parkinson 's disease ( pd ) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta , resulting in the subsequent loss of function of the basal ganglia circuit .
the molecular pathogenesis of pd is believed to be associated with mitochondrial dysfunction , oxidative stress , and activation of the apoptotic cascade .
the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) induces permanent parkinsonism in humans via its metabolite mpp ( 1-methyl-4-phenylpyridinium ) [ 2 , 3 ] .
mpp has been shown to induce a pd - like pathology in animals and cellular models by selective and potent inhibiting of complex 1 of the mitochondrial electron transport chain [ 4 , 5 ] .
mpp - induced neuronal death is mediated by impairment of the mitochondrial membrane potential and opening of the mitochondrial permeability transition pore [ 6 , 7 ] .
elevation in the reactive oxygen species ( ros ) level has also been involved in mpp - induced cytotoxicity [ 810 ] .
activation of the apoptotic cascade may play a role in mpp - induced cell death by altering mitochondrial membrane permeability and controlling the release of cytochrome c from mitochondria [ 11 , 12 ] .
caspase-3 activation by released cytochrome c has been shown to involve mpp - induced apoptosis [ 9 , 13 , 14 ] .
once activated , caspase-3 will induce nuclear dna condensation and fragmentation and , ultimately , apoptosis . a number of antioxidants , such as xanthones ,
have been demonstrated to have a protective effect on vulnerable neurons under oxidative stress conditions [ 1618 ] .
the fruit hull of mangosteen ( garcinia mangostana linn . ) , a tropical fruit , has been demonstrated to exert an antioxidative effect .
alpha - mangostin was shown to induce a protective effect in cardiac reperfusion damage by attenuation of oxidative stress .
neuroprotective activities of alpha - mangostin against h2o2-induced oxidative stress have been demonstrated in ng108 - 15 neuroblastoma cells .
this xanthone ameliorated iodoacetate - induced cell death in primary cultures of cerebellar granule neurons by reducing ros formation .
alpha - mangostin was also shown to attenuate the neurotoxicity induced by beta - amyloid oligomers in sk - n - sh neuroblastoma cells and primary rat cerebral cortical neurons [ 22 , 23 ] .
the antioxidative property of alpha - mangostin is probably mediated by its modulatory effect on the activity of glutathione peroxidase . although alpha - mangostin has been reported to possess potential neuroprotective properties , there is insufficient information on its protective effects in a pd cellular model .
this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp - induced apoptosis and the possible underlying mechanisms .
the sh - sy5y human neuroblastoma cells were cultured in a 1  :  1 mixture of dulbecco 's modified eagle medium ( dmem ) and nutrient mixture ham 's f12 medium and supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) , 1  mm sodium pyruvate , 0.1  mm nonessential amino acid , 1.5  g / l sodium bicarbonate , 100 units / ml penicillin , and 100  g / ml streptomycin .
all media and supplements were purchased from gibco ( gaithersburg , md , usa ) . cells were maintained at 37c in a humidified atmosphere of 5% co2 . in the experiment
the number of cells to be subcultured was assessed under a phase - contrast microscope based on the exclusion of trypan blue dye .
the cultured cells were maintained for 2 days to allow for adhering on the plates .
louis , mo , usa ) , or a combination of -mangostin and mpp according to the experiment design .
sh - sy5y cells were seeded onto a 96-well plate at a density of 8  10  cells / well in 200  l of medium and incubated at 37c under 5% co2 in a humidified incubator for 2 days .
after exposure to mpp ( 1000  m ) , -mangostin ( 10  m ) , or a combination of both for 24 hours , cell viability was measured by mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) colorimetric assay ( sigma - aldrich , st .
this method was based on the reduction of tetra ring of mtt by mitochondrial dehydrogenases with nadh in the active mitochondria , yielding a blue formazan product , which can be measured spectrophotometrically .
after incubation , 20  l of mtt ( 5  mg / ml ) was added to each well and the cells were cultured for another 4 hours , then medium was removed , and 100  l of dmso ( sigma - aldrich , st .
louis , mo , usa ) was added to each well to dissolve the formazan .
the color reaction was measured at wavelength 570  nm with a reference at 690  nm using the versamax tunable microplate reader with softmax pro software ( molecular devices , sunnyvale , ca , usa ) .
sh - sy5y cells were seeded on coverslips and fixed with 4% paraformaldehyde in 0.01  m pbs ( ph 7.4 ) for 20 minutes at room temperature and then rinsed three times with pbs . after being washed with pbs ,
the cells were stained with 1  mg / ml hoechst 33258 ( sigma - aldrich , st .
louis , mo , usa ) in pbs for 20 minutes at room temperature and then washed again .
hoechst 33258 ( bisbenzimide ) preferentially binds to triplet adenine and thymine base pairs in the minor groove outside the double helix , which allows one to observe the morphological change in the nuclei of apoptotic cells .
nuclear morphology was examined under a laser scanning confocal microscopy ( olympus fv1000 , olympus , tokyo , japan ) with excitation wavelength 556  nm and emission wavelength 573  nm .
sh - sy5y cells were seeded onto 6-well plates at a density of 12  10  cells / well in 2  ml of medium and then incubated at 37c under 5% co2 in a humidified incubator for 24 hours .
after exposure to mpp ( 1000  m ) , -mangostin ( 10  m ) , or the combination of mpp ( 1000  m ) and -mangostin ( 10  m ) for 24 hours , cells were trypsinized and centrifuged at 2,500  rpm , 4c for 5 minutes .
the pellet was washed twice with cold pbs and resuspended at a concentration 1  10  cells / ml .
apoptotic cells were detected using an fitc annexin - v / dead cell apoptosis kit ( molecular probes , eugene , or , usa ) . cells were incubated with 5  l of fitc annexin - v and 1  l of the 100  g / ml propidium iodide ( pi ) at room temperature for 15 minutes . the apoptotic cells were analyzed with a facscalibur flow cytometer ( bd biosciences , san jose , ca , usa ) , measuring the fluorescence emission at 530  nm ( fl1 ) and > 575  nm ( fl3 ) .
both early apoptotic ( annexin - v - positive and pi - negative ) and late apoptotic ( annexin - v - positive and pi - positive ) cells were included in cell death determinations .
the production of ros was determined by measuring the intensity of fluorescence emitted by cell - permeable fluorescent dyes , dihydrorhodamine 123 ( dhr123 ) and dihydroethidium ( dhe ) , purchased from invitrogen ( eugene , or , usa ) .
dhr123 is oxidized to a fluorescent rhodamine-123 by intracellular hydrogen peroxide and peroxynitrite , while dhe is oxidized by superoxide anion to ethidium .
sh - sy5y cells were seeded on 6-well plates at a density of 12  10  cells / well and incubated at 37c under 5% co2 in a humidified incubator for 48 hours .
after exposure to mpp ( 1000  m ) , -mangostin ( 10  m ) or the combination of mpp ( 1000  m ) and -mangostin ( 10  m ) for 48 hours , cells were washed twice with hanks buffer salt solution ( hbss ) and trypsinized by 0.25% trypsin / edta and centrifuged at 2,000  rpm for 7 minutes .
the pellets were incubated with 15  m dhr123 and 10  m dhe for 20 minutes at 37c in the dark .
thereafter , cells were washed with hbss before cell fixation in 1% paraformaldehyde and the mean fluorescence intensity ( mfi ) of green ( fl1 , dhr ) or red ( fl2 , dhe ) fluorescence was determined using a facscalibur flow cytometer .
sh - sy5y cells were seeded onto 6-well plates at a density of 12  10  cells / well and incubated at 37c under 5% co2 in a humidified incubator for 24  h. after exposure to mpp ( 1000  m ) , -mangostin ( 10  m ) or the combination of mpp ( 1000  m ) and -mangostin ( 10  m ) for 24 hours , cells were trypsinized and centrifuged at 2,500  rpm , 4c for 5 minutes .
total mrna was extracted from the pellet using paris kit according to the supplier 's instructions .
the quantity and purity of rna were determined by optical density measurements at od a260/a280 ratio with 1.8 or above using nanodrop 2000 spectrophotometer ( thermo fisher scientific inc . , wilmington , de , usa ) .
the integrity of rna was confirmed by running 0.5  g of rna samples on 1% agarose gel .
later , the cdna was synthesized from 1  g of rna using masterscript rt - pcr system ( 5 prime , gaithersburg , md , usa ) , according to the manufacturer 's instruction and stored at 20c until assay .
kapa sybr fast qpcr kit ( kapa biosystems , woburn , ma , usa ) was used for real - time pcr quantification .
the 20  l real - time pcr reaction mixture contained 20  ng cdna template , 10  l of 1x kapa sybr fast qpcr master mix , 200  nm of forward and reverse primers , and pcr - grade water .
the sequences of the primers for the qrt - pcr are as follows : p53 : sense , 5-ggaggttgtgaggcgctgg-3 ; antisense , 5-cacgcacctcaaagctgttc-3 ; bax : sense , 5-cccgagaggtctttttccgag-3 ; antisense , 5-ccagcccatgatggttctgat-3 ; bcl-2 : sense , 5-catgtgtgtggagagcgtcaa-3 ; antisense , 5-gccggttcaggtactcagtca-3 ; -actin : sense , 5-tgcagaggatgattgctgac-3 ; antisense , 5-gaggactccagccacaaaga-3. the reaction was performed in performed in the applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) with the pcr cycling conditions as follows : 3 minutes enzyme activation at 95c , 40 cycles of 3 seconds initial denaturation at 95c and annealing / extension at 58c for 32  sec .
melting curve analysis was performed for verify specificity of each primer after pcr to ensure amplification specificity .
the threshold cycle ( ct ) number was determined and used in the comparative ct method .
sh - sy5y cells were plated into 6-well plates at a density of 12  10  cells / well and incubated overnight . after exposure to mpp ( 1000  m ) ,
-mangostin ( 10  m ) or the combination of mpp ( 1000  m ) and -mangostin ( 10  m ) for 24 hours under 5% co2 in a humidified incubator at 37c , cells were trypsinized by 0.25% trypsin / edta and centrifuged at 4,000  rpm , 4c for 5 minutes .
the pellets were washed with cold pbs , resuspended and incubated on ice for 30 minutes
. then cells were vortexed and centrifuged at 15,000  rpm for 15 minutes at 4c .
the supernatant was collected and total protein concentration was determined using a bca protein assay .
an equal amount of 30  g proteins from each experiment group were separated by 15% sds - polyacrylamide gel electrophoresis .
the membrane was blocked with a 5% skim milk for 2 hours , washed with tbst buffer , and incubated with 1  :  1000 dilution of cleaved caspase-3 primary antibodies ( cell signaling technology , denvers , ma , usa ) or 1  :  5000 dilution of monoclonal -actin primary antibodies ( sigma - aldrich , st .
after washing , the membrane was incubated with 1  :  10000 dilution of hrp - conjugated goat polyclonal anti - rabbit igg ( abcam , cambridge , uk ) as the secondary antibody for cleaved caspase-3 and 1  :  5000 hrp - conjugated goat anti - mouse igg ( invitrogen , eugene , or , usa ) as the secondary antibody for -actin for 2 hours at room temperature before being developed using ecl prime western blotting detection ( ge healthcare , buckinghamshire , uk ) . the membrane was reproved with anti -actin antibody to control for equal loading of protein .
the signal intensities were determined by densitometry using image - j software ( national institutes of health , bethesda , maryland , usa ) .
statistical analyses were performed with one - way anova test followed by a post hoc analysis ( tukey 's multiple comparison test ) using graphpad prism 5 software for windows ( graphpad software , inc . , san diego , ca , usa ) .
all values were presented as mean  standard error of the mean ( mean  sem ) for each group .
to investigate the influence of alpha - mangostin on neuronal cell viability , we treated sh - sy5y cells with various concentrations of alpha - mangostin for 24 hours and examined cell viability with the mtt assay .
exposure of sh - sy5y cells to alpha - mangostin induced a reduction in cell viability in a concentration - dependent manner .
cell viability significantly decreased when cells were treated with 10  m alpha - mangostin for 24 hours , compared with that of control ( p < 0.01 ; figures 1 and 2 ) .
treatment of 20 and 40  m alpha - mangostin led to loss of cell viability by more than 50% ( p < 0.001 ; figure 1 ) . when sh - sy5y cells were exposed to 2.510  m alpha - mangostin in the presence of 1000  m mpp for 48 hours
, the co - treated cells showed a significant increase in cell viability in a concentration - dependent manner , compared to those treated with 1000  m mpp alone ( figure 3 ) .
based on these results , 10  m alpha - mangostin was utilized in later experiments .
apoptotic cells were quantified with pi and annexin - v dual staining using flow cytometry .
the annexin - v / pi population consisted primarily of normal healthy cells , while annexin - v / pi cells were considered to be in the early stage of apoptosis , and annexin - v / pi cells were those considered to be in the necrosis / late apoptosis stage ( figure 4(a ) ) .
after exposure to 1000  m mpp for 24 hours , the percentage of apoptotic cells increased ( p < 0.001 ; figure 4(b ) ) .
cotreatment with 10  m alpha - mangostin in the presence of 1,000  m mpp for 24 hours significantly alleviated the apoptosis when compared to those treated with mpp only ( p < 0.001 ) .
treatment with 10  m alpha - mangostin decreased the number of apoptotic nuclei ( figure 5 ) .
alpha - mangostin significantly decreased the percentage of apoptotic nuclei when compared to mpp treatment alone ( p < 0.001 ; figure 5(e ) ) . to evaluate whether ros play an important role in the attenuation effect of alpha - mangostin on mpp - induced apoptosis in sh - sy5y cells , cells were exposed to 1000  m mpp and
10  m alpha - mangostin plus 1000  um mpp for 6 hours , and intracellular ros production was assessed as dhe and dhr123 fluorescence using flow cytometry .
the result shows that cotreatment with 10  m alpha - mangostin in the presence of 1000  m mpp significantly decreased intracellular ros levels as measured by the mean fluorescence intensity ( mfi ) of dhe ( p < 0.001 ; figure 6(a ) ) and dhr123 ( p < 0.001 ; figure 6(b ) ) , compared to mpp treatment alone .
expression of mrna of bax and bcl-2 was investigated using real - time quantitative rt - pcr analysis .
bax expression significantly increased in 1000  m mpp - treated cells , compared to that of control cells ( figure 7(a ) ) .
cotreatment of 10  m alpha - mangostin and 1000  m mpp for 24 hours significantly decreased the bax expression , compared to mpp treatment alone ( p < 0.01 ) .
treatment with 1000  m mpp significantly decreased bcl-2 expression , while cotreatment with 10  m alpha - mangostin significantly increased bcl-2 expression ( p < 0.01 ; figure 7(b ) ) . as a result
, bax / bcl-2 expression was significantly increased in sh - sy5y cells treated with 1000  m mpp for 24 hours ( p < 0.001 ; figure 7(c ) ) , whereas bax / bcl-2 expression was significantly lowered in cells cocultured in 10  m alpha - mangostin and 1000  m mpp ( p < 0.001 ) .
the expression of p53 mrna was significantly increased in 1000  m mpp - treated sh - sy5y cells after 24-hour exposure when compared to unexposed cells ( p < 0.001 ; figure 7(d ) ) .
cotreatment of 10  m alpha - mangostin and 1000  m mpp significantly reduced p53 expression when compared to cells cultured in mpp alone ( p < 0.01 ) . to investigate the effect of alpha - mangostin on the caspase-3 protein , a critical executioner of apoptosis
activation of caspase-3 requires cleavage of the protein at asp175 into the activated p17 and p19 fragments . after a 24-hour treatment of sh - sy5y cells with 1000  m mpp , a significant increase in activated caspase-3 protein
when sh - sy5y cells were exposed to 10  m alpha - mangostin in the presence of 1000  m mpp , the cotreated cells showed a significant decrease in activated caspase-3 compared to the mpp treatment alone ( p < 0.001 ) .
the present study has , for the first time , demonstrated that alpha - mangostin rescues apoptosis in dopaminergic sh - sy5y cells treated with mpp , a cellular model of parkinson 's disease .
the results suggest that cytoprotection of alpha - mangostin against mpp - induced apoptosis may be associated with the reduction of ros production , modulating the balance of pro- and antiapoptotic genes , and suppression of caspase-3 activation .
mpp is a neurotoxin used to generate animal and cellular models of parkinson 's disease .
mpp has been shown to increase ros in neuroblastoma cells [ 9 , 25 , 26 ] and induce superoxide anion ( o2 ) production via inhibition of mitochondrial complex i [ 10 , 27 ] .
the presence of antioxidant enzymes such as superoxide dismutase protects against mpp toxicity in neuronal cell lines   and dopaminergic neurons in primary culture .
previous studies showed that alpha - mangostin reduced ros formation [ 21 , 24 , 30 ] . in the present study , dhe and dhr123
dhe is used extensively to monitor superoxide production , perhaps the most specific dye that is retained well by cells [ 31 , 32 ] .
our results suggest that alpha - mangostin attenuated the oxidative effect of mpp through the reduction of superoxide production .
dhr123 is used for the detection of peroxide and peroxynitrite ( onoo ) . in vitro ,
our dhr123 oxidation assay suggests that treatment with alpha - mangostin also decreased the production of h2o2 and onoo induced by mpp .
ros are elevated in cells undergoing apoptosis   and antioxidants have been shown to protect neuronal cells against apoptosis induced by a variety of apoptotic agents .
ros activate caspase-3 and caspase-3-like proteases in various cell types including neuronal cells , leading to nuclear condensation and dna fragmentation [ 36 , 37 ] .
ros also induce apoptosis , probably by decreasing expression of bcl-2 , an antiapoptotic molecule [ 9 , 38 ] .
bcl-2 and bax may control the mitochondrial permeability transition pore , which can influence the passage of cytochrome c and other apoptosis - inducing factors that trigger the activation of caspase cascade and result in apoptosis .
mangosteen extract has been shown to protect sk - n - sh neuroblastoma cells against an a-induced increase in caspase-3 activity . here , we showed that alpha - mangostin decreased caspase-3 activation , decreased bax mrna expression , and increased bcl-2 mrna expression induced by mpp in dopaminergic sh - sy5y neuroblastoma cells .
a previous study on cisplatin - induced apoptotic death showed that alpha - mangostin attenuates the increase in p53 expression induced by cisplatin .
proapoptotic bax , directly induced by p53 , can overcome the antiapoptotic effect of bcl-2 , whereas p53 can directly inhibit bcl-2 .
in the present study , treatment with 1000  m mpp significantly increased the expression of p53 . as a result , increased p53 induced bax expression and inhibited bcl-2 expression , leading to the apoptosis of sh - sy5y cells .
our results suggest that alpha - mangostin might reduce the effect of mpp on bax and bcl-2 expression by attenuating p53 expression .
caspase-3 has been shown to be involved in the apoptotic events occurring in the mitochondrial - dependent pathway , which are associated with nuclear condensation and dna cleavage .
caspase-3 also induces phosphatidylserine externalization from the internal to external leaflets of the plasma membrane .
phosphatidylserine exposure on the external leaflet of the plasma membrane can be bound with annexin - v and is widely observed during apoptosis .
our dna staining and pi / annexin - v dual staining supported the role of alpha - mangostin in protection against nuclear changes and phosphatidylserine externalization induced by mpp treatment in sh - sy5y neuroblastoma cells .
alpha - mangostin is an antioxidant among the most abundant bioactive xanthones found in the mangosteen pericarp , which has long been used in traditional medicine to treat diarrhea , dysentery , infected wounds , and chronic ulcers .
it has a broad range of bioactivities , such as antioxidant , anti - inflammation , anticancerogenic , and antihistaminergic effects [ 39 , 4447 ] .
alpha - mangostin has a cardioprotective effect against myocardial infarct and reperfusion injury in rats ; such an effect was related to low levels of lipid peroxidation , a decrease in protein carbonylation and the preservation of high glutathione content [ 19 , 48 ] .
recently , alpha - mangostin has been shown to inhibit and dissociate the beta - amyloid aggregation , which could contribute to its effect of attenuating beta - amyloid oligomers - induced neurotoxicity in a cellular model of alzheimer 's disease .
this study indicates that alpha - mangostin protects sh - sy5y cells against mpp - induced apoptosis .
the underlying mechanism could be attributed to its antioxidative properties and thus modulating the apoptotic process .
the results showed that alpha - mangostin potentially possesses neuroprotective effects in a cellular model of pd .
alpha - mangostin is a small lipid - soluble molecule with the potential to pass the blood - brain barrier .
 
 in vitro studies have shown that extracts from mangosteen ( garcinia mangostana linn . ) 
 act as antioxidants and cytoprotective agents against oxidative damage . 
 the protective effect of alpha - mangostin , the major xanthone found in the pericarp of the mangosteen , in cellular models of parkinson 's disease ( pd ) , has not been investigated . 
 this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp+-induced apoptosis . 
 the effects of alpha - mangostin on mpp+-induced cell death were evaluated with a cell viability assay , staining for nuclear dna morphology , flow cytometry for apoptotic cells and reactive oxygen species ( ros ) production , quantitative real - time pcr for the expression of p53 , bax , and bcl-2 , and western blot analysis for cleaved caspase-3 . 
 concomitant treatment with alpha - mangostin attenuated the effect of mpp+ on cell viability and apoptotic cell death . 
 alpha - mangostin reduced ros formation induced by mpp+ . 
 bax / bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha - mangostin and mpp+ . 
 the cotreated cells showed a significant decrease in activated caspase-3 compared with mpp+ treatment alone . 
 our data suggest that cytoprotection of alpha - mangostin against mpp+-induced apoptosis may be associated with the reduction of ros production , modulating the balance of pro- and antiapoptotic genes , and suppression of caspase-3 activation . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion