The Protective Effects Of Hydroxysafflor Yellow A On Glutamate-induced Oxidative Neurotoxicity

Backgroud:Glutamic acid,now known to be a neurotransmitter of central nervous system(CNS),it plays an important role in CNS.However,Glutamic acid will be a potent neurotoxin when it presents in excess,thereby leading to the damage of neurons and many neurological degenerative diseases.Hydroxysafflor yellow A(HSYA)is a monomer of Cart hamus tinctorius L,which is soluble in water.It has benn reported that HSYA is a central component in counteracting oxidative toxicity. Previous stuies have centralized in its neuroprotective effects on cerebral ischemia injuries in animals;but,as an antioxidant,little information has been reported on cell line,With respect to its effect and mechanism on glutamate-induced oxidative neurotoxicity,no reports has been appeared in literatures.Objective:To evaluate the neuroprotective effect and its signal pathway of HSYA on glutamate-induced oxidative neurotoxicity,so as to enrich the theory system applying in anti-neurotoxicity study as well as in clinical therapy research.Methods:In this study,primary cortical cultures,cortices from embryonic day 14～15 Wistar rat fetuses,were cultured in vitro,and 24 h later the experimental agents were administered.1.MAP2 fluorescent antibody was used to analyse the purity of neurons;2.To obtain morphology of cells when observing them with inverted phase contrast microscope at the right moment;3.The morphology of cells were measured by acridine orange staining;4.Cell viability was assayed by MTT assay,12 h after the addition of glutamate;5.The way of cell death was measured by Ho33342 and PI double staining;6.Intracellular GSH and SOD were measured via the biochemical assays;7.Apoptosis of different groups was examined by flow cytometry(FCM);8.The changes of Ca²⁺in different groups,staining with Flou-3/AM,were detected. using laser scanning confocal microscopy;9.Bcl-2/Bax,caspase-3 and caspase-9,which were related to apoptosis were detected by Westem-blot;Results:1.MAP2 immunofluorescence staining observation:results showed that 90%cells were positive to MAP2;2.Morphological observation:the embryotic cortical neurons,when cultured 48 hours later in vitro,most were spread around adhered to the dishes with a triangular or polygonal plump body.After exposure to glutamate(2mmol/L)for 12 h,almost all of the synapses between neurons were destroyed with broken-up fragments scattered all around.Cells pre-treated with HSYA(160μg/ml)remained favorable growth,distinctly contrasted to the condition of glutamate-intoxicated cells. 3.Acridine orange:neurons in normal group were intact,karyons presented Kelly,while cytoplasm was jacinth;Neurons nucleus in Glu were smaller and broken into fragments,and there were nearly no cytoplasm surrounding it.Most neurons in HSYA protected group were similar to neurons in control group.4.MTT assay:cell viability in glutamate group reduced markedly(36.31%),HSYA can enhance cell viability evidently,and in concentration 0.4～160μg/ml,along with higher concentration,its protective effect showed better and better,160μg/ml is the best protective concentration,its cell ability was 89.49%;5.After Ho33342 and PI double staining,neurons karyons in control group were round or oval,dyed into blue.The cells,disposaled with Glu for 12 h,had two cellular death pathways:necrosis and apoptosis.30%cell membrane was broken and cells were dyed into red.cells dyed into blue showed pycnosis or agglutination clearly,and their nucleus crimpled apparently and apoptotic bodies appeared.Most neurons in HSYA protected group were dyed into blue as cells in control group. Apoptosis and necrosis were seldom seen;6.Detection of GSH content and SOD activity:HSYA(160μg/ml)can enhance GSH content and SOD activity evidently(GSH:normal group 116.3394mg/L,Glu damaged group 47.6664 mg/L,HSYA group 94.3452mg/L;SOD:normal group 46.9966 U/mgprot,Glu damaged group 20.697 U/mgprot,HSYA group 39.4981U/mgprot);7.Flow cytometry showed apoptosis of defferent groups:Glu damaged group was 20.56%,HSYA protective group was 7.94%,while normal neurons can not be detected apoptosis evidently,with only 0.43%apoptosis;8.According to the characteristic of Flou-3/AM,using laser scanning confocal microscopy, Ca²⁺was found increased markedly,HSYA(160μg/ml)can counteract with Glu, depress the release of Ca²⁺(fluorescence intensity:normal group 10.147,Glu damaged group 64.059,HSYA protective group 23.344);9.Bcl-2/Bax,caspase-3 and caspase-9,which were related to apoptosis,were detected by Western-blot.Results showed that,compaired with normal neurons(151.18),in Glu damaged neurons,Bcl-2,was weakened evidently(7.8),HSYA(160μg/ml)can counteract with Glu,keeped the express of Bcl-2(119.38);and in normal group,Bax was 78.05,in Glu damaged group was enhenced evidently(122.88),while HSYA can keep low level of Bax(102.19);about active caspase-3,in normal group,it was 50.37,which was quite low to Glu damaged neurons(132.4),and at the same time, HSYA(160μg/ml)neurons was 91.96,showing that HSYA can contact with Glu by lowing active caspase-3 content;caspase-9 had the same changes with caspase-3,its relative level:normal was 34.28,Glu damaged neurons was 187.07,HSYA (160μg/ml)neurons was 80.34.This proved the same question with caspase-3.Conclusion:1.HSYA protects cells from glutamate-induced oxidative stress,the effect is related to concentration of HSYA nearly,and the best concentration is 160μg/ml,its cell ability is 89.49%;2.Glu results in the depletion of intracellular GSH and SOD,by enhancing of GSH and SOD,HSYA plays its protective effects; 3.HSYA can conteract with Glu in apoptosis;4.By inhibition of Ca²⁺and proteins related to apoptosis,such as Bcl-2,Bax, caspase-3 and caspase-9,HSYA conteracts with Glu;5.This study reported the protective effect of HSYA on glutamate oxidative toxicity for the first time,and studied its signal pathway,this contributes to the study of antioxidants to antagonize glutamate-induced neuronal degenerative disorders,and offers theory conducting to the therapy of neurological degenerative diseases.