Protective Effects Of Dexmedetomidine On Oxygen-glucose Deprivation/reperfusion-induced Neuronal Apoptosis

Background Dexmedetomidine is a highly selective and specific ?2 adrenoreceptor agonist,which exhibits a broad spectrum of effects,including sedation,hypnosis,anesthesia and analgesia.Dex favours ? 2 over ? 1 by 1 600:1.Although it has been found that Dex can inhibit apoptosis and has neuroprotective effect in the developing brain by a large number of clinical and basic experiments,there was not very clear about its mechanism in cell and molecular level.Endoplasmic reticulum stress is not only a protective stress reaction of eukaryotic cells but also a subcellular pathological process of impairment in ER homeostasis.A number of insults have been shown to induce protein misfolding in the ER and cause ER stress,such asnutrient deprivation,alterations in calcium concentrations,oxidative stress and ischemia reperfusion.As one of the significant ways to initiate cell apoptosis,studies had shown the ER stress was involved in pathophysiological process of important organs such as heart,brain and kidney induced by ischemia-reperfusion injury.This experiment aimed to investigate protective effects of different concentrations Dex on oxygen-glucose deprivation/reperfusion-induced neuronal apoptosis in the cellular level,and discuss the neuroprotection of the underlying mechanisms involved in the ER stress.Objective To investigate and study protective effects of Dexmedetomidine on oxygen-glucose deprivation/reperfusion(OGD/R)-induced neuronal apoptosis.Methods SH-SY5 Y cells were differentiated to neurons with ATRA and followed by TPA.According to the results of preliminary experiment,OGD/R modle was constructed by oxygen-glucose deprivation(OGD)for 12 h and reperfusion(R)for another 12 h.During the period of the OGD,neurons were divided into six groups:Group D0(0?M Dex),Group D1(0.1?M Dex),Group D2(1?M Dex),Group D3(10?M Dex),Group D4(100 ?M Dex),Group D5(1000?M Dex).After reperfusion 12 h,the cell viability was evaluated by the method of MTT.The cellular apoptosis was observed by Flow Cytometry method.Then we investigate the protective effects of different concentration Dex on OGD/R-induced neuronal apoptosis.Then chosen the exact group of protective effects,the endoplasmic reticulum stress specific protein MANF and pro-apoptotic protein caspase-3 and CHOP were detected by Western Blot method.Results 1.ATRA and TPA sequentially induced the SH-SY5 Y cells to the differentiation of mature neurons.Compared with the normal and untreated SH-SY5 Y cells,the SH-SY5 Y cells induced by ATRA for 3 days cells appeared to have a more polar morphology and began to extend long and slender neurites in the both ends of cell body.The SH-SY5 Y cells sequentially induced by TPA had a further differentiation and not only stretched longer neurites of cell body but also formed extensive connection with neurites among cells.Moreover,compared with the normal,it appeared to have reduced proliferationa and the number of cells.2.The cell viability of neurons induced by OGD12h/R12 h is nearly 50%. Compared with different OGD/R time,such as 4h/12 h,8h/12 h,16h/12 h,MTT method showed that the cell viability of neurons decreased gradually with the prolongation of OGD time.The cell viability decreased to 89.63% ± 2.77 when exposured to OGD4h/12 h.After the 8h/12 h and 12h/12 h OGD/R time,cell viability decreased to 81.68%±3.86 and 53.13%±1.54.The cell viability decreased to 21.24%±1.49 when induced by OGD16h/12 h.The results showed that the cell viability of OGD12h/R12 h was close to 50%,which could be used to built a standard time of OGD/R model.3.10?M Dex can obviously increase the cell viability of OGD/R-induced neurons and inhibit apoptosis.After OGD/R-induced neurons effected by Dex,MTT method showed that the cell viability of group D0,D1,D2,D3,D4 and D5 was 52.15%±0.96,51.72%±1.96,51.97%±0.39,64.78%±2.76,45.69%±2.30 and 19.93%±4.22.Compared with group D0 and the others,D3 obviously decreased the mortality rate and significantly increased the cell viability(P<0.05).The result showed by Flow cytometry were consistent with the MTT method and indicated that 10?M Dex can obviously increase the cell viability of OGD/R-induced neurons and inhibite apoptosis.4.10?M Dex can regulate the expression of endoplasmic reticulum stress-related protein.Based on the results of MTT method and Flow cytometry,10?M Dex was selected to further investigate the relationship between the neuroprotection of Dex and ER stress,MANF and pro-apoptosis protein in the experiment.The Westernblot showed that 10?M Dex remarkably up-regulated MANF(P<0.01)and inhibited up-regulation of caspase-3 and CHOP(P<0.01).Conclusion These data suggest that 10?M Dex had neuroprotective effect on OGD/R-induced neuronal apoptosis and significantly increased cell viability.Our results also indicate that up-regulation of ER stress specific protein MANF and inhibition of CHOP and caspase-3 by MANF are involved in the neuroprotective effects of Dex.