相关热词搜索：
ABSTRACT Oxidative damage and redox metalhomeostasis loss are two contributing factors in brain aging and widelydistributed neurodegenerative diseases. Oxidative species in company withexcessive amounts of intracellular free iron result in Fenton-type reactionwith subsequent production of highly reactive hydroxyl radicals which initiateperoxidation of biomolecules and further formation of non-degradable toxicpigments called lipofuscin that amasses in long-lived postmitotic cells such asneurons. Dietary flavonoid baicalein can counteract the detrimentalconsequences through exertion of a multiplicity of protective actions withinthe brain including direct ROS scavenging activity and iron chelation. In thisstudy, we evaluated the neuroprotective effects of baicalein in menadione(superoxide radical generator)-treated SK-N-MC neuroblastoma cell line. Ourresults showed that treatment of cells with menadione led to lipofuscinformation due to elevated intracellular iron contents and accumulation ofoxidative products such as MDA and PCO. Also, menadione caused apoptotic celldeath in SK-N-MC cells. However, pretreatment with baicalein (40 μM) reversedthe harmful effects by chelating free iron and preventing biomoleculesperoxidations. Moreover, baicalein prevented cell death through modulation ofkey molecules in apoptotic pathways including suppression of Bax and caspase-9activities and induction of bcl2 expression. Key structural features such aspresence of hydroxyl groups and iron-binding motifs in baicalein make it theappropriate candidate in antioxidant-based therapy in age-relatedneurodegenerative diseases. Keywords: Aging; Baicalein; Lipofuscin; Menadione;Neurodegenerative Disease; Oxidative Stress 1. Introduction The key precept ofthe oxidative stress theory of aging is that senescence-related loss offunction is due to the progressive and irreparable accrual of molecularoxidative damage which is brought about by powerful pro-oxidant speciesincluding reactive oxygen species (ROS) [1,2]. ROS include a broad range ofpartially reduced metabolites of oxygen (e.g. superoxide, hydrogen peroxide andhydroxyl radical) having higher reactivity than molecular oxygen [3]. Theirraison d’être remains unclear. Putative explanations for their occurrence rangefrom inadvertent by-products of aerobic metabolism to highly regulated andintricate signaling mechanisms [4]. Free radical or oxidative stress theory ofaging was first proclaimed by Denham Harman demonstrating the role of oxidativespecies in aging process acceleration and cell death [5,6]. This theory canexplain many of the senescent changes including accumulation of brown-yellow,electron-dense, autofluorescent bodies in cells called lipofuscin pigments orage pigments [5,7,8]. Correlation of lipofuscin with aging is not only becausethe amount of lipofuscin elevates with age, but also, more significantlybecause the rate of lipofuscin accumulation negatively correlates to longevity.High consumption of oxygen via brain makes it susceptible to oxidative damage[9,10]. Reactive oxygen species which are generated by mitochondria throughdifferent ways, diffuse into lysosomes which encompass a variety ofmacromolecules under degradation as well as redox-active low molecular ironwhich would be released from different sorts of metalloproteins. Based onFenton reaction, hydroxyl radical can be generated through the reaction ofhydrogen peroxide with iron, bringing about the cross-linking of adjacentmacromolecules and resultant lipofuscin formation [7,8,10-12]. There is adebate on the function of lipofuscin pigments formed during exposure of cellsto oxidative agents. Some researchers believe that lipofuscin formation doesnot have any serious effects on normal func- * Corresponding author. Copyright© 2013 SciRes. CellBio 36 M. MOSLEHI, R. YAZDANPARAST tion of cells. On the contrary,some scientists state that although lipofuscin cannot react directly withextralysosomal constituents because of the lysosomal membrane, the high contentof iron within lipofuscin granules may promote generation of ROS, sensitizingcells to oxidative injury through lysosomal destabilization. Destabilization oflysosomal membrane results in leaking of hydrolytic enzymes into the cytosol.Hence, oxidative species and redox-active transition metals homeostasisimpairments which facilitate further formation of active and hazardous reactiveoxygen species might be two main characteristics of age-relatedneurodegenerative diseases [13]. Human’s aspiration for greater longevity haslong been a strong motivation for a lot of studies in the field of aging and age-relateddisorders. Escalating body of evidence implies that lifestyle factors, andspecially the diet, may counteract oxidative damage [2,14]. Dietary flavonoidswith blood-brain barrier ability were shown to have potential anti-aging andbrain-protective activities [5, 15-18]. Baicalein(5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), one of the naturallyoccurring flavonoids in Scutellaria baicalensis GEORGI known as “Huang qin” inChina and “Ogon” in Japan, is prescribed for oxidative stress-related diseases[19]. Numerous studies have shown that baicalein protects neurons fromoxidative damage via multiple bio-effects ranging from classic radicalscavenging activities to modulation of signaling pathways involved instress-associated diseases. Moreover, recent studies have denoted thatbaicalein mitigates formation of hydroxyl radical through its iron-binding(anti-Fenton) and strong chelation properties [20-23]. In this study, wescrutinize the effect of baicalein on menadione (superoxide anion generator)-inducedlipofuscin formation in human neuroblastoma SK-N-MC cell line to comprehend themechanism by which baicalein protect SK-N-MC cells against oxidative damages.2. Materials and Methods 2.1. Materials The cell culture medium (RPMI-1640),penicillin-streptomycin and fetal bovine serum (FBS) were purchased from GibcoBRL (Life technology, Paisely, Scotland). The culture plates were purchasedfrom Nunc (Brand products, Denmark). dimethyl sulfoxide (DMSO), FeCl3 and KMnO4were obtained from Merck (Darmstadt, Germany). Ethidium bromide, acridineorange, Baicalein and Triton X-100 were purchased from Pharmacia LKBBiotechnology (Sweden). MTT [3-(4,5-dimethyl tiazol-2, 5-diphenyl tetrazoliumbromide], phenylmethylsulphonyl fluoride (PMSF), leupeptin, pepstatin,aprotinin, monochlorobimane (mBCL), dithionitrobenzoic acid (DTNB), GSH,ascorbic acid, ferrozine and pan-caspase inhibitor (Z-VAD-fmk) were purchasedfrom Sigma Chem. Co. (Germany). 2’,7’-dichlorofluorescein diacetate (DCFHDA)was obtained from Molecular Probe (Eugene, Oregon, USA).Ethylenediaminetetraacetic acid (EDTA) was from Aldrich (Germany). Human SK-N-MC neuroblastoma cells were obtained from Pasteur Institute (Tehran, Iran). Allantibodies including anti-Bax, anti-Bcl-2, anticleaved caspase-9, anti-tubulinand mouse/rabbit horseradish peroxidase-conjugated second-dary antibodies werepurchased from Biosource (Nivelles, Belgium). Chemiluminescence detectionsystem was purchased from Amersham-Pharmacia (Piscataway, NJ, USA). 2.2. CellCulture Human neuroblastoma cell line SK-N-MC was cultured in RPMI-1640 mediumsupplemented with FBS (10%, v/v), streptomycin (100 μg/ml) and penicillin (100U/ml) and incubated in 5% CO humidified atmosphere at 37˚C. To induce oxidativestress, menadione was freshly prepared from a stock solutions (10 mM), prior toeach experiment. Menadione and baicalein were dissolved in a minimum amount ofdimethyl Sulfoxide (DMSO) and then diluted with the culture medium to thedesired concentration. The concentration of DMSO in the culture medium keptlower than 0.1% and the control cells were treated with the vehicle solutioncontaining the same amount of DMSO. 2.3. Determination of Cell Viability Cellviability was assessed by the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) reduction assay. Viable cells with active mitochondria reduce theyellow tetrazolium salt MTT giving dark blue water insoluble formazan crystals.To perform the assay for evaluation of the cytoprotective effects of baicaleinand caspase inhibitor (z-VAD-fmk) on menadione-treated SK-N-MC cells, SK-N-MCcells were suspended in medium and seeded at a density of 5 × 104 cells/well in96 well plates for a day. Cells were pretreated with various concentrations ofbaicalein (10, 20, 40, 50 μM) and pancaspase inhibitor (50 μM) and then treatedwith menadione (35 μM) for additional 24 h at 37˚C. MTT was dissolved at aconcentration of 5 mg/ml in PBS and stored at 4˚C, protected from light andtightly capped. After incubation, cells were treated with the 10 μl MTTsolution for 4 h. Then, the medium was removed and 200 μl DMSO was added toeach well. The formazan dye crystals were solubilized in 30 min, and absorbancewas measured at 570 nm using an ELISA reader (Exert 96, Asys Hitch, Ec Austria).Results were expressed as the percentage of MTT reduction, assuming that theabsorbCopyright © 2013 SciRes. CellBio M. MOSLEHI, R. YAZDANPARAST 37 ance ofthe control cells was 100%. 2.4. Measurement of Intracellular ROS Oxidation of2’,7’-dichlorofluorescein diacetate (DCFHDA) to fluorescent DCF is taken as anindex of overall oxidative stress in biological system according to LeBelmethod [24]. Cells were pre-treated with various concentrations of baicalein(10, 20, 40 μM) and 50 μM caspase inhibitor for 3 h followed by menadionetreatment (35 μM) for 12 h at 37˚C. Then the cells were incubated with 10 μMDCFH-DA for 1 h followed by washing twice with phosphate buffer saline andsuspension in the same buffer. Finally, the fluorescent intensity was monitoredusing a varian-spectrofluorometer with excitation and emission wavelength of485 and 530 nm, respectively. 2.5. Determination of Lipid PeroxidationMalondialdehyde (MDA) levels were measured by the double heating method [25].The method is based on spectrophotometric measurement of the purple colorgenerated by the reaction of thiobarbituric acid (TBA) with MDA. Briefly, 0.5ml of cell lysate was mixed with 2.5 ml of trichloroacetic acid (TCA, 10%, w/v)solution followed by boiling in a water bath at 95˚C for 15 min. After coolingto room temperature, the samples were centrifuged at 3000 rpm for 10 min and 2ml of each sample supernatant was transferred to a test tube containing 1 ml ofTBA solution (0.67% w/v). Each tube was then placed in a boiling water bath for15 min. After cooling to room temperature, the absorbance was measured at 532nm with respect to the blank solution. The protein concentration was determinedby Lowry’s method [26]. The concentration of MDA was calculated based on theabsorbance coefficient of the TBA-MDA complex (ε = 1.56 × 105 cm−1 ·M−1 ) andit was expressed as nmol/ mg of protein. 2.6. Determination of Protein CarbonylFormation The assessment of protein carbonyl content is a widelyused marker foroxidative protein modification. Protein carbonyls (PCOs) were measured usingReznick and Packer method [27]. Briefly, 1 ml of 10 mM DNPH in 2 M HCl wasadded to the cell lysates. Samples were incubated for 1 hr at room temperatureand were vortexed every 15 min. Then, 1 ml of trichloroacetic acid (TCA 10%w/v) was added to each reaction mixture and centrifuged at 3000 rpm for 10 min.The pellets were washed twice with 2 ml of ethanol/ethyl acetate (1:1, v/v) andeach dissolved in 1 ml of guanidine hydrochloride (6 M, pH 2.3) and incubatedfor 10 min at 37˚C whilst mixing. The carbonyl content was calculated based onthe molar extinction coefficient of DNPH (ε = 2.2 × 104 cm−1 ·M−1 ). 2.7.Fluorescence Microscopy Evaluation of Apoptotic Cells Acridine orange/ethidiumbromide double staining was applied to observe the morphological changes amongmenadione-treated cell. Using this technique, cells can be distinguished asnormal cells (uniformly stained green) and apoptotic cells that are stainedorange because of cell membrane destruction and the intercalation of ethidiumbromide between the nucleotide bases of DNA. After treatment, cells were washedtwice with phosphate buffer saline and adjusted to a cell density of 1 × 104cells/ml of phosphate solution (1:1 v/v). The nuclear morphology was evaluatedby Axoscope 2 plus fluorescence microscope from Zeiss (Germany). The cells withcondensed or fragmented nuclei were counted as apoptotic cells. All experimentswere repeated three times, and the number of stained cells was counted in 10 randomlyselected fields. 2.8. Evaluation of Intracellular Formation of LipofuscinPigments Extraction of intracellular lipofuscin was achieved following lysis ofeach sample according to a published procedure with slight modification [28].The cells were seeded in triplicate into 24-well plates for 24 h prior topretreatments. After pretreatment with different doses of baicalein (10, 20, 40μM) for 3 h, each cell sample was treated with 35 μM menadione for 24 h. Theattached cells in each well were trypsinized with trypsin-EDTA solutionfollowed by cell counting using a hemocytometer. Each plate was thencentrifuged, the cell pellet was washed with PBS, and the cell content waslysed with lysis buffer containing 1% Triton X-100, 1 mM EDTA and 1 mM PMSF.Each cell lysate was harvested and its fluorescence intensity was monitored ona varian spectrofluometer, model Cary Eclipse, with an excitation wavelength of310 nm and emission wavelength of 620 nm [29]. The fluorescence intensities ofthe samples were then normalized for equal cell numbers. 2.9. Measurement ofIntracellular Iron Contents via Ferrozine-Based Colorimetric Assay The assaywas performed directly in 24-well plates. Cells were lysed by addition of 200μl iron releasing reagent (a freshly mixed solution of equal volumes of 1.4 MHCl and 4.5% (w/v) KMnO4 in H2O2) to each well. The plates were sealed withfoil and incubated for 2 h at 60˚C, after which 60 μl of the detection reagent(6.5 mM ferrozine, 6.5 mM EDTA, 2.5 M ammonium acetate and 1 M ascorbic aciddissolved in water) was added. After further incubation for 30 min at roomtemperature, 280 μl of the mixture was transferred to a well of a 96-well plateand its absorbance recorded at 550 nm and compared to the absorbance of theFeCl3-treated standards under all Copyright © 2013 SciRes. CellBio 38 M.MOSLEHI, R. YAZDANPARAST equal experimental conditions. The determinedintracellular iron concentration for each well was normalized against theprotein content of replicate wells [30]. 2.10. Western Blot Analysis SK-N-MCcells were seeded at a density of 105 cells/ml in 12-well plates for 24 h. Thecells were pretreated with baicalein (40 μM) and caspase inhibitor (50 μM).After 3 h, menadione (35 μM) was added to the cells and incubated at 37˚C for anadditional 24 h. Then, the cells were harvested and lysed using lysis buffercontaining 1% Triton X-100, 1% SDS, 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mMEGTA, 1 mM EDTA, 20 mM sodium pyrophosphate, 2 mM Na3VO4, 1 mM NaF, 0.5% sodiumdeoxycholate, 10% glycerol, 1mM phenylmethylsulphonyl fluoride, 10 μg/mlleupeptin, 1 μg/ml pepstatin and 60 μg/ml aprotinin. Protein concentration ofeach sample was determined using Lowry’ method (Lowry et al., 1951). Equalquantities of protein (40 μg) were subjected to 12.5% SDS-polyacrylamide gelelectrophoresis (PAGE) and were transferred to PVDF membranes. The blots wereblocked with 5% (w/v) non-fat dry milk in Tris-buffered saline buffercontaining 0.1% Tween-20 (TBS/T) for an overnight at 4˚C. The blocked blotswere incubated with primary antibodies for 2 hr at room temperature usingantibody dilutions as recommended by the manufacturer in Tris-buffered salinepH 7.4 containing 0.1% Tween-20. After 1-hr incubation with anti-rabbit oranti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies(Biosource), the proteins were detected by an enhanced chemiluminescencedetection system (Amersham-Pharmacia, Piscataway, NJ, USA) according to themanufacturer’s instructions. Blots were stripped at 50˚C for 30 min in 100 mM2-mercaptoethanol, 2% SDS, 62.2 mM Tris-HCl pH 6.7 and reprobed for furtherinvestigations. For analysis of the western blotting data, densitometricanalysis was performed using Image.J software, and the densities werenormalized with respect to β-tubulin as the internal control. 2.11. StatisticalAnalysis Data were expressed as percent of values of untreated control cells,and each value represents the mean ± SD (n = 3). The significant differencesbetween the means of the treated and untreated cells were calculated byunpaired Student’ t-test, and p-values < 0.05 were considered significant.3. Results 3.1. Baicalein and Pan-Caspase Inhibitor (Z-VAD-Fmk) Shield SK-N-MCCells against Menadione-Induced Cytotoxicity Menadione is a quinone known to inducean oxidative stress generated primarily by superoxide radicals leading to celldeath [31]. We found that menadione at 35 μM caused 55% cell death amongSK-N-MC cells (Figure 1). In our previous study, we ascertained that noremarkable changes were seen among the cells in range of 10 - 50 μM ofbaicalein after 24 h [32]. Thus, cytoprotcetive effects of different doses ofbaicalein (10, 20, 40, 50 μM) on menadione (35 μM)-induced cytotoxicity inSK-N-C cells were investigated. The detrimental effects of menadione on SK-N-MCcells were considerably blocked by pretreatment with baicalein. The same resultwas observed for Z-VAD-fmk. As shown in Figure 1, the extent of survival wasrestored to 67%, 84%, 89% and 71% by pretreatment of cells with different dosesof baicalein (10, 20, 40, 50 μM) for 3 h followed by treatment with 35 μMmenadione for 24 h. Baicalein at a concentration of 40 μM, provided utmostprotection against menadione insult producing a 44% increase in cell survival.Moreover, Z-VAD-fmk (50 μM) increased cell viability to 86% (Figure 1). 3.2.Baicalein but Not Z-VAD-Fmk, Mitigates Menadione-Induced Increase inIntracellular ROS Generation Increase in ROS generation was measured as one ofthe indicators of menadione-induced oxidative stress in cells. As shown inFigure 2, generation of intracellular ROS (in term of DCF fluorescentintensity) in SK-N-MC cells increased by almost a factor of 6.2 after 12-htreatment Figure 1. Cts of menadione, baicalein, and Z-VAD-fmk on viability ofSK-N-MC cells. SK-N-MC cells were treated with different concentrations ofmenadione (20, 35, 50 μM) to find IC50 of menadione for further experiments (35μM). Then, SK-N-MC cells were pretreated with different concentrations ofbaicalein (10, 20, 40, 50 μM) and Z-VAD-fmk (50 μM) for 3h and then incubatedwith menadione (35 μM) for 24 h. Cell viability was examined by MTT assay.Values correspond to means ± SD of three independent experiments. *significantly different from control cells (p < 0.05), # significantly differentfrom menadione-treated cells (p < 0.05). Copyright © 2013 SciRes. CellBio M.MOSLEHI, R. YAZDANPARAST 39 Figure 2. Effects of baicalein and Z-VAD-fmk onintracellular ROS level in menadione-treated SK-N-MC cells. SKN-MC cells werepretreated with baicalein (10, 20, 40 μM) and Z-VAD-fmk (50 μM) for 3 h andthen incubated with menadione for 12 h. ROS levels were monitored using 2", 7"dichlorofluorescein diacetate (DCFH-DA) staining. The fluorescence intensitywas monitored on a varian-spectrofluorometer with excitation and emissionwavelengths of 485 and 530 nm, respectively. Values correspond to means ± SD ofthree independent experiments. * significantly different from control cells (p< 0.05), # significantly different from menadione-treated cells (p < 0.05).with menadione (35 μM) compared to ROS level of the untreated control cells.Pretreatment of the cells with different doses of baicalein (10, 20, 40 μM)attenuated ROS production in SK-N-MC cells by factors of 2.3, 3.6 and 4.3,respectively. However, pretreatment with ZAD-mk (50 μM for 3 h) did notsignificantly change the ROS level in menadione-treated SK-N-MC cells. 3.3.Baicalein but Not Z-VAD-Fmk, Curbs Menadione-Induced Lipid PeroxidationMenadione-induced oxidative stress causes oxidation of intracellularbiomolecules such as lipids. MDA is produced while lipid peroxidation happens.So, MDA level measurement is used as a marker of menadione-induced oxidativestress. As shown in Figure 3, baicalein repressed lipid peroxidation in SK-N-MCcells. After 12 h of incubation with 35 μM menadione, MDA levels weresignificantly increased relative to the untreated control cells (0.41 nmol/mgprotein in control cells versus 2.33 nmol/mg protein in menadione-treatedcells). Pretreatment of cells with different doses of baicalein (10, 20, 40 μM)for 3 h followed by a 12 h treatment with menadione (35 μM) reduced MDAformation to 1.64, 1.01, and 0.66 nmol/mg protein, respectively, indicatingthat baicalein had quenched lipid peroxidation of the SK-NMC cells. However,pretreatment with 50 μM Z-VADfmk did not significantly alter MDA contents inmenadione-treated SK-N-MC cells. Figure 3. Effects of baicalein and Z-VAD-fmkon intracellular lipid peroxidation and protein carbonyl formation inmenadione-treated SK-N-MC cells. SK-N-MC cells were pretreated with baicalein(10, 20, 40 μM) and Z-VAD-fmk (50 μM) for 3 h and then incubated with menadionefor 12 h. lipid and protein oxidations were measured by analysis of MDA andPCO. Values correspond to means ± SD of three independent experiments. *significantly different from control cells (p < 0.05), # significantlydifferent from menadionetreated cells (p < 0.05). 3.4. Baicalein but NotZ-VAD-Fmk, Diminishes Menadione-Induced Protein Carbonyl Formation Protein carbonylis a marker of protein oxidation in oxidative stress condition. We evaluatedthe effects of different doses of baicalein (10, 20, 40 μM) and Z-VADfmk (50μM) on protein carbonyl formation in SK-N-MC cells. After treatment withmenadione (35 μM), the amount of protein carbonyl increased to 4.03 nmol/mgprotein compared to 0.65 nmol/mg protein of control cells. Pretreatment withbaicalein (10, 20, 40 μM) reduced protein carbonyl formation to 2.6, 1.7 and1.1 nmol/mg protein, respectively (Figure 3). However, pretreatment with 50 μMZ-VAD-fmk did not significantly alter PCO contents in menadione-treated SK-N-MCcells. 3.5. Baicalein and Z-VAD-Fmk Prevent Menadione-Induced Caspase-DependentApoptotic Cell Death To study the protective effect of baicalein on SK-N-MCcells, acridine orange/ethidium bromide double staining technique was used toevaluate the occurrence of apoptosis in cells. As shown in Figure 4, thenon-apoptotic control cells were stained green and the apoptotic cells hadorange particles in their nuclei due to nuclear DNA fragmentation. Themenadione treatment increased the extent of apoptosis relative to untreatedcontrol cells and pretreatment with baicalein (40 μM, 3 h) diminished apoptosiscompared to menadione-treated cells (Figure 4). We also pretreated SK-N-MCcells with Z-VAD-fmk (50 μM) for 3 h followed by exposure to menadione (35Copyright © 2013 SciRes. CellBio 40 M. MOSLEHI, R. YAZDANPARAST (a) (b) Figure4. Effect of baicalein and Z-VAD-fmk treatments on menadione-induced apoptosisin SK-N-MC cells. (a) SKN-MC cells were treated with baicalein (40 μM) andZ-VAD-fmk (50 μM) for 3 h followed by exposure to menadione (35 μM) for 24 h.cell pretreatment with baicalein and Z-VAD-fmk clearly decreased the number ofapoptotic cells relative to cells treated only with menadione. Valuescorrespond to means ± SD of three independent experiments. * significantlydifferent from control cells (p < 0.05), # significantly different frommenadione-treated cells (p < 0.05); (b) morphological analysis of SK-N-MCcells by double staining method. White arrow indicates live cells, dashed arrowshows apoptotic cells. Scale bar: 40 μM. μM) for 24 h. As shown in Figure 4,Z-VAD-fmk reduced the extent of apoptosis relative to menadionetreated cells,confirming the caspase-dependent apoptosis of cells. 3.6. Effect of Baicaleinon Menadione-Induced Lipofuscin Formation Exposure of the cells to 35 μMmenadione for 24 h caused 374% increase in the intracellular level oflipofuscin relative to menadione-untreated control cells. Pretreatment of thecells with baicalein (10, 20, 40 μM) diminished the formation of lipofuscinpigments by 155%, 192% and 214% after 24 h of exposure (Figure 5). 3.7.Baicalein Decreases Iron Accumulation in Menadione-Induced SK-N-MC Cells Iron isimportant for electron transport in the respiratory chain and for variousenzymatic reactions. When present in excess, however, iron can harm biologicalsystems since in redox-active form it catalyzes the generation of highlyreactive oxygen species [33]. Since both iron deficiency and overload impairedcellular functions, the quantitation of iron in cells and extracellular fluidsis of considerable interest [34,35]. As shown in Figure 6, treatment of SK-N-MCcells with menadione elevated free iron contents compare to basal iron level inthe control samples (2.17 nmol/mg proteins compare to 1.1 nmol/mg protein ofcontrol). However, pretreatments with different doses of baicalein (10, 20, 40μM) diminished the iron contents to 1.75, 1.54 and 1.33, respectively. 3.8.Effects of Baicalein and Z-VAD-Fmk on Menadione-Induced Cell Death Previousstudies have shown that menadione-induced Figure 5. Inhibitory effect ofbaicalein on the menadionetreated accumulation of intracellular lipofuscinpigments. SK-N-MC cells were exposed to baicalein (10, 20, 40 μM) for 3 hfollowed by exposure to menadione (35 μM) for 24 h. Then, the extent oflipofuscin in cell lysates were evaluated using a varian spectrofluorometer,model Cary Eclipse, set at an excitation wavelength of 310 nm and an emissionwavelength of 620 nm. * significantly different from control cells (p <0.05), # significantly different from menadionetreated cells (p < 0.05).Figure 6. Effect of baicalein on intracellular iron contents inmenadione-treated SK-N-MC cells. SK-N-MC cells were exposed to baicalein (10,20, 40 μM) for 3 h followed by exposure to menadione (35 μM) for 24 h. Ironcontents were evaluated by colorimetric ferrozine-based assay. * significantlydifferent from control cells (p < 0.05), # significantly different frommenadione-treated cells (p < 0.05). Copyright © 2013 SciRes. CellBio M.MOSLEHI, R. YAZDANPARAST 41 apoptosis is associated with changes inapoptosis-related Bcl-2 family of regulatory proteins. Bax is a pro-apoptoticmember of the Bcl-2 family which forms mitochondrial permeability pores forrelease of cytochrome c to the cytosol via binding to the anti-apoptotic Bcl-2member. This event in turn will lead to cleavage of procaspase-9 and furtheractivation of procaspase-3 and cell death through apoptosis [36]. Pretreatmentof cells with baicalein prior to menadione treatment, reduced Bax/ Bcl2 ratioand pretreatment of cells with baicalein and Z-VAD-fmk decreased cleavedcaspase-9 in SK-N-MC cells which showed that baicalein inhibitedcaspase-dependent apoptosis in this cell line (Figure 7). 4. Discussion One ofthe well-accepted theories for explicating the aging process is the freeradical theory proposed by Denham Harman [5]. This theory illustrates thatthere is a causal relationship between oxidative stress and pathogenesis ofage-related disorders [6]. Lipofuscin, a histological index of aging, is ahighly oxidized cross-link aggregate consisting of oxidized proteins (30% -58%) and lipids (19% - 51%) clusters accrues mostly in postmitotic cells suchas neurons, cardiac myocytes, skeletal muscle fibers and retinal pigments [7].Since oxidative reactions are compulsory components of normal life processes,the incidence of reactive oxygen species with ensuing lipofuscin formation is aninexorable side effect of life [10]. Many studies have signified that manyROSinduced diseases such as neurodegenerative disorders are associated withhigh levels of lipofuscin within neuronal cells [37,38]. It has been widelyreported that loosely bound iron in the cellular iron pool can react withendogenous hydrogen peroxide to produce the short-lived and highly reactivehydroxyl radicals through the Fenton reaction. These hydroxyl radicals, inturn, can oxidize nucleic acids, proteins or lipids leading to lipofuscinformation [23]. Oxidized proteins within lipofuscin are linked byintermolecular cross-links. Many of these cross-links are caused by nonproteineous compounds including oxidized lipids such as Malondialdehyde (MDA)and 4-hydroxy-2-nonenal by means of reactions with lysine amino groups,cysteine sulfhydryl groups and histidine imidazole groups of proteins [39].Thus, preventing biomolecules peroxidations and maintaining iron homeostasisplay major roles in blocking lipofuscin formation. Menadione (2-methyl-1,4naphthoquinone) in the cells converts to menadione semiquinone radical viaNADPH cytochrome c reductase activity. Then, semiquinone radical is recycledback to menadione through rapid reaction with molecular oxygen. This can resultin the formation of superoxide radical which causes oxidative stress [40].Although superoxide is chemically incapable of (a) (b) Figure 7. Analysis ofBcl-2, Bax and procaspase-9 activation in SK-N-MC cells treated with menadione,baicalein and Z-VAD-fmk. SK-N-MC cells were pretreated with baicalein (40 μM)and Z-VAD-fmk (50 μM) for 3 h and then incubated with menadione (35 μM) for 24h. (a) bcl-2, Bax and the (b) procaspase-9 expression were estimated byimmunoblots using relevant specific antibodies, and intensity of each band wasestimated by densitometric analysis. Equal sample loadings were confirmed bytubulin band. Values correspond to means ± SD of three independent experiments.* significantly different from control cells (p < 0.05), # significantly differentfrom menadione-treated cells (p < 0.05). affecting biomolecules directly, itis assumed to do so indirectly by participating in the production of hydroxylradicals through Fenton reaction. Superoxide radicals can provide free iron tocatalyze peroxidation from two sources: release iron from ferritin and oxidizesthe [4Fe - Copyright © 2013 SciRes. CellBio 42 M. MOSLEHI, R. YAZDANPARAST 4S]clusters of enzymes such as dehydratases, precipitating the release of one ormore iron atoms [41]. Thus, menadione as a Fenton catalyst, assisted theproduction of free iron for production of hydroxyl radicals to ignite crosslink of oxidized proteins and lipids in order to form lipofuscin. There is anaccumulating evidence denoting that lipofuscin can induce neurotoxicity via itscapacity for binding metals such as iron, copper, zinc and calcium whichstimulates generation of excessive ROS and decrease proteasomal and lysosomaldegradation by inhibition of the proteasomal turnover [7]. Numerous studieshave shown that intracellular iron accumulation contributes to the developmentof several common neurodegenerative diseases such as Alzheimer’s disease (AD)and Parkinson’s disease (PD) [33-35]. In order to restrain the destructiveeffects of ROS including superoxide radicals in neuronal cells, dietaryflavonoids are shown to have potential anti-aging and brain-protectiveactivities. Baicalein (5, 6, 7-trihydroxy- 2-phenyl-4H-1-benzopyran-4-one), anaturally occurring flavonoid, is the major bioactive compounds found intraditional Chinese medicinal herb, Baikal Skullcap (Scutellaria baicalensisGEORGI) [22]. Baicalein produces promising results as a strong antioxidant. Itsability to cross blood brain barrier (BBB), hydrophobicity, presence ofhydroxyl groups at C-5 and C-7, a double bond between C-2 and C-3, high troloxequivalent antioxidant capacity (TEAC) and DPPH free radical scavengingactivity make baicalein a good ROS scavenger in neurons [20,21]. Presence ofhydroxyl groups in baicalein structure results in scavenging of charged speciessuch as superoxide radicals and hydroxyl radicals more efficiently compared tonon-charged oxidant species [20]. On the other hand, baicalein can inhibit theproduction of endogenous hydroxyl radicals produced through the Fenton reactionby forming stable and inert complexes with iron [23]. Iron-binding motifs insome phenolic compounds can clarify the potential ability of them to modulateiron homeostasis in the body. Baicalein contains these motifs and thus expectedto chelate iron. Some recent studies have shown that two hydroxyl groups at the6 and 7 positions on the A ring seems to be the powerful metal binding site[20,23]. In support of what we have explained before, our studies showed thatbaicalein reduced the harmful effects of menadione by scavenging superoxideradicals which led to increased cell viability and decreased intracellular MDAand PCO. In addition, our results confirmed that baicalein has anti-Fentonproperties since it decreased the free iron contents of SK-N-MC cells exposedto menadion treatment. We also observed that baicalein strongly inhibitedlipofuscin formation in menadione-treated SKN-MC cells and displays anti-agingfeatures. Morphological analysis and western blot results implied thatbaicalein prevented apoptotic cell death through inhibition of Bax andprocaspase-9 activations and induction of bcl2 expression which avertedactivation of further caspases and transcription factors, release of cytochromec and resultant cell death. The results were confirmed by applying pan-caspaseinhibitor (Z-VAD-fmk). Moreover, our experiments have shown that Z-VAD-fmkprevented cell death in SK-N-MC cells through inhibition of caspases and didnot have any significant antioxidant characteristics. Overall, flavonoidbaicalein can be considered as a strong and auspicious antioxidant which couldprotect neuronal cells and hence, baicalein is a reliable option forantioxidant therapy in treatment of age-related and neurodegenerativedisorders, pending further in vivo and clinical investigations. 5.Acknowledgements The author appreciates the financial support of thisinvestigation by the Research Council of University of Theran.
