Neuroprotective Effects Of Salidroside In The PC12 Cell Model Exposed To Hypoglycemia And Serum Limitation

Objective To observe the protective effects of Salidroside on hypoglycemia and serum limitation-induced cell death in PC12 cellsMethods1. PC12 cells were cultured under hypoglycemia and serum limitation environment to establish a hypoglycemia and serum limitation model in PC12 cell line. MTT assay was used to detect the celluar viability after incubation different periods(12h, 24h, 36h, 48h).2. Cultured PC12 cells were pre-incubated with different concentrations (80μg/ml, 160μg/ml, 320μg/ml) of Salidroside for 24h, after which hypoglycemia and serum limitation was applied to the cells for 24h, phase contrast microscope observation and MTT assay were used to identify the effect of Salidroside against hypoglycemia and serum limitation induced injury.3. Hoechst 33342 staining and FCM were applied to detect the effect of Salidroside against hypoglycemia and serum limitation induced apoptosis.4. RT-PCR and Western blot were performed to examine the effect of Salidroside on the mRNA and protein expression levels of Bcl-2 and Bax.5. RT-PCR and Caspase3 activity assay were performed to examine the effect of Salidroside on the mRNA and protein activity levels of Caspase3.6. Cultured PC12 cells labeled with lipophilic cation fluorescent dye Rhodamine 123 were collected and quantified by confocal laserscanning microscope to determine the effect of Salidroside on the mitochondrial transmembrane potential after 24h stimulation with hypoglycemia and serum limitation.7. Cultured PC12 cells labeled with molecular probes DCFH-DA were collected and quantified by FCM to determine the fluorescence intensity of DCF and observe the effect of Salidroside on the ROS levels.Results1. The results of MTT indicated that hypoglycemia and serum limitation could evoked significant cytotoxicity to the cultured PC12 cells, with the incubation time increased, the cell viability decreased.2. Morphalogical observation and MTT assay showed that Salidroside could protect cultured PC12 cells against hypoglycemia and serum limitation-induced cell viability decrease in a dose-effect manner ,with the most obvious changes at the concentration of 320μg/ml(P<0.05).3. Hoechst 33342 staining and FCM showed that Salidroside could attenuate PC12 cells'apoptosis induced by hypoglycemia and serum limitation(P<0.05).4. The results of RT-PCR and Western blot indicated that Salidroside could antagonize the down-regulation of Bcl-2/Bax induced by hypoglycemia and serum limitation, and the effect showed a dose-effect manner, with the best concentration of 320μg/ml(p<0.05).5. The results of RT-PCR and Caspase3 activity detection indicated that Salidroside could antagonize the up-regulation of Caspase3 mRNA and protein activity induced by hypoglycemia and serum limitation, and the effect showed a dose-effect manner, with the best concentration of 320μg/ml(p<0.05) .6. After exposure to hypoglycemia and serum limitation for 24h, the mitochondrial membrane potential of PC12 cells significantly decreased, Salidroside could protect cells against the hypoglycemia and serum limitation-induced lowering of mitochondrial membrane potential(p<0.05).7. After exposure to hypoglycemia and serum limitation for 6h, the production of ROS significantly increased, Pretreatment with Salidroside could protect cells against the hypoglycemia and serum limitation-induced elevation of ROS production(p<0.05).Conclusions1. Salidroside may protect cultured PC12 cells against hypoglycemia and serum limitation-induced injury in a dose-effect character.2. The protection effect of Salidroside against hypoglycemia and serum limitation-induced injury is probably related to the modulation of apoptosis-related gene expression, the restoration of mitochondrial membrane potential and the inhibition of the intracellular ROS production.