although neurological insults are diverse in nature , there are common mechanisms of cell injury , and glutamate toxicity plays an integral role in a variety of neurobiology of disorders such as parkinson 's disease , alzheimer 's disease , epilepsy ischemic stroke , anxiety , and depression [ 1 , 2 ] . glutamate is the major fast excitatory neurotransmitter in the mammalian central nerve system . thus far besides , it has been proposed that the combination of antidepressant drugs that elevate the noradrenergic neurotransmission with drugs that modify the glutamatergic system could be an option in treating depression . 3,6-disinapoyl sucrose ( diss , figure 1(a ) ) is the active oligosaccharide ester component found in the root of polygala tenuifolia willd ( radix polygala ) . recorded as  yuanzhi  in the pharmacopoeia of the people 's republic of china , the root has been used in traditional medicine as , among other things , an expectorant , tonic , tranquillizer , and antipsychotic agent . results from previous studies indicated that diss had notable antidepressant effects in pharmacological depression models , an action closely related to the potentiation of central 5-hydroxytryptamine ( 5-ht ) and norepinephrine ( ne ) systems . our recent study also found that diss increased expression in the hippocampus of three noradrenergic - regulated plasticity genes ( laminin , cam - l1 , and creb ) and one neurotrophic factor ( bdnf ) . thus , the authors used the human neuroblastoma sh - sy5y cell line , with a low degree of differentiation and metabotropic glutamate receptors , utilized glutamate as an insult to induce sh - sy5y neuronal cell injury , and aimed to investigate the neuroprotective effects of diss on glutamate - induced apoptosis as well as its mechanisms . diss , used in present study , with a purity of over 90% , was extracted from the roots of p. tenuifolia , which were purchased from traditional chinese medicinal ( tcm ) pharmacy , chinese people 's liberation army ( pla ) general hospital ( beijing , china ) ; a voucher specimen ( nu-80617 ) was deposited in the herbarium there . three - month air - dried roots ( 965.27  g ) were extracted with 60% etoh ( 8  :  1 ) at room temperature for 2 weeks . the dry extract obtained was then subjected to open column chromatography ( cc ) packed with macroporous resin ( 1300 version ) . the 50% aqueous - ethanol fraction was concentrated under reduced pressure using a rotary evaporator and lyophilized into powders , further chromatographed on the silica gel column , and eluted by chcl3meoh the structures were identified by a combination of spectral methods ( uv , ir , ms , and nmr ) , with purity of over 90% . the human neuroblastoma sh - sy5y was provided by department of obstetrics and gynecology in chinese pla general hospital . the human neuroblastoma sh - sy5y cells were maintained in dulbecco 's modified eagle medium ( dmem ) supplemented with 10% fetal calf serum , 100  u / ml penicillin , and 100  u / ml streptomycin in a humid atmosphere of 5% co2 and 95% air at 37c . confluent sh - sy5y cells were washed twice with d - hanks solution before the addition of 0.25% trypsin - edta . the flask was left for 2 - 3  min at room temperature(close to 20c ) , after which the cells were detached , resuspended in full medium , counted , and seeded into 96-well plates at a density of 1  10  cells / well in normal growth medium . . then cells were exposed to l - glutamate ( glu ) for 0.5  h , followed by treatment with various concentrations of disss ( 0.6 , 6 , and 60  mol / l ) and desipramine as a positive drug . diss and glutamate were dissolved in dimethyl sulfoxide ( dmso ) before added in cell . final concentrations of dmso were always less than 0.01% , which was proved to have no effects on cell viability . briefly , after 0.5  h exposure to glu , 20  l of mtt ( 2  mg / ml in pbs ) were added to each well and the cells were incubated at 37c for 4  h. the supernatants were aspirated carefully , 150  l of dmso were added to each well to dissolve the precipitate , and absorbance was measured at 570  nm using a microplate reader ( spectra mr , dynex , usa ) . cell death was determined by measuring the lactate dehydrogenase ( ldh ) activity using commercially available kits from the nanjing jiancheng bio - company ( nanjing , china ) . after treatment , 5  10  cells were trypsinized , washed in pbs , and centrifuged at 1000  g for 5  min . the pellet was rinsed twice and resuspended in 0.5  ml pbs ( containing 50  g / ml rnasea ) , incubated for 30  min at 37c . pi ( 50  g / ml ) was added , mixed gently , and incubated for 30  min at 4c in dark . the samples were then read in a becton dickinson flow cytometer ( usa ) at 488  nm excitation . ten thousand cells in each sample were analyzed , and the percentage of apoptotic cells accumulating in the sub - g1 peak was calculated by cellquest software . total rna was extracted from cells cultured in the 25  cm plastic flasks with 5  10  cells using trizol reagent ( gibco brl ) as described by the manufacturer . and its reverse transcribtion to cdna was performed using iscript cdna synthesis kit ( bio - rad , calif , usa ) , according to the manufacturer 's protocol . human -actin , bcl-2 , and bax primers were synthesized by bm ( biomed , china ) according to the following sequences . -actin : forward 5- ggacatccgcaaagacctgta-3 , reverse 5- acatctgctggaaggtggaca-3 ; bcl-2 : forward 5- tttgagttcggtggggtcatc-3 , reverse 5-ccaggaga - aatcaaacagagg -3 ; bax : forward 5-tttgcttcagggtttcatcc -  3 , reverse 5-gccactcggaaaaagacctc-3. ssofast evagreen supermix ( bio - rad , calif , usa ) was used for real - time pcr to detect abundance of pcr products among samples . standard curves were generated for each gene , and transcript values were calculated relative to dilution series of cdna as described in bio - rad iq5 system ( calif , usa ) . target quantities were normalized to 18s ribosomal rna , calibrated using control values , and defined as a value of  1.0  . all quantities were expressed as n - fold relative to the calibrator ( control ) . statistical comparisons were made by one - way anova followed by tukey 's post hoc test . as shown in figures 1(b ) and 1(c ) , 8  mm of glu was used to induce sh - sy5y cell injury , whose group was only 67.28  1.2% viable cells and increased the ldh level as compared to control cells , while treating the cells with diss at different concentrations ( 0.6 , 6 , and 60  mol / l ) increased the viability of cells and inhibited ldh release , both with dose - dependent . moreover the effects of diss could also be confirmed by the morphological observation ( figure 1(d ) ) . there was a significant injury in sh - sy5y cells after treatment with glu , including the disappearance of cellular processes , decrease of the refraction , and falling to pieces . the nuclear staining assay was used to evaluate the morphological changes of apoptosis in sh - sy5y cells . as shown in figure 2 , control cells without the treatment with glu exhibited uniformly dispersed chromatin and intact cell membrane . the cells , treated with 8  mm glu , increased the percentage of apoptotic cells from 0.96% to 4.93% , compared to control cells . however , in 0.6 , 6 , and 60  mol / l diss - treated cells , cell apoptosis induced by glu was markedly decreased to 2.58% , 2.36% , and 1.95% , respectively . the effects of diss on the expression of the apoptotic genes bax and bcl-2 were also examined in glu - injured sh - sy5y cells . as shown in table 1 , glu enhanced 2-fold expression of bax increased , on the contrary however , diss ( 60  mol / l ) treatment inhibited the increase of bax and the decrease of bcl-2 dramatically at 12  h of glutamate exposure . glutamate , a major excitatory amino acid neurotransmitter in central nervous system , mediates several physiological processes . an increasing body of evidence indicates the important role of the glutamatergic system in the pathophysiology of depression . firstly , depressed patients exhibit elevated levels of glutamate both in plasma and the limbic brain areas , which are believed to be involved in mood disorders . additionally , it has been shown that chronic treatment with antidepressants of different mechanisms reduces the glutamate release in rats [ 12 , 13 ] . diss is the active oligosaccharide ester component and has been proven to have antidepressant [ 5 , 6 ] , cerebral protective , and cognition - improving effects . in the present study , we demonstrated that cotreatment with diss protected sh - sy5y neuronal cells against glutamate insult , with maximal neuroprotection being observed at 60  mol / l . similarly , flow cytometry detects apoptotic cells with fragmented nuclei , which are also called sub - g1 cells . we also investigated whether diss has any effect on the transcriptional level of bax and bcl-2 in glu - treated cells . proapoptotic gene bax , antiapoptotic gene bcl-2 mrna expression levels were measured by real - time pcr . it was reported that bax and bcl-2 , the two main members of bcl-2 family , play a key role in the mitochondrial pathway of apoptosis . bax has been implicated in promoting cell apoptosis , whereas bcl-2 in inhibiting apoptosis [ 15 , 16 ] . our results indicate that diss provides neuroprotection partly by the dramatic inhibition of bax overexpression induced by glutamate and increasing antiapoptotic bcl-2 gene expression . in summary the protective effects of diss were related to modulating apoptosis - related gene expression , by downregulating the synthesis of proapoptotic bax and upregulating antiapoptotic bcl-2 expression . further studies on the neuroprotection effects of diss in primary neuronal cell injury model , mediated via mapk - creb - bdnf / trkb signaling pathway , are currently under way to evaluate the detail neuroprotective mechanism of therapeutic efforts of diss .
the neuroprotective effects of 3,6-disinapoyl sucrose ( diss ) from radix polygala against glutamate - induced sh - sy5y neuronal cells injury were evaluated in the present study . 
 sh - sy5y neuronal cells were pretreated with glutamate ( 8  mm ) for 30  min followed by cotreatment with diss for 12  h. cell viability was determined by ( 3,4,5-dimethylthiazol-2-yl)-2,5-diphenylte - trazolium bromide ( mtt ) assay , and apoptosis was confirmed by cell morphology and flow cytometry assay , evaluated with propidium iodide dye . 
 treatment with diss ( 0.6 , 6 , and 60  mol / l ) increased cell viability dose dependently , inhibited ldh release , and attenuated apoptosis . 
 the mechanisms by which diss protected neuron cells from glutamate - induced excitotoxicity included the downregulation of proapoptotic gene bax and the upregulation of antiapoptotic gene bcl-2 . 
 the present findings indicated that diss exerts neuroprotective effects against glutamate toxicity , which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases .