Oxidative damage and redox metal homeostasis loss are two contributing factors in brain aging and widely distributed neurodegenerative diseases. Oxidative species in company with excessive amounts of intracellular free iron result in Fenton-type reaction with subsequent production of highly reactive hydroxyl radicals which initiate peroxidation of biomolecules and further formation of non-degradable toxic pigments called lipofuscin that amasses in long-lived postmitotic cells such as neurons. Dietary flavonoid baicalein can counteract the detrimental consequences through exertion of a multiplicity of protective actions within the brain including direct ROS scavenging activity and iron chelation. In this study, we evaluated the neuroprotective effects of baicalein in menadione (superoxide radical generator)-treated SK-N-MC neuroblastoma cell line. Our results showed that treatment of cells with menadione led to lipofuscin formation due to elevated intracellular iron contents and accumulation of oxidative products such as MDA and PCO. Also, menadione caused apoptotic cell death in SK-N-MC cells. However, pretreatment with baicalein (40 μM) reversed the harmful effects by chelating free iron and preventing biomolecules peroxidations. Moreover, baicalein prevented cell death through modulation of key molecules in apoptotic pathways including suppression of Bax and caspase-9 activities and induction of bcl2 expression. Key structural features such as presence of hydroxyl groups and iron-binding motifs in baicalein make it the appropriate candidate in antioxidant-based therapy in age-related neurodegenerative diseases.
The key precept of the oxidative stress theory of aging is that senescence-related loss of function is due to the progressive and irreparable accrual of molecular oxidative damage which is brought about by powerful pro-oxidant species including reactive oxygen species (ROS) [1,2]. ROS include a broad range of partially reduced metabolites of oxygen (e.g. superoxide, hydrogen peroxide and hydroxyl radical) having higher reactivity than molecular oxygen [
Human’s aspiration for greater longevity has long been a strong motivation for a lot of studies in the field of aging and age-related disorders. Escalating body of evidence implies that lifestyle factors, and specially the diet, may counteract oxidative damage [2,14]. Dietary flavonoids with blood-brain barrier ability were shown to have potential anti-aging and brain-protective activities [5, 15-18]. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), one of the naturally occurring flavonoids in Scutellaria baicalensis GEORGI known as “Huang qin” in China and “Ogon” in Japan, is prescribed for oxidative stress-related diseases [
In this study, we scrutinize the effect of baicalein on menadione (superoxide anion generator)-induced lipofuscin formation in human neuroblastoma SK-N-MC cell line to comprehend the mechanism by which baicalein protect SK-N-MC cells against oxidative damages.
The cell culture medium (RPMI-1640), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco BRL (Life technology, Paisely, Scotland). The culture plates were purchased from Nunc (Brand products, Denmark). dimethyl sulfoxide (DMSO), FeCl3 and KMnO4 were obtained from Merck (Darmstadt, Germany). Ethidium bromide, acridine orange, Baicalein and Triton X-100 were purchased from Pharmacia LKB Biotechnology (Sweden). MTT [3-(4,5-dimethyl tiazol-2, 5-diphenyl tetrazolium bromide], phenylmethylsulphonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin, monochlorobimane (mBCL), dithionitrobenzoic acid (DTNB)GSH, ascorbic acid, ferrozine and pan-caspase inhibitor (Z-VAD-fmk) were purchased from 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-NMC neuroblastoma cells were obtained from Pasteur Institute (Tehran, Iran). All antibodies including anti-Bax, anti-Bcl-2, anticleaved caspase-9, anti-tubulin and mouse/rabbit horseradish peroxidase-conjugated second-dary antibodies were purchased from Biosource (Nivelles, Belgium). Chemiluminescence detection system was purchased from Amersham-Pharmacia (Piscataway, NJ, USA).
Human neuroblastoma cell line SK-N-MC was cultured in RPMI-1640 medium supplemented with FBS (10%, v/v), streptomycin (100 μg/ml) and penicillin (100 U/ml) and incubated in 5% CO humidified atmosphere at 37˚C. To induce oxidative stress, menadione was freshly prepared from a stock solutions (10 mM), prior to each experiment. Menadione and baicalein were dissolved in a minimum amount of dimethyl Sulfoxide (DMSO) and then diluted with the culture medium to the desired concentration. The concentration of DMSO in the culture medium kept lower than 0.1% and the control cells were treated with the vehicle solution containing the same amount of DMSO.
Cell viability was assessed by the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay. Viable cells with active mitochondria reduce the yellow tetrazolium salt MTT giving dark blue water insoluble formazan crystals. To perform the assay for evaluation of the cytoprotective effects of baicalein and caspase inhibitor (z-VAD-fmk) on menadione-treated SK-N-MC cells, SK-N-MC cells were suspended in medium and seeded at a density of 5 × 104 cells/well in 96 well plates for a day. Cells were pretreated with various concentrations of baicalein (10, 20, 40, 50 µM) and pancaspase inhibitor (50 µM) and then treated with menadione (35 µM) for additional 24 h at 37˚C. MTT was dissolved at a concentration of 5 mg/ml in PBS and stored at 4˚C, protected from light and tightly capped. After incubation, cells were treated with the 10 µl MTT solution for 4 h. Then, the medium was removed and 200 µl DMSO was added to each well. The formazan dye crystals were solubilized in 30 min, and absorbance was 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 the absorbance of the control cells was 100%.
Oxidation of 2’,7’-dichlorofluorescein diacetate (DCFHDA) to fluorescent DCF is taken as an index of overall oxidative stress in biological system according to LeBel method [
Malondialdehyde (MDA) levels were measured by the double heating method [
The assessment of protein carbonyl content is a widelyused marker for oxidative protein modification. Protein carbonyls (PCOs) were measured using Reznick and Packer method [
Acridine orange/ethidium bromide double staining was applied to observe the morphological changes among menadione-treated cell. Using this technique, cells can be distinguished as normal cells (uniformly stained green) and apoptotic cells that are stained orange because of cell membrane destruction and the intercalation of ethidium bromide between the nucleotide bases of DNA. After treatment, cells were washed twice with phosphate buffer saline and adjusted to a cell density of 1 × 104 cells/ml of phosphate solution (1:1 v/v). The nuclear morphology was evaluated by Axoscope 2 plus fluorescence microscope from Zeiss (Germany). The cells with condensed or fragmented nuclei were counted as apoptotic cells. All experiments were repeated three times, and the number of stained cells was counted in 10 randomly selected fields.
Extraction of intracellular lipofuscin was achieved following lysis of each sample according to a published procedure with slight modification [
The assay was performed directly in 24-well plates. Cells were lysed by addition of 200 µl iron releasing reagent (a freshly mixed solution of equal volumes of 1.4 M HCl and 4.5% (w/v) KMnO4 in H2O2) to each well. The plates were sealed with foil 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 acid dissolved in water) was added. After further incubation for 30 min at room temperature, 280 µl of the mixture was transferred to a well of a 96-well plate and its absorbance recorded at 550 nm and compared to the absorbance of the FeCl3-treated standards under all equal experimental conditions. The determined intracellular iron concentration for each well was normalized against the protein content of replicate wells [
SK-N-MC cells were seeded at a density of 105 cells/ml in 12-well plates for 24 h. The cells 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 an additional 24 h. Then, the cells were harvested and lysed using lysis buffer containing 1% Triton X-100, 1% SDS, 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM sodium pyrophosphate, 2 mM Na3VO4, 1 mM NaF, 0.5% sodium deoxycholate, 10% glycerol, 1mM phenylmethylsulphonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin and 60 μg/ml aprotinin. Protein concentration of each sample was determined using Lowry’ method (Lowry et al., 1951). Equal quantities of protein (40 μg) were subjected to 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and were transferred to PVDF membranes. The blots were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline buffer containing 0.1% Tween-20 (TBS/T) for an overnight at 4˚C. The blocked blots were incubated with primary antibodies for 2 hr at room temperature using antibody dilutions as recommended by the manufacturer in Tris-buffered saline pH 7.4 containing 0.1% Tween-20. After 1-hr incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Biosource), the proteins were detected by an enhanced chemiluminescence detection system (Amersham-Pharmacia, Piscataway, NJ, USA) according to the manufacturer’s instructions. Blots were stripped at 50˚C for 30 min in 100 mM 2-mercaptoethanol, 2% SDS, 62.2 mM Tris-HCl pH 6.7 and reprobed for further investigations. For analysis of the western blotting data, densitometric analysis was performed using Image.J software, and the densities were normalized with respect to β-tubulin as the internal control.
Data were expressed as percent of values of untreated control cells, and each value represents the mean ± SD (n = 3). The significant differences between the means of the treated and untreated cells were calculated by unpaired Student’ t-test, and p-values < 0.05 were considered significant.
Menadione is a quinone known to induce an oxidative stress generated primarily by superoxide radicals leading to cell death [
Increase in ROS generation was measured as one of the indicators of menadione-induced oxidative stress in cells. As shown in
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 not significantly change the ROS level in menadione-treated SK-N-MC cells.
Menadione-induced oxidative stress causes oxidation of intracellular biomolecules such as lipids. MDA is produced while lipid peroxidation happens. So, MDA level measurement is used as a marker of menadione-induced oxidative stress. As shown in
Protein carbonyl is a marker of protein oxidation in oxidative stress condition. We evaluated the effects of different doses of baicalein (10, 20, 40 μM) and Z-VADfmk (50 µM) on protein carbonyl formation in SK-N-MC cells. After treatment with menadione (35 μM), the amount of protein carbonyl increased to 4.03 nmol/mg protein compared to 0.65 nmol/mg protein of control cells. Pretreatment with baicalein (10, 20, 40 μM) reduced protein carbonyl formation to 2.6, 1.7 and 1.1 nmol/mg protein, respectively (
To study the protective effect of baicalein on SK-N-MC cells, acridine orange/ethidium bromide double staining technique was used to evaluate the occurrence of apoptosis in cells. As shown in
µM) for 24 h. As shown in
Exposure of the cells to 35 µM menadione for 24 h caused 374% increase in the intracellular level of lipofuscin relative to menadione-untreated control cells. Pretreatment of the cells with baicalein (10, 20, 40 µM) diminished the formation of lipofuscin pigments by 155%, 192% and 214% after 24 h of exposure (
Iron is important for electron transport in the respiratory chain and for various enzymatic reactions. When present in excess, however, iron can harm biological systems since in redox-active form it catalyzes the generation of highly reactive oxygen species [
Previous studies have shown that menadione-induced
apoptosis is associated with changes in apoptosis-related Bcl-2 family of regulatory proteins. Bax is a pro-apoptotic member of the Bcl-2 family which forms mitochondrial permeability pores for release of cytochrome c to the cytosol via binding to the anti-apoptotic Bcl-2 member. This event in turn will lead to cleavage of procaspase-9 and further activation of procaspase-3 and cell death through apoptosis [
One of the well-accepted theories for explicating the aging process is the free radical theory proposed by Denham Harman [
Menadione (2-methyl-1,4 naphthoquinone) in the cells converts to menadione semiquinone radical via NADPH cytochrome c reductase activity. Then, semiquinone radical is recycled back to menadione through rapid reaction with molecular oxygen. This can result in the formation of superoxide radical which causes oxidative stress [
affecting biomolecules directly, it is assumed to do so indirectly by participating in the production of hydroxyl radicals through Fenton reaction. Superoxide radicals can provide free iron to catalyze peroxidation from two sources: release iron from ferritin and oxidizes the [4Fe - 4S] clusters of enzymes such as dehydratases, precipitating the release of one or more iron atoms [
There is an accumulating evidence denoting that lipofuscin can induce neurotoxicity via its capacity for binding metals such as iron, copper, zinc and calcium which stimulates generation of excessive ROS and decrease proteasomal and lysosomal degradation by inhibition of the proteasomal turnover [
In order to restrain the destructive effects of ROS including superoxide radicals in neuronal cells, dietary flavonoids are shown to have potential anti-aging and brain-protective activities. Baicalein (5, 6, 7-trihydroxy- 2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, is the major bioactive compounds found in traditional Chinese medicinal herb, Baikal Skullcap (Scutellaria baicalensis GEORGI) [
In support of what we have explained before, our studies showed that baicalein reduced the harmful effects of menadione by scavenging superoxide radicals which led to increased cell viability and decreased intracellular MDA and PCO. In addition, our results confirmed that baicalein has anti-Fenton properties since it decreased the free iron contents of SK-N-MC cells exposed to menadion treatment. We also observed that baicalein strongly inhibited lipofuscin formation in menadione-treated SKN-MC cells and displays anti-aging features. Morphological analysis and western blot results implied that baicalein prevented apoptotic cell death through inhibition of Bax and procaspase-9 activations and induction of bcl2 expression which averted activation of further caspases and transcription factors, release of cytochrome c and resultant cell death. The results were confirmed by applying pan-caspase inhibitor (Z-VAD-fmk). Moreover, our experiments have shown that Z-VAD-fmk prevented cell death in SK-N-MC cells through inhibition of caspases and did not have any significant antioxidant characteristics.
Overall, flavonoid baicalein can be considered as a strong and auspicious antioxidant which could protect neuronal cells and hence, baicalein is a reliable option for antioxidant therapy in treatment of age-related and neurodegenerative disorders, pending further in vivo and clinical investigations.
The author appreciates the financial support of this investigation by the Research Council of University of Theran.
MDA: Malondialdehyde;
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;
PBS: Phosphate buffer saline;
PCO: Protein carbonyl;
AD: Alzheimer’s disease;