.L. Astaxanthin Attenuates Homocysteine-Induced Cardiotoxicity in Vitro and in Vivo by Inhibiting Mitochondrial Dysfunction and Oxidative Damage. Front. Physiol. 2017, 8, 1041. [CrossRef] 39. Zhang, Y.; Wang, W.; Hao, C.; Mao, X.; Zhang, L. Astaxanthin protects PC12 cells from glutamate-induced neurotoxicity through multiple signaling pathways. J. Funct. Foods 2015, 16, 137–151. [CrossRef] 40. Qi, H.; Shuai, J. Alzheimer’s disease via enhanced calcium signaling caused by the decrease of endoplasmic reticulum-mitochondrial distance. Med. Hypotheses 2016, 89, 28–31. [CrossRef] 41. Robert, K.; Pages, C.; Ledru, A.; Delabar, J.; Caboche, J.; Janel, N. Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus. Neuroscience 2005, 133, 925–935. [CrossRef][PubMed] 42. Magi, S.; Castaldo, P.; Macri, M.L.; Maiolino, M.; Matteucci, A.; Bastioli, G.; Gratteri, S.; Amoroso, S.; Lariccia, V. Intracellular Calcium Dysregulation: Implications for Alzheimer’s Disease. Biomed. Res. Int. 2016, 2016, 6701324. [CrossRef][PubMed] 43. Pierce, G.N. Advanced Bioactive Compounds Countering the Effects of Radiological, Chemical and Biological Agents: Strategies to Counter Biological Damage; Springer: New York, NY, USA, 2013. 44. Kidd, P. Astaxanthin, cell membrane nutrient with diverse clinical benefits and anti-aging potential. Altern. Med. Rev. 2011, 16, 355–364. [PubMed] 45. Chang, C.H.; Chen, C.Y.; Chiou, J.Y.; Peng, R.Y.; Peng, C.H. Astaxanthine secured apoptotic death of PC12 cells induced by beta-amyloid peptide 25–35: Its molecular action targets. J. Med. Food 2010, 13, 548–556. [CrossRef] 46. Shen, H.; Kuo, C.C.; Chou, J.; Delvolve, A.; Jackson, S.N.; Post, J.; Woods, A.S.; Hoffer, B.J.; Wang, Y.; Harvey, B.K. Astaxanthin reduces ischemic brain injury in adult rats. FASEB J. 2009, 23, 1958–1968. [CrossRef] 47. Yamagishi, R.; Aihara, M. Neuroprotective effect of astaxanthin against rat retinal ganglion cell death under various stresses that induce apoptosis and necrosis. Mol. Vis. 2014, 20, 1796–1805. 48. Galasso, C.; Orefice, I.; Pellone, P.; Cirino, P.; Miele, R.; Ianora, A.; Brunet, C.; Sansone, C. On the Neuroprotective Role of Astaxanthin: New Perspectives? Mar. Drugs 2018, 16, 247. [CrossRef] 49. Eskes, R.; Antonsson, B.; Osen-Sand, A.; Montessuit, S.; Richter, C.; Sadoul, R.; Mazzei, G.; Nichols, A.; Martinou, J.C. Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J. Cell Biol. 1998, 143, 217–224. [CrossRef] Molecules 2020, 25, 214 17 of 17

50. Gomez-Crisostomo, N.P.; Lopez-Marure, R.; Zapata, E.; Zazueta, C.; Martinez-Abundis, E. Bax induces cytochrome c release by multiple mechanisms in mitochondria from MCF7 cells. J. Bioenerg. Biomembr. 2013, 45, 441–448. [CrossRef] 51. Jiang, J.M.; Wang, L.; Gu, H.F.; Wu, K.; Xiao, F.; Chen, Y.; Guo, R.M.; Tang, X.Q. Arecoline Induces Neurotoxicity to PC12 Cells: Involvement in ER Stress and Disturbance of Endogenous H2S Generation. Neurochem. Res. 2016, 41, 2140–2148. [CrossRef] 52. Garcia de la Cadena, S.; Massieu, L. Caspases and their role in inflammation and ischemic neuronal death. Focus on caspase-12. Apoptosis 2016, 21, 763–777. [CrossRef][PubMed] 53. Nakagawa, T.; Zhu, H.; Morishima, N.; Li, E.; Xu, J.; Yankner, B.A.; Yuan, J. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 2000, 403, 98–103. [CrossRef][PubMed] 54. Liu, H.; Wang, Z.; Nowicki, M.J. Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice. World J. Gastroenterol. 2014, 20, 18189–18198. [CrossRef][PubMed] 55. Zhang, Q.; Liu, J.; Chen, S.; Liu, J.; Liu, L.; Liu, G.; Wang, F.; Jiang, W.; Zhang, C.; Wang, S.; et al. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress. Apoptosis 2016, 21, 432–442. [CrossRef] 56. Matsui, A.; Kaneko, H.; Kachi, S.; Ye, F.; Hwang, S.J.; Takayama, K.; Nagasaka, Y.; Sugita, T.; Terasaki, H. Expression of Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells Induced by Amyloid-beta Is Depressed by an Endoplasmic Reticulum Stress Inhibitor. Ophthalmic Res. 2015, 55, 37–44. [CrossRef] 57. Cultured for Neuronal PC12 Cells: A Model Function, Differentiation, and Survival. p. Available online: https://biocyclopedia.com/index/cell_biology_methods/cultured_for_neuronal_pc12_cells.php (accessed on 5 December 2019). 58. Bolin, A.P.; Macedo, R.C.; Marin, D.P.; Barros, M.P.; Otton, R. Astaxanthin prevents in vitro auto-oxidative injury in human lymphocytes. Cell Biol. Toxicol. 2010, 26, 457–467. [CrossRef] 59. Zhou, W.; Chai, H.; Lin, P.H.; Lumsden, A.B.; Yao, Q.; Chen, C. Ginsenoside Rb1 blocks homocysteine-induced endothelial dysfunction in porcine coronary arteries. J. Vasc. Surg. 2005, 41, 861–868. [CrossRef] 60. Kawakami, Z.; Kanno, H.; Ueki, T.; Terawaki, K.; Tabuchi, M.; Ikarashi, Y.; Kase, Y. Neuroprotective effects of yokukansan, a traditional Japanese medicine, on glutamate-mediated excitotoxicity in cultured cells. Neuroscience 2009, 159, 1397–1407. [CrossRef] 61. Sul, D.; Kim, H.S.; Lee, D.; Joo, S.S.; Hwang, K.W.; Park, S.Y. Protective effect of caffeic acid against beta-amyloid-induced neurotoxicity by the inhibition of calcium influx and tau phosphorylation. Life Sci. 2009, 84, 257–262. [CrossRef] 62. Zhang, Y.; Peng, F.; Gao, B.; Ingram, A.J.; Krepinsky, J.C. High glucose-induced RhoA activation requires caveolae and PKCbeta1-mediated ROS generation. Am. J. Physiol. Renal. Physiol. 2012, 302, F159–F172. [CrossRef] 63. Chang, C.H.; Chen, Y.; Yew, X.X.; Chen, H.X.; Kim, J.X.; Chang, C.C.; Peng, C.C.; Peng, R.Y. Improvement of erinacine A productivity in Hericium erinaceus mycelia and its neuroprotective bioactivity against the glutamateinsulted apoptosis. LWT Food Sci. Technol. 2016, 65, 1100–1108. [CrossRef] 64. Chang, X.; Wang, J.; Yang, S.; Chen, S.; Song, Y. Antioxidative, antibrowning and antibacterial activities of sixteen floral honeys. Food Funct. 2011, 2, 541–546. [CrossRef][PubMed]

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