(The Study Of The Protective Effects And Mechanism Of Edaravone On Neuronal Injury Induced By Hypoxia And Acidosis/Reoxygenation)

Part Ⅰ To investigate the molecular mechanism of edaravone of mitigating the neurotoxicity induced by hypoxia-acidosis/reoxygenationAbstractObjective:1To evaluate the effects of edaravone on cytotoxicity induced by hypoxia/reoxygenation combined with acidosis injury;2To investigate the molecular mechanism how edaravone reduces cytotoxicity caused by hypoxia with acidosis and protects the neurons.Methods:1Establish ischemia model--oxygen and glucose deprivation combined with acidosis/reoxygenation model(OGD-A/R).To eliminate the activation of glutamate and voltage-dependent Ca2+channels,all experiments were conducted in the presence of blockers for NMDA and AMPA receptors and voltage-gated Ca2+channels.2Cells were divided into several groups:control group,OGD-A/R group,OGD-A/R and edaravone of diferent concentration groups, OGD-A/R and blocker of ASICs channels groups. The levels of LDH releasing、Caspase-3activity and Hoechst33258staining are detected at different time point of reoxygenation after OGD-A/R to evaluate the neuroprotective effect of edaravone against hypoxia/reoxygenation combined with acidosis induced injury; 3The appropriate concentration of edaravone and ASIC1a blockers and MEK/ERK antagonists were used on cells of OGD-A/R model in different groups individully; The expression levels of ERK、 AKT and Bcl-2were detected with Western-blot, while the expression levels of BDNF and Bcl-2mRNA detected with RT-PCR, the expression levels of BDNF protein detected with ELISIA,the levels of LDH releasing and Hoechst33258staining detected with homologue assay kit or regents. The data above were analyzing to probe the possible protective mechanism of edaravone in OGD-A/R model.Results:1OGD-A/R model is established successfully, and OGD-A/R for4h and8h induced a robust increase in LDH release;2Pretreatment with PcTX or amiloride remarkably reduced the LDH release after4h and8h of OGD-A/R.Relative to ASICs blockade, the pretreatment of edaravone followed by OGD-A/R elicited a similar but greater protection by a dose-dependent suppression of LDH release after reperfusion;3Compared to OGD-A/R,edaravone pretreatment markedly promoted BDNF and Bcl-2mRNA expression at each time point,and the most pronounced effects occured at8h after OGD-A/R.ELISIA analysis showed that BDNF protein levels were significantly elevated after edaravone pretreatment at the same point. Western-blot analysis indicted that edaravone pretreatment significantly enhanced Bcl-2expression at8h after OGD-R. Results from caspase-3activity assay indicated that OGD-A/R insults greatly elevated caspase-3activity which could be relieved by edaravone in a dose-dependent manner. This protective effects were comparable to those from blockade of ASICs similarly.4Neurons exposed to OGD-A/R had a marked increase in the p-AKT level compared to the normal control,p<0.01. However, the high activity of p-AKT was significantly inhibited by edaravone or PcTX pretreatment during OGD-A/R,p<0.01;5Compared to the normal control, OGD-A/R or OGD-A/R combined with PcTX treatment induced a mild increase in p-ERK1/2, whereas edaravone administration resulted in a dramatic increase in p-ERK1/2during OGD-A/R.The effect was blocked by the MEK (MAPK/ERK kinase) antagonists PD98059(10μM) and U0126(5μM).6The data from Hoechst33258staining and LDH release assay showed that OGD-A/R-induced cell death triggered fragmentation of nuclear chromatin and augmentation of LDH release. Edaravone pretreatment largely prevented nuclear morphological changes and LDH release in injured cells. However, the MEK inhibitors PD98059and U0126administration30min prior to OGD-A/R, almost completely abrogated the antiapoptotic ability of edaravone on neurons exposed to OGD-A/R.Conclusions: 1Edaravone is capable of attenuating hypoxia-acidosis/reoxygenation-mediated neurotoxicity and the effect,at least partially,depends on the enhanced ERK1/2activation.2Edaravone confers neuroprotective effects through enhancing BDNF and Bcl-2expression and supressing caspase-3activity in the presence of hypoxia-acidosis/reoxygenation.3Acidosis and ASICs play a critical role in the process of acido-toxicity induced by hypoxia-acidosis/reoxygenation.The neuroprotective effects of Edaravone may be related to ASICs blockade. Part Ⅱ To investigate the molecular mechanism of edaravone of alleviating the neurotoxicity induced by hypoxia/reoxygenationAbstractObjective1To evaluate the neuroprotective effect of edaravone on neurons damaged by hypoxia/reoxygenation-induced injury;2To investigate the molecular mechanism how edaravone reduces cytotoxicity caused by hypoxia/reoxygenation-induced injury and protect the neurons. Methods1Establish ischemia model--hypoxia/reoxygenation model which is also called oxygen and glucose deprivation/reoxygenation model (OGD-R);2Cells were divided into several groups:control group,OGD-R group,OGD-R with edaravone of diferent concentration groups.The levels of LDH releasing、Caspase-3activity and Hoechst33258staining were detected at different time point of reoxygenation after OGD to evaluate the neuroprotective effect of edaravone against hypoxia/reoxygenation induced injury;3Cells of OGD-R model were pretreated with the appropriate concentration of edaravone and edaravone combine with the specific channel blockers LY294002and MK2206individually, the expression levels of ERK NAKT and Bcl-2were detected with Western-blot, while the expression levels of BDNF and Bcl-2mRNA detected with RT-PCR, the expression levels of BDNF protein detected with ELISIA,the levels of LDH releasing and Hoechst33258staining detected with homologue assay kit or regents. The data above were analyzing to probe the possible protective mechanism of edaravone in OGD-R model.Results1OGD-R model is established successfully, significant pathological changes can form in four hours and eight hours;2The pretreatment of cultured neurons with edaravone alleviates hypoxia/reoxygenation induced neuronal injury in a dose-dependent manner(LDH releasing reduced,Caspase-3activity and ratio of nuclear pyknosis declined).3The levels of LDH releasing detection is determined as a simple and reliable quantitative measure for neuronal cell injury, and this conclusion is verified by detecting Caspase-3activity and Hoechst33258staining.4The edaravone pretreatment did not increase the p-ERK expression levels significantly, but the amount of p-AKT expression was significantly increased, p<0.01.5Pretreatment of edaravone(10μM) remarkably reduced the LDH release and declined the ratio of nuclear pyknosis after reoxygenation (p<0.01),which was inhibited by MK2206.6Compared to OGD-R,edaravone pretreatment markedly promoted BDNF and Bcl-2mRNA expression at each time point,and the most pronounced effects occured at8h after OGD-R.ELISIA analysis showed that BDNF protein levels were significantly elevated after edaravone pretreatment at the same point. Western-blot analysis indicted that edaravone pretreatment significantly enhanced Bcl-2protein expression at8h after OGD-R,which was blocked by MK2206.Conclusions 1Edaravone alleviates hypoxia/reoxygenation induced neuronal injury by enhancing BDNF and Bcl-2expression and inhibiting Caspase-3activity,that is to say,Edaravone has already the neuroprotective effects during the period of hypoxia with no acidosis;2Edaravone protects neurons against hypoxia/reoxygenation induced cytotoxicity maybe through activation of the PI3K/Akt pathway.