parkinson 's disease ( pd ) is characterized by the selective degeneration of nigrostriatal dopaminergic ( daergic ) neurons , resulting in dopamine ( da ) depletion . while the etiology of pd is not completely clear , several pathology studies have demonstrated that , in postmortem samples of substantia nigra pars compacta ( snpc ) , daergic neurons exhibit markers of oxidative stress , such as lipid peroxidation , dna oxidative damage , and carbonyl modifications of soluble proteins [ 2 , 3 ] . the pd brain is also characterized by oxidative damage and functionally impaired and misassembled mitochondrial complex i , which affirm the involvement of oxidative stress in the pathophysiology of pd . indeed , recent data have shown that mn - dependent superoxide dismutase ( sod ) level and activity are increased in pd brains [ 3 , 5 ] . further evidence implicating oxidative stress in pd comes from studies with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) , which has been widely used as a daergic neurotoxin because it causes a severe pd - like syndrome in humans as well as in monkeys and mice . its administration to c57bl mice leads to a decline of striatal da and tyrosine hydroxylase ( th ) levels in the snpc [ 7 , 8 ] as well as the death of daergic neurons . mptp crosses the blood - brain barrier and is converted into its toxic metabolite 1-methyl-4-phenylpyridinium ( mpp ) in astrocytes by the enzyme monoamine oxidase b. mpp is then selectively taken up by daergic neurons via the high - affinity da transporter ( dat ) and is actively transported into mitochondria where it interferes with mitochondrial respiration through complex i inhibition [ 911 ] , elevating reactive oxygen species ( ros ) levels and increasing sod , catalase ( cat ) , and glutathione peroxidase ( gpx ) activities in mptp - treated mice and mpp - treated neuronal cells [ 1214 ] . also , mptp and its active metabolite mpp induce th nitration , that is associated with the degeneration of daergic neurons . on the other hand , h2o2 , o2   and oh have been detected after complex i inhibition with mpp . in addition , the decrease of complex i and subsequent oxidative stress evoked by mpp administration elicit neuronal cell death by apoptosis [ 17 , 18 ] . brassinosteroids ( brs ) are steroidal plant growth regulators found in several vegetables , including vicia faba ( broad bean ) seed and pollen [ 1921 ] . brs , considered to be a new group of plant hormones , are currently being studied intensively to understand their role in plant metabolism . their main physiological effects in plants include the regulation of hormonal balance , activation of protein and nucleic acid synthesis , enzyme activity , growth promotion , increased size and quantity of fruits , and , most interestingly , augmented resistance to unfavorable environmental factors , stress , and disease ( for review see ) . it has also been demonstrated that the exogenous application of natural brs to other vegetables has a specific antioxidative effect . the natural br 24-epibrassinolide ( 24-epi ) occurring in vicia faba   increases the enzymatic antioxidant activities of sod , cat , and gpx in lycopersicon esculentum ( tomato ) leaves and brassica juncea l. ( indian mustard ) plants [ 24 , 25 ] . 24-epi also reduces lipid peroxidation in oryza sativa l. ( rice ) and indian mustard plants [ 25 , 26 ] . the antioxidative properties of brs , clearly apparent in vegetables , strongly suggest that these compounds exert an antioxidant and neuroprotective role in mammals by curbing apoptosis , as reported recently for other natural molecules ( for review see [ 13 , 2734 ] ) . indeed , in mammals , the effects of brs are just starting to be elucidated . brs are known to exert anticancer and antiproliferative activities on human cell lines [ 3538 ] . antiviral activity has also been identified in natural brs and synthetic analogs [ 3941 ] . moreover , beans from vicia faba also contain l-3,4-dihydroxyphenylalanine ( l - dopa ) [ 42 , 43 ] , the amino acid precursor of da , which is nowadays the most effective symptomatic treatment of pd . clinical reports indicate that the consumption of vicia faba has a beneficial outcome in pd patients [ 45 , 46 ] . however , l - dopa concentrations in vicia faba are not sufficient to explain the magnitude of the responses observed in pd patients and raise the possibility that other compounds from vicia faba , such as brs , may complement the l - dopa effect by their antioxidative activities . the aim of our study was to examine , in detail , the influence of 24-epi , a br present in vicia faba , on mpp - induced oxidative stress in a well - known model of pd , nerve growth factor- ( ngf- ) differentiated pc12 cells [ 47 , 48 ] . we showed that 24-epi reduces apoptotic cellular death as well as protein markers of apoptosis , modulates sod , cat , and gpx activities , and decreases intracellular ros concentrations . overall , our findings clearly demonstrate that 24-epi is a new , efficient , protective molecule against mpp - induced oxidative stress and might thus be regarded as a novel agent in complementary and/or preventive therapies of neurodegenerative diseases . all reagents were purchased from sigma ( st . louis , mo ) unless stated otherwise . pc12 cells , obtained from the american type culture collection ( rockville , md ) , were maintained in a controlled environment at 37c and in 5% co2 atmosphere . they were grown in rmpi-1640 medium supplemented with 5% fetal bovine serum ( fbs ) , 10% horse serum , and gentamicin ( 50  g / ml ) . the culture medium was changed every 2 days and the cells were seeded at a density of 30,000 cells / cm . neuronal differentiation was induced for 4 days with 50  ng / ml ngf in rpmi-1640 medium supplemented with 1% fbs . to examine the effects of 24-epi on mpp - induced cellular death and oxidative stress , neuronal pc12 cells were pretreated with 24-epi ( 10  m ) or vehicle ( culture medium ) for 3  h and then exposed to mpp 5  mm for 1 , 3 , 15 , or 24  h [ 49 , 50 ] ( figure 1 ) . in apoptosis experiments , we used 500  m of mpp for 24  h , as reported elsewhere [ 4951 ] . after kinetics and dose - response studies [ 49 , 50 ] , the final concentration of 10  m 24-epi ( see figure 2 for chemical structure ) was chosen as the lowest dose capable of rescuing cells from mpp - induced cellular death ( data not included ) . all experiments were performed in phenol red - free medium and charcoal - stripped serum to remove steroids from the medium . cytotoxicity was evaluated by colorimetric assay based on the measurement of lactate dehydrogenase ( ldh ) activity released from damaged cells into the supernatant . ldh , a stable cytoplasmic enzyme present in all cells , is rapidly released into the cell culture supernatant upon plasma membrane injury . the amount of enzyme activity detected in the culture supernatant correlates with the proportion of lysed cells [ 53 , 54 ] . ngf - differentiated pc12 cells were grown and treated in collagen - coated 96-well plates . then , 100  l of ldh substrate mixture was added to 50  l of cell - free supernatant , as described elsewhere . absorbance was measured at a wavelength of 490  nm in a microplate reader ( thermolab system , franklin , ma ) . total cellular ldh was determined by lysing the cells with 1% triton x-100 ( high control ) ; the assay medium served as a low control and was subtracted from all absorbance measurements : 
 ( 1)cytotoxicity  ( % )  = ( experimental  valuelow  control )  ( high  controllow  control)100 . the antioxidative effect of 24-epi against mpp - induced ros was evaluated by dihydrorhodamine ( dhr ) 123 assay and mitosox red ( invitrogen , toronto , on , canada ) , according to a previously - described method [ 13 , 55 ] . briefly , to detect oh , no2  , co3  , h2o2 , hocl , and onoo by dhr [ 5659 ] , ngf - differentiated pc12 cells were grown and treated on collagen - coated slides in 24-well plates . a stock solution of dhr was prepared in dimethylsulfoxide under nitrogen , to a concentration of 10  mm and stored at 80c . after 3-hour pretreatment with 24-epi , mpp was added for 3-hour or 24-hour treatment ( figure 1 ) . then , the   neuronal pc12 cells were quickly washed with pbs 0.1  m and exposed to 250  l of dhr at 37c for 20  min . slides with live cells were immediately examined under a leitz orthoplan fluorescence microscope ( leica , wetzlar , germany ) and photographed with a qimaging camera ( nikon , mississauga , on , canada ) . then , the antioxidative effect of 24-epi against mpp - induced o2   was evaluated with mitosox red , according to the manufacturer 's protocol . to show the selectivity of mitosox red , 80  m of n , n - diethyldithiocarbamate ( ddc ) , an inhibitor of sod , was used as a positive control . after 3-hour pretreatment with 24-epi , mpp was added for 3-hour or 24-hour treatment ( figure 1 ) . pc12 cells were washed with hanks ' buffered salt solution and incubated for 10  min with mitosox red 5  m solution at 37c . they were then counterstained in blue with hoechst 33342 for 10  min at 37c , fixed for 15  min in 4% paraformaldehyde at 37c , and finally mounted with prolong antifade kits ( invitrogen ) . the slides were examined under a leitz orthoplan fluorescence microscope ( leica ) and photographed with a qimaging camera ( nikon ) . ngf - differentiated pc12 cells were grown and treated in collagen - coated 6-well plates . after 3-hour pretreatment with 24-epi , mpp was added for 1 , 3 , 15 , or 24  h ( figure 1 ) . neuronal cells were harvested mechanically and collected by centrifugation at 2,000  g for 10  min at 4c . for sod and gpx activities , the pellets were homogenized in 1  ml of cold pbs and centrifuged at 2,000  g for 10  min at 4c . the supernatants were discarded and the freeze - thaw method was performed to break the cells ( 20c for 20  min , followed by a 37c bath for 10  min , repeated twice ) . the pellets were homogenized in cold pbs and centrifuged at 10,000  g for 15  min at 4c . finally , the supernatant was analyzed according to the manufacturer 's protocol ( sod assay kit - wst , dojindo molecular technologies , gaithersburg , md ; gpx assay kit , cayman chemical , ann arbor , mi ) . the reaction was monitored at 450  nm for sod activity and 340  nm for gpx activity in a microplate reader ( thermolab system ) . for cat activity , the pellets were homogenized in 1  ml of cold buffer ( 50  mm potassium phosphate , ph 7.0 , containing 1  mm edta ) and sonicated ( 3 times , 5  s ) . the samples were centrifuged at 10,000  g for 15  min at 4c , and the supernatant was assayed according to the manufacturer 's protocol ( catalase assay kit , cayman chemical ) . the reaction was monitored at 540  nm in a microplate reader ( thermolab system ) . apoptotic neuronal cells were detected by both terminal deoxynucleotidyl transferase dutp nick end labeling ( tunel , roche diagnostics , laval , qc , canada ) and activated caspase-3 immunofluorescence . neuronal pc12 cells were grown and treated on collagen - coated circular glass coverslips in 24-well plates ( fischer scientific , ottawa , on , canada ) . after 3-hour pretreatment with 24-epi , mpp ( 500  mm ) was added for 24  h ( figure 1 ) . the cells were fixed for 15  min in 4% paraformaldehyde at 37c , washed , and incubated in a blocking and permeabilizing solution ( containing 1% bsa , 0.18% fish skin gelatin , 0.1% triton - x , and 0.02% sodium azide ) for 30  min at room temperature ( rt ) . they were then incubated with anticleaved caspase-3 antibody ( new england biolabs , pickering , on , canada ) diluted 1  :  500 , for 2  h at rt , followed by 90-minute incubation with a cy3-conjugated secondary antibody ( medicorp , montreal , qc , canada ) diluted 1  :  500 for 1  h at 4c . the coverslips were then transferred to the tunel reaction mixture in a humidified atmosphere at 37c . the cells were rinsed with pbs , nuclei were counterstained in blue with dapi for 10  min at 37c , and mounted with prolong antifade kits ( invitrogen ) . images were acquired with a leitz orthoplan fluorescence microscope ( leica ) and photographed with a qimaging camera ( nikon ) . neuronal cells were considered to be apoptotic when they were positive for cleaved caspase-3 and their nuclei were stained by tunel . z - devd - fmk ( bachem , torrance , ca ) , a cell - permeable caspase-3 inhibitor , was used on specific wells of neuronal pc12 as internal control for caspase-3 activation ( figure 7 ) . the number of apoptotic neuronal cells among 300 randomly chosen neuronal cells was counted on 10 different optical fields from 3 slides per group , as already reported , with nis elements 2.2 software ( nikon ) . in addition , dna fragmentation was assessed with single - stranded dna ( ssdna ) apoptosis elisa kits ( chemicon international , temecula , ca ) according to the manufacturer 's instructions , to quantify ssdna present in apoptotic cells . this procedure is based on the ability of a monoclonal antibody to detect ssdna , which occurs in apoptotic cells but not in necrotic cells or in cells with dna breaks in the absence of apoptosis . the assay involves the binding of cells to 96-well plates and treatment of the attached cells with formamide which selectively denaturates dna in apoptotic cells . a mixture of anti - ssdna monoclonal antibody and peroxidase - conjugated secondary antibody served to specifically identify apoptotic cells . the reaction was stopped and ssdna fragmentation was quantified by measuring absorbance at 405  nm in a microplate reader ( thermolab system ) . the amount of ssdna was calculated with reference to control conditions . to confirm assay specificity , positive ( ssdna fragment ) and negative ( s1 nuclease - treated cells ) controls ngf - differentiated pc12 cells were grown and treated in collagen - coated 6-well plates . after 3-hour pretreatment with 24-epi , mpp was added for 24  h ( figure 1 ) . total cellular proteins were extracted with nuclear extraction kits ( active motif , carlsbad , ca ) , diluted in 50  l of lysis solution , and their concentrations quantified by protein assay ( bca protein assay kit ; pierce , rockford , il ) . after electrophoretic separation ( 180  v , 1  h ) , the polyacrylamide gels were transferred onto nylon pvdf membranes ( 0.22-m pore size , biorad , hercules , ca ) at 60  v for 2  h. the membranes were blocked with 5% nonfat powder milk for 1  h at rt . rabbit anti - bax antibody ( delta biolabs , gilroy , ca ) was diluted 1  :  1,000 , and rabbit anti - bcl2 antibody ( santa cruz biotechnology , santa cruz , ca ) , 1  :  50 . the membranes were washed the following day , and antirabbit horse - radish peroxidase - conjugated secondary antibody diluted 1  :  10,000 was added for 2  h at rt . immunopositive signals were visualized by enhanced chemiluminescence with the alphaease fc imaging system ( alpha innotech , san leandro , ca ) and analyzed with alphaease fc software ( alpha innotech ) . significant differences between groups were determined by 1-way anova , followed by tukey 's post hoc analysis with the graphpad instat program , version 3.06 , for windows ( san diego , ca , http://www.graphpad.com/welcome.htm ) . all data , analyzed at the 95% confidence interval , are expressed as means  s.e.m . from 3 independent experiments . asterisks indicate statistical differences between the treatment and respective control condition ( * * * p < .001 , * * p < .01 , and * p < .05 ) , full circles show statistical differences between the treatment and mpp condition ( p < .001 , p < .01 , and p < .05 ) , and diamonds denote statistical differences between the treatment and ddc condition ( p < .001 , p < .01 , and p < .05 ) . the ability of 24-epi to reverse mpp - induced cytotoxicity was investigated by ldh colorimetric assay [ 13 , 51 ] . cytotoxicity measurements revealed significant cell death in neuronal pc12 cells after exposure to mpp for 24  h ( figure 2 , mpp ) . specifically , mpp induced 22% cell death whereas 24-epi , when used alone , did not cause any cellular mortality ( figure 2 , 24-epi ) . three - hour pretreatment with 24-epi before the induction of oxidative stress significantly decreased mpp - induced cytotoxicity . specifically , 24-epi partially protected neuronal pc12 cells against mpp toxicity by decreasing cellular death by 60% ( figure 2 , 24-epi + mpp ) . figure 3 depicts the preventive effect of 24-epi against mpp - induced oxidative stress measured by dhr assay . dhr , a nonfluorescent dye , is oxidized to highly fluorescent rhodamine in the presence of several free radicals ( oh , no2  , co3  , h2o2 , hocl , and onoo ) [ 5659 ] . figure 3(a ) illustrates low levels of rhodamine fluorescence in control neuronal pc12 cells , treated only with vehicle ( figure 3(a ) , ctrl ) , as well as in cells exposed to 24-epi alone ( figure 3(a ) , 24-epi ) . in contrast , a marked signal was detected in neuronal cells treated with mpp for 24  h ( figure 3(a ) , mpp ) . pretreatment with 24-epi prior to mpp revealed a dampened signal in comparison to mpp alone ( figure 3(a ) , 24-epi + mpp ) , indicating a preventive role of 24-epi in mpp - induced ros production . semiquantitative image analysis ( figure 3(b ) ) disclosed high levels of fluorescent rhodamine only in neuronal pc12 cells treated with mpp for 24  h and a statistically significant reduction ( p < .001 ) when they were preincubated with 24-epi prior to the induction of oxidative stress ( figure 3(b ) , 24  h ) . a modest increment of fluorescence was evident after 3  h of mpp administration ( figure 3(b ) , 3  h ) , indicating that longer exposure to mpp is needed to show the presence of several free radicals , such as oh , no2  , co3  , h2o2 , hocl , and onoo . in addition , the selective detection of o2   by the fluorogenic dye mitosox red is illustrated in figure 4(a ) , in neuronal pc12 cells after 3  h of pretreatment with 24-epi or vehicle ( ctrl ) , and then 3-hour or 24-hour treatment with mpp . it should be noted that we performed complete kinetics analysis at 3 , 15 , and 24  h ( figure 1 ) . however , we detected considerable levels of fluorescence , that is , o2  , only at 3  h of treatment ( figure 4(a ) ) . fluorescence pictures revealed high fluorescence intensity in mpp- and ddc - treated cells after 3-hour treatment . low levels of oxidized mitosox red were detected in control neuronal pc12 cells ( figure 4(a ) , ctrl ) as well as in cells receiving only 24-epi ( figure 4(a ) , 24-epi ) . pretreatment with 24-epi provoked a marked reduction of the red fluorescence signal induced by mpp or ddc ( figure 4(a ) , 24-epi + mpp and 24-epi + ddc ) . semiquantitative image analysis disclosed high levels of fluorescent mitosox red in neuronal pc12 cells treated with mpp or ddc and a considerable reduction ( p < .001 ) of fluorescence intensity when these cells were pretreated with 24-epi prior to mpp or ddc ( figure 4(b ) ) for 3  h. marginal or slightly detectable levels of fluorescence were apparent at 15  h ( data not included ) and at 24  h of treatment ( figure 4(b ) , 24  h ) . sod catalyzes the dismutation of superoxide anion radical by converting it to peroxide , which can be destroyed by cat or gpx [ 60 , 61 ] . we , therefore , investigated the effect of mpp , 24-epi , and mpp + 24-epi on sod , cat , and gpx activities in neuronal pc12 cells . figures 5(a)5(c ) , illustrating sod , cat , and gpx activities , respectively , reveal that the exposure of neuronal pc12 cells to mpp for 3  h significantly increased the levels of these 3 antioxidant enzymes , corroborating a cell stress response to mpp - induced ros production . at 15  h , mpp administration decreased sod and gpx activities ( figures 5(a ) and 5(c ) , mpp ) , with cat activity remaining stable relative to 3  h activity ( figure 5(b ) , mpp ) whereas at 24  h , sod activity was strongly reduced , cat activity remained stable , and gpx was increased . our results show that the administration of 24-epi alone also induced a very significant rise of sod , cat , and gpx activities at 3  h , supporting an antioxidant role for this br ( figures 5(a)5(c ) , 24-epi ) . specifically , 24-epi elicited a significant increment of sod , cat , and gpx activities , with a maximal increase apparent after 3  h of treatment . afterward , at 15  h and later , sod and cat activities declined when exposed to 24-epi ( figures 5(a ) and 5(c ) , 24-epi ) . on the other hand , figure 5(c ) illustrates that gpx activity declined at 15  h and then peaked after incubation with 24-epi for 24  h. we also analyzed whether 24-epi administration prior to mpp could modulate the activities of these antioxidant enzymes . we found that exposure to 24-epi before the induction of mpp oxidative damage significantly increased sod , cat , and gpx at 3  h , to levels higher than those obtained with mpp alone but lower than those detected with 24-epi alone . at 15  h , our results demonstrate that 24-epi + mpp still elevated sod activity over control values ( figure 5(a ) , 24-epi + mpp ) while cat activity was similar to that observed with 24-epi alone ( figure 5(b ) , 24-epi + mpp ) , and gpx activity declined significantly . finally , at 24  h , sod and cat activities decreased markedly , while gpx activity increased to control levels ( figures 5(a)5(c ) , 24-epi + mpp ) . to determine whether 24-epi protects neuronal pc12 cells from mpp - induced apoptosis , we undertook dna fragmentation measurement ( figure 6 ) , tunel assay , and immunofluorescence investigation with an antibody to activated caspase-3 ( figures 7(a ) and 7(b ) ) . dna fragmentation is a marker of late apoptosis , and exposure to 500  m mpp for 24  h resulted in its 60% increase in neuronal pc12 cells ( figure 6 ) . pretreatment with 24-epi significantly ( p < .01 ) prevented the mpp - induced increment of dna fragmentation , indicating a powerful role of 24-epi in reducing apoptosis in our cell paradigm . next , caspases are central initiators and executioners of the complex biochemical events associated with apoptotic cell death [ 62 , 63 ] . as caspase-3 activation has been shown to be one of the concluding effectors of the apoptosis process , we investigated whether 24-epi has the ability to prevent mpp - induced caspase-3 activation ( figure 7 ) . immunofluorescence ( figure 7(b ) ) clearly illustrated the presence of simultaneous tunel- and caspase-3-positive cells ( appearing in light blue and indicated by arrows ) when mpp was administered alone . furthermore , pretreatment of neuronal pc12 cells with a cell - permeable caspase-3 inhibitor ( z - devd - fmk ) for 1  h prior to mpp significantly decreased mpp - induced apoptosis , demonstrating that caspase-3 activation is a key factor in mpp - induced apoptosis . as already depicted in figure 6 , 3-hour preincubation with 24-epi prior to mpp revealed a considerable reduction ( p < .001 ) in the number of apoptotic neuronal pc12 cells ( figure 7(b ) ) . these results strongly suggest an antiapoptotic effect of 24-epi and indicate that mpp - induced apoptosis is associated with caspase-3 activation . we also studied the modulation of protein expression of the proapoptotic gene bax and the antiapoptotic gene bcl-2 by 24-epi . the ratio of proapoptotic bax to antiapoptotic bcl-2 ( bax / bcl-2 ) has been reported to be correlated with apoptosis [ 51 , 65 ] . our results reveal that the administration of 24-epi alone did not significantly modulate the bax / bcl-2 ratio ( figure 8 , triangles on a continuous line ) . treatment with mpp alone significantly increased the bax / bcl-2 ratio , indicating that mpp - induced apoptosis of pc12 cells may be mediated by the mitochondrial pathway . the mpp - induced increase of the bax / bcl-2 ratio was considerably attenuated in cells pretreated with 24-epi ( figure 8 , 24-epi + mpp ) to control levels , suggesting , for the first time , that the br 24-epi is a strong modulator of proapoptotic and antiapoptotic gene expression . in this paper , we demonstrated , for the first time , that 24-epi , a br found in a variety of vegetables as well as in vicia faba , can exert antioxidative and consequent antiapoptotic actions in mammalian neural cells . in particular , we studied pc12 cells , a known , reliable , and efficient model for the investigation of oxidative stress and neuroprotection of da neurons [ 49 , 66 ] . after ngf administration , pc12 cells differentiate into a neuronal - like phenotype that secretes high da levels and expresses th , dat , neurofilaments as well as estrogen receptor - alpha and -beta ( er and er ) [ 49 , 6668 ] . recent studies have reported the powerful properties of various natural polyphenols against oxidative stress in several cellular and in vivo paradigms of neurodegenerative disease [ 3034 ] . currently , many natural polyphenols are under intense investigation for their antioxidative effects and their possible use as complementary and/or preventive therapies of diseases [ 33 , 34 ] . our aim was to demonstrate that brs , contained in a wide variety of vegetables , indeed exert antioxidative as well as neuroprotective properties in neuronal pc12 cells , a cellular model of pd [ 47 , 48 ] .   at present , phytosterols are recognized antioxidants , and some of them possess antioxidative properties associated with neuroprotective effects [ 70 , 71 ] . others , such as -sitosterol , modulate sod , gpx , and cat activities , and the ginsenoside rg1 , a   phytosterol derived from ginseng , is also reported to be antiapoptotic in neuronal pc12 cells after oxidative stress [ 73 , 74 ] . however , brs , in particular , are much less studied , even if the recent literature is pointing to their interesting potential in mammalian systems , such as antiviral , anticancer , and antiproliferative activities [ 3537 ] . at present , no data on a possible antioxidative and antiapoptotic role of brs are available in mammalian neurons in vitro or in vivo . as such , in this study , we examined , for the first time , the neuroprotective , antioxidant , and antiapoptotic consequences of low - dose 24-epi ( 10  m ) , a common br , against oxidative damage induced by treatments with mpp , the active metabolite of mptp , a known parkinsonian toxin . the positive outcomes we reported in daergic neuronal culture , using nanomolar doses of 24-epi , on parameters of neuroprotection , oxidative metabolism , and apoptosis , are supported by the fact that brs may be considered the plant equivalent of steroid hormones in vertebrates , sharing similar metabolic pathways . thus , brs could easily pass through the blood - brain barrier and are likely to accumulate in the brain and serum , as demonstrated for plant sterol and stanol esters in watanabe rabbits . in particular , we established that 24-epi can protect da neuronal cells from mpp - induced cellular death by reducing intracellular ros production . indeed , nonfluorescent dhr has the capacity to enter cells and , once inside them , it is oxidized by oxygen species ( superoxide anion , peroxynitrite ) to fluorescent rhodamine . accordingly , our results show increased rhodamine fluorescence after mpp treatment and reduced fluorescence when 24-epi is administered to neuronal pc12 cells prior to mpp . dhr has been deployed extensively to measure intracellular ros , but it does not quantify o2   production . mitosox red is a selective indicator of mitochondrial o2   production and becomes highly fluorescent when oxidized by this ros but not by other oxidants . with mitosox red , we illustrated an increase of fluorescence when mpp was administered alone for 3  h , and a substantial reduction with 24-epi treatment given prior to mpp for 3  h ,   suggesting a potent scavenging role for 24-epi . as o2   is a highly reactive ros , we could barely detect its presence by mitosox red at 15 or 24  h of treatment . however , at the cellular level , since antioxidant enzymes are the primary defense mechanisms of protection against ros damage , sod , cat , and gpx are pivotal in preventing cellular injury and apoptosis . in our study , augmented sod activity demonstrated that 24-epi may enhance the ability to eliminate ros during various oxidative stresses and may indicate a protective role in pretreatment experiments . besides , several other natural and synthetic molecules are reported to heighten sod activity in various cellular systems [ 13 , 7779 ] . mpp augmented sod activity in our experiments , as described in recent literature in vitro   and in vivo , where mptp increased sod activity by generating superoxide ions . this apparent contrasting result should be analyzed by comparing it with those obtained by fluorescent rhodamine and mitosox red . indeed , low ros levels , illustrated by low rhodamine and low mitosox red fluorescence ( 24-epi and 24-epi + mpp , figures 3 and 4 ) , sustain the ability of 24-epi to induce sod activity , as demonstrated by our data . when mpp was administered , rhodamine and mitosox red fluorescence indicated high ros levels and , consequently , the cells responded by augmenting sod activity , as already reported [ 13 , 14 ] . our findings clearly show that sod activity may be induced by 2 different mechanisms , a protective mechanism ( 24-epi ) and a response - to - stress mechanism ( mpp ) . more importantly , pretreatment with 24-epi prior to mpp administration indicates low ros levels , as revealed by rhodamine and mitosox red fluorescence , suggesting that the relatively low sod activity induced by 24-epi pretreatment may have already scavenged mpp - generated ros before 24  h.   cat activity is another parameter of oxidative stress . our results point out that at 15  h and 24  h , 24-epi pretreatment reduces the mpp - induced increase in cat activity , confirming a scavenging role for 24-epi in the pretreatment experimental condition , as already reported for another natural antioxidant molecule , sesamin . gpx is a selenium - dependent enzyme involved in antioxidant defense and intracellular redox regulation and modulation . cardiovascular and neuroprotective effects of the trace element selenium have been observed , although long - term supplementation has a  ying - yang  effect . the glutathione response after mpp treatments has already been described in a daergic cell line and is in accordance with our results , demonstrating an increase in gpx activity at 24  h after toxin administration . our data and other findings suggest a change in glutathione regulatory enzyme activities during the kinetics of mpp administration . more interestingly , our data show a significant increase in gpx activity after 24-epi pretreatment , indicating that this molecule may augment stock of the antioxidant enzyme above control levels . in addition , our results demonstrate a clear neuroprotective and antiapoptotic role of 24-epi against cellular death induced by mpp administration . we also document that 24-epi is a potent modulator of apoptosis , opposing mpp - induced dna fragmentation and decreasing mpp - evoked apoptotic / antiapoptotic protein expression , namely , the bax / bcl-2 ratio . altogether , our data establish that oxidative stress - induced apoptosis in daergic cells can be reversed by preadministration of 24-epi . thus , 24-epi may be accounted for by another natural molecule interacting with intracellular apoptotic pathways [ 51 , 82 ] . recent studies have reported the anticancer and antiproliferative activities of 2 brs , 28-homocastasterone and 24-epi , supporting their cytotoxic and apoptotic role . this is not the first time that natural neuroprotective and antioxidant molecules appear to act as double agents  on apoptotic parameters , depending on the cell lines studied and the concentrations tested . first , it should be noted that to demonstrate the anticancer and antiproliferative activities of brs , these authors used micromolar concentrations , and toxicity was apparent at 10  m and higher dose levels . in our study , we tested nanomolar concentrations of 24-epi since micromolar levels would likely be difficult to sustain in vivo in the human brain . on the other hand , neuronal pc12 cells are differentiated cells expressing a neuronal phenotype as well as dat , neurofilament proteins , and er and er , in contrast to native mitotic pc12 cells , where 17- estradiol or several polyphenols do not counteract mpp - induced cellular death [ 50 , 55 ] . it is certainly important in future work to study in vivo models of pd to better understand the role of plant steroids in mammalian neuronal systems . finally , this is the first investigation to highlight 24-epi 's powerful function in parameters of neuronal cell distress , apoptosis , and cellular death . other studies should be performed to elucidate the possible modulation of 24-epi on the intrinsic parameters of daergic neurotransmission . in addition , we can not completely exclude a role of 24-epi on mpp uptake , in particular via the modulation of dat and vmat expression , that could mimic downstream events . though , altogether these results can open the way to further document , in an animal model of pd , the importance of brs as natural molecules in preventive or / and complementary strategies to control neurodegeneration .
oxidative stress and apoptosis are frequently cited to explain neuronal cell damage in various neurodegenerative disorders , such as parkinson ' s disease 
 . brassinosteroids ( brs ) are phytosterols recognized to promote stress tolerance of vegetables via modulation of the antioxidative enzyme cascade . 
 however , their antioxidative effects on mammalian neuronal cells have never been examined so far . 
 we analyzed the ability of 24-epibrassinolide ( 24-epi ) , a natural br , to protect neuronal pc12 cells from 1-methyl-4-phenylpyridinium- ( mpp+- ) induced oxidative stress and consequent apoptosis in dopaminergic neurons . 
 our results demonstrate that 24-epi reduces the levels of intracellular reactive oxygen species and modulates superoxide dismutase , catalase , and glutathione peroxidase activities . 
 finally , we determined that the antioxidative properties of 24-epi lead to the inhibition of mpp+-induced apoptosis by reducing dna fragmentation as well as the bax / bcl-2 protein ratio and cleaved caspase-3 . 
 this is the first time that the potent antioxidant and neuroprotective role of 24-epi has been shown in a mammalian neuronal cell line .