Study On The Protective Effect And Mechanism Of Salidroside On Neuronal Damage

During ischemic brain damage, excitatory amino acids, glutamate (Glu)-induced excitotoxicity in neurons play a rather important role. Thereby protecting neurons from the Glu-induced excitotoxicity in ischemic brain damage has become one of the important treatment strategies. This study aimed to evaluate the neuroprotective effects of salidroside on excitotoxicity and investigate the possible mechanism involved in the neuroprotection.Part I Protective effects and mechanism of salidroside on primary hippocampal neurons damage induced by GluObjective: To investigate the protective effects and underlying mechanism of salidroside on primary rat hippocampal neurons damage induced by Glu.Methods:1. Phase contrast microscope and MTT assay were used to observe the effects of salidroside on morphology and viability of primary cultured hippocampal neurons.2. To build an excitatory neurotoxic model of hippocampal neurons, MTT colorimetric assay and LDH (in medium) release assay were applied to detect cell viability after incubation with different concentrations of Glu for different periods.3. Phase contrast microscope observation and MTT assay were used to identify the effect of salidroside against Glu-induced cell damage.4. Hoechst staining, TUNEL assay and flow cytometry analysis, were applied to detect the effect of salidroside against Glu-induced apoptosis of hippocampal neurons.5. Western blotting analysis were used to observe the effect of salidroside on the total amount of anti-apoptotic protein Bcl-2 or pro-apoptotic protein Bax in cultured hippocampal neurons after exposure to Glu.6. To investigate whether caspase-3-like enzymes involved in Glu-induced excitotoxicity, we detect cell viability of Glu-injuried hippocampal neurons after preincubation with Z-DEVD-FMK, a cell-permeable irreversible inhibitor of caspase-3-like enzymes.7. Caspase-3 activity assay were performed to examine the effect of salidroside on Caspase-3 activity after exposure to Glu.8. Cultured hippocampal neurons labeled with fluo-4/AM were collected and quantified by confocal laserscanning microscope to determine whether NRF could protect the neurons against Glu-induced excitotoxicity by attenuating Glu-induced rise in [Ca²⁺]_i.Results:1. Co-incubition with different concentrations of salidroside resulted in neither cell viability loss nor morphological alteration of hippocampal neurons.2. The results of MTT, Hoechst staining, TUNEL assay, flow cytometry analysis and detection of caspase-3 activity indicated that Glu could evoke neurotoxicity in cultured hippocampal neurons, with the concentrations of Glu and the incubation time increased, the neurotoxicity augmented. Salidroside could significantly prevent cultured hippocampal neurons from Glu-induced neuronal apoptosis, showing an excellent dose-effect relationship.3. Neither treatment with 125μmol/L Glu for 15min nor pretreatment with salidroside affected the expressions of Bcl-2 and Bax protein.4. Salidroside could significantly inhibit the up-regulation of [Ca²⁺]_i induced by Glu. Conclusion: salidroside efficiently protects hippocampal neurons against Glu-induced apoptosis. The protective effects are mediated by inhibiting the increased caspase-3-like activity and excessive Ca²⁺ influx triggered by Glu. Further studies need to be done to explore the underlying mechanisms.Part II Protective effects and mechanism of salidroside on primary hippocampal neurons damage induced by NMDAObjective: To investigate the protective effects and underlying mechanism of salidroside on primary rat hippocampal neurons damage induced by NMDA.Methods:1. To build an excitatory neurotoxic model of hippocampal neurons, MTT colorimetric assay and LDH (in medium) release assay were applied to detect cell viability after incubation with different concentrations of NMDA. 2. Phase contrast microscope observation and MTT assay were used to identify the effect of salidroside against NMDA-induced injury.3. NF staining and TUNEL assay were applied to detect the effect of salidroside against NMDA-induced apoptosis of hippocampal neurons.4. RT-PCR were used to observe the effect of salidroside on mRNA expressions of NR2A,NR2B,NR2C and NR2D.5. Western blotting analysis was used to observe the effect of salidroside on protein expression of PSD-95 in cultured hippocampal neurons after exposure to NMDA.6. Western blotting analysis were used to observe the effect of salidroside on protein expressions of phosphorylated p38 MAPK and p38 MAPK.7. Whole-cell patch-clamp technique was applied to detect the effect of salidroside on NMDA-induced current (INMDA).8. NO production and NOS activity were measured by the NO2/3 assay kit and the NOS assay kit according to the instructions provided with.9. MTT colorimetric assay cell viability was used to observe the effect of salidroside on SNP (NO donor)-induced cell viability in hippocampal neurons.Results:1. MTT and LDH assays, together with NF staining and TUNEL assay, flow cytometric analysis and dictation of caspase-3 activity indicated that NMDA induced the dose-dependent excitotoxicity in cultured hippocampal neurons. Salidroside pretreatment attenuated NMDA-induced apoptosis in primary cultured hippocampal neurons, showing a dose-dependent pattern.2. Salidroside pretreatment does not affect the mRNA expression of NR2 subunit and protein expression of PSD95.3. Salidroside pretreatment significantly inhibited the NMDA-induced NO production and NOS activity.4. Salidroside pretreatment significantly inhibited the SNP-induced increase of cell viability.5. NMDA-induced apoptosis is associated with p38MAPK phosphorylation. Salidroside significantly inhibited p38MAPK protein phosphorylation.Conclusion: Salidroside efficiently protected hippocampal neurons against NMDA-induced apoptotic cell death. Its protective mechanism related to the inhibition of NMDA-induced caspase-3 activity and NOS activity increased NO production and NO-induced damage, and expression of phosphorylation of p38MAPK protein.Togather, salidroside could efficiently protect hippocampal neurons against Glu-induced excitotoxicity. The protective effects may be related to the inhibition of NMDA receptor mediated excitotoxicity which is related to the activiation of the NMDA receptor-Ca²⁺-NO pathway and phosphorylation of p38MAPK protein. Its neuroprotection may be used to develop a potential therapeutic approach for preventing and/or treating ischemic brain damage.