| OBJECTIVE:To investigate the effects of pharmacological preconditioning with different plasma concentrations of salvia miltiorrhiza beg.f.alba on cell viability and expression of apoptosis modulating proteins Bcl-2 and Bax of Wistar neonatal rats hippocampal neurons cultured in vitro following ischemic/hypoxic injury.To explore the protective effects of pharmacological preconditioning with salvia miltiorrhiza beg.f.alba on the neurons and the optimal dosage.METHOD:Established apoptotic model of Wistar neonatal rats hippocampal neurons cultured in vitro following ischemic/hypoxic injury by the OGD.Before the apoptotic models,the cultured Wistar neonatal rats hippocampal neurons were randomly devided into four groups:control group(C,n=6),low dose group(L,n=6), middle dose group(M,n=6),and high dose group(H,n=6).24 hours after culturing following OGD, the cell viability was detected by MTT,expression of apoptosis modulating proteins Bcl-2 and Bax by immunofluorescence staining with immu- nofluorescent microscope and apoptosis by Annexin V-FITC/PI double staining.RESULTS:①detection results of cell viability by MTT: Compared with the control group,measured OD values of high dose of salvia miltiorrhiza beg.f.alba group,middle dose of salvia miltiorrhiza beg.f.alba group and low dose of salvia miltiorrhiza beg.f.alba group were 0.703±0.135(p <0.05),0.580±0.118(p <0.05), 0.417±0.130(p>0.05)respectively;survival rates were 195.28(p <0.05),161.11 (p <0.05),115.83(p>0.05).The differences between the control group and the prior two groups were significant(p <0.05).②Expression of apoptosis modulating proteins Bcl-2 and Bax by immuno- fluorescence staining with immunofluorescent microscope:Bcl-2 positive cells were mainly stained with green fluorescence in cytoplasm,nuclear membrane and apophysis. Compared with the control group, the expression of Bcl-2,the strength of green fluorescence and the optical density values were higher in high dose of salvia miltiorrhiza beg.f.alba group,middle dose of salvia miltiorrhiza beg.f.alba group and low dose of alvia miltiorrhiza beg.f.alba group, the differences were significant(P<0.05).On the other hand, the Bax positive cells were mainly stained with green fluorescence in cytoplasm,nuclear membrane and apophysis. Compared with the control group, the expression of Bax,the strength of green fluorescence and the optical density values were lower in high dose of salvia miltiorrhiza beg.f.alba group,middle dose of salvia miltiorrhiza beg.f.alba group and low dose of salvia miltiorrhiza beg.f.alba group, the differences were significant(P<0.05).③The results of Annexin V-FITC/PI double staining by immunofluorescent microscope:the cell membrane expressed green fluorescence at the beginning of apoptosis;the cell nucleus expressed red fluorescence and the cell membrane expressed green fluorescence in middle and late stages of apoptosis cells;Only the cell nucleus expressed red fluorescence and the cell membrane disaggregated at the end of apoptosis as well as dead neurons.A large number of middle and late stages of apoptosis cells and dead neurons were observed in the control group.A lot of early apoptotic cells with little middle and late stages of apop- tosis cells and dead neurons were observed in high dose of salvia miltiorrhiza beg.f.alba group,middle dose of salvia miltiorrhiza beg.f.alba group and low dose of salvia miltiorrhiza beg.f.alba group.Conclusion:1.Preconditioning by different doses of salvia miltiorrhiza beg.f.alba could protect the Wistar neonatal rats hippocampal neurons cultured in vitro and the protective effects were significant in the high dose of salvia miltiorrhiza beg.f.alba group and the middle dose of salvia miltiorrhiza beg.f.alba group.2.The mechanism of neuroprotective effect of preconditioning by different doses of salvia miltiorrhiza beg.f.alba on the Wistar neonatal rats hippocampal neurons cultured in vitro may be involved with the increase of Bcl-2 expression and the decrease of Bax expression. |
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