The Protection And Possible Mechanism Of Oxygen-glucose Deprivation Postconditioning In Oxygen-glucose Deprivation-reperfusion Injury

Cerebral infarction is one of ischemic cerebrovascular diseases with the characteristic of highmorbidity and disability, which is mainly pathophysiological manifestation is neurons ischemic deathcaused by thrombus blocking blood vessels, to which most effective therapy is thrombolytic therapy torestore the blood supply of the ischemic tissue, but this also leads to cerebral ischemia-reperfusion(I/R)injury. Ischemic postconditioning(IPostC) refers to a mode conducted by several brief episodes of sublethalischemia–reperfusion after ischemic damage and before reperfusion in tissues and organs, which has beenconfirmed that it had anti-I/R injury in a number of animal experiments and clinical studies, which isperformed before reperfusion, and is more practical. It has been presumed that apoptosis and PI3K/Aktsignaling pathway were the main targets of IPostC, but the mechanisms have not yet been clarified. Atpresent, few studies focused on IPostC of I/R injury after stroke, most of which were based on animalmodels, and not on cell models.Therefore, SK-N-SH cells were cultured in vitro, and tried to make improved oxygen-glucosedeprivation-reperfusion(OGD/R) model to simulate the I/R injury in vivo, simultaneously, oxygen-glucosedeprivation postconditioning(OGDP) was performed by a series of brief oxygen-glucose deprivation(OGD)and re-oxygen-glucose (ROG), and the influence and influential factors of OGDP on OGD/R injury wereobserved. We could further discuss the anti-OGD/R injury mechanism of OGDP with optimal modality soas to provide reliable experimental basis for in vitro studies of brain I/R injury mechanisms and therapeutictargets. our study is divided into two following parts:(1) The protective effect and influential factors of OGDP on OGD/R injury.(2) The effect and possible mechanism of OGDP on apoptosis in OGD/R injury.Chapter1The protective effect of OGDP in OGD/R injuryObjective: To observe the neuroprotection and influential factors of OGDP in OGD/R injury.Methods: OGD/R injury model was made by OGD4h and ROG20h for SK-N-SH cells, then OGDPwas operated after OGD. The cells were divided into control group(normal culture group), OGD/R group, and OGDP group. OGDP group were further divided into1,5,10,15,20,30,60min subgroups accordingto different time of OGDP; and5,10,15,20,30,60min subgroups according to different time windows ofOGDP; and1,2,3,4,5,6cycles subgroups according to different cycles of OGDP. Cell viability wasdetected by MTT (2-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide) staining, and cellmorphological changes were observed by inverted phase contrast microscope in the different groups.Results:(1) Cell viability of different time for OGDPCell viability of OGD/R group decreased by approximately44.5%, compared to control (P<0.01).Compared to OGD/R group, the increase of cell viability for10,15min groups was statistically significant(P<0.01). There was no significant difference between10and15min groups (P>0.05), and cell viabilitycould not futher improve in20,30,60min groups (Compared to OGD/R group, P>0.05).(2) Cell viability of different time windows for OGDPCompared to OGD/R group, there was significantly improved cell viability of time windows10,15min groups (P<0.01), while cell viability was not improved in5,20,30,60min groups (P>0.05).(3) Cell viability of different cycles for OGDPCompared to OGD/R group, there was significantly improved cell viability of1,2,3,4, cycle groups(P<0.01). Cell viability of3cycles group was significantly increased by approximately8.5%(P=0.02),Compared to1cycle group, while viability of2,4cycles groups could not further improve (P>0.05).(4) Cell morphology was observed by inverted phase contrast microscopeCell population of control group was sufficient, presented irregular and adheren growth, cone-shapedor polygonal cell body, its boundary was clearly and obvious halo, and highly refractive, coarse and long ofcell ecphyma. Cell population of OGD/R group decreased significantly, sparse and decline of the refraction,poor adherence and retractive ecphyma, the cell body became shrunken, smaller and round. Cell populationof OGDP group significantly increased, compared to OGD/R group, the majority morphological of themwas normal, adherence was better and refraction rised, most cell body was polygon and a few becameshrinkage and round.Conclusion: There were protective effect on the OGD/R injury of OGDP, which is correlate with time,time windows and cycles of OGDP, and the effect of OGDP with3cycles of10min was outstanding. Chapter2The effect and possible mechanism of OGDP on apoptosis in OGD/R injuryObjective: To investigate the anti-apoptosis of OGDP in OGD/R, and to explore the role and possiblemechanism of the PI3K/Akt signaling pathway in the anti-apoptosis effect of OGDP in OGD/R injury.Methods: The method of cell model and OGDP was same to chapter1. The cells were divided intocontrol, OGD/R, OGDP groups. Apoptotic morphology and apoptosis count were detected byHoechst33258fluorescence staining, the expression of protein caspase-3, p-Akt and Akt were detected byWestern blot assay.Results:(1) The results of Hoechst33258stainingCell nuclear fluorescence of control group showed a uniform light blue, and morphological rules ofnucleus. Nucleus of OGD/R group was a higher and brighter blue fluorescent, it showed the characteristicsof apoptosis: nucleus fragmentation, chromatin condensation and marginalization. Compared to OGD/Rgroup, nuclear fluorescence of OGDP group was significantly darker, nuclear morphology tended to rule,chromatin condensation reduced.The rate of apoptosis for OGD/R and OGDP groups were significantly higher than that of controlgroup (P<0.01), however, the rate of apoptosis for OGDP group was significantly lower than that ofOGD/R group (P=0.023).(2) The expression of protein Akt and p-AktThere was no significant difference on the expression of Akt of the groups (P>0.05). Compared tocontrol, the expression of p-Akt for OGD/R and OGDP groups were significantly increased (P<0.01). andcompared to OGD/R group, the expression of p-Akt for OGDP group was a significant increased (P=0.04).(3) The expression of caspase-3proteinThe expression of caspase-3for OGD/R and OGDP groups were significantly higher than controlgroup (P<0.01), whereas compared to OGD/R group, the expression of caspase-3for OGDP groupdecreased significantly (P=0.02). Conclusion: There were anti-apoptosis effects on OGD/R injury of OGDP, and this effects werecorrelated with the expression of caspase-3was inhibited, PI3K/Akt signaling pathways was involvedin the process that OGDP against apoptosis.