Proteomics Studies Of Apoptosis In SH-SY5Y Cells Induced By OGD/R Model

Cerebral ischemia and reperfusion insult frequently occurs as a kind ofcommon nervous system disorder whose clinical manifestations are causedessentially by pathologic structure transmutation of neuron named necrosis andapoptosis. More studies have indicated, in recent years, nerves afunction andabnormity aggravated with prolonged time after I/R were mainly induced byneuronal apoptosis, inhibitation of which was considerd as an effective andfeasible strategy in clinical therapy. Nevertheless, neuronal apoptosis wasregulated by complicated network s system in which multiple elements and linkswere conformed with unidentified mechanisms of interaction especially inneuronal proteomic studies.Objective: To build OGD/R model in vitro to mimic ischemia andreperfusion inducing apoptosis of SH-SY5Y cells, in which to detect differentialproteins by employing proteomics technique and reveal the potentialmechanisms of neuronal apoptosis.Methods: SH-SY5Y cells was employed to construct OGD with differenttime (2h,4h,6h,8h,10h,12h,16h,20h) and reperfusion model. Cell viabilitywas calculated by the method of Trypan blue exclusion and MTT conversion.The OGD10h/R groups in which cell viability decreased approximately 50%after reperfusion 24h and control groups were selected to make further assay.Observation with light microscope , fluorescence microscope and transmission electron microscope showed the variation of structure and morphous in cells atdifferent time(0h,24h,48h). Changes of oxidative stress enzyme and metabolinincluding LDH,MDA,SOD level were detected at different time of reperfusion(0h,6h,12h,24h,48h,72h). Flow cytometry was used to analyse the cell cycledistribution and apoptotic percentage at different time of reperfusion (0h,6h,12h,24h,48h,72h).With the proteins extracted from SH-SY5Y cells in controland OGD10h/R24h groups, 2D-DIGE and MALDI-TOF MS techniques wereused to detect differential proteins.Results: Trypan blue exclusion and MTT conversion showed the dead cellssignificantly increased with prolonged OGD time. At different time ofreperfusion, the cell viability varied from 70% to 100% in OGD 2h,4h,6h,8h groups, and decreased significantly in OGD10h above groups. At reperfusion24h, the cell viability was 44.91±1.21% after OGD 10h by MTT, and decreasedto 20%～0% in OGD16h above groups. The cell viability in other differentOGD groups, versurs OGD 2h groups, was significantly differential (P < 0.05 orP < 0.01). Inverted microscope showed, cells density decreased and some cellsturned round with axon retracting or disrupted following varied shape andincreased metabolic product in OGD10h/R groups at R0h,24h,48h,72h. H&Estaining showed, in cells of control groups, nuclei took on blue round-alikewith chromatin spots uniformly distributed and cytoplasm took on pink withclear contrast to nucleus, however, i n cells of OGD10h/R groups, nuclei showeddeep blue pycnotic with some of likely fragment, cytoplasm were stainedsuperficially or loss of color. Hoechst33342 staining indicated, in control groups,nuclei were round-alike with uniformly blue staining, the density of cellsincreased with prolonged time. In OGD10h/R groups, nuclei took on sapphirineor pycnotic,round pearl and fragment of apoptotic view with the densitydecreased and apoptotic body emerged. AO/EB staining showed, in controlgroups, nuclei took on green with chromatin spots uniformly distributed and cytoplasm took on shallow green. In OGD10h/R group, nuclei took on compactround,pycnotic and fragment of apoptotic view with nuclear fluoresceinstaining enhanced and apoptotic body emerged. The counting of apoptotic cellsstained by AO/EB under fluorescence microscope showed, in control groups,there were a certain ratio of apoptotic cells. In OGD/R groups, apoptotic rate atdifferent reperfusion time increased gradually with prolonged reperfusion timeand reached its peak of 25.35±1.71% at R24h, compared with control groups atthe same time the contrast was significant (P < 0.001). Under electronmicroscope, the cells in control groups showed fake villi on membrane surface ,abundant mitochondria and free ribosome, rough endoplasmic reticulumexpanding in cytoplasm, anomal nuclei with clear nucleoli andmulti-euchromatin. In contrast, the cells in OGD10h/R groups showed fake villidisappeared, bulk decreased, ground substance of nuclei and cytoplasmconcentrated with vacuolar structure, membrane integrity or disrupt,heterochromatin fringe agglutinated in nucleus, which were viewed as theappearance of apoptosis and autophagia. The level of LDH in culture fluid andthe contents of intracytoplasm MDA at different reperfusion time, in OGD10h/Rgroups, increased with prolonged time and at reperfusion 24h respectivelyreached peak and platform phase, shown as 1908.57±127.97U/L and75.83±24.31nmol/mgprot , the contrast with control groups was significant (P <0.01). Intracytoplasm SOD activity at different reperfusion time declined at firstand recovered later with prolonged reperfusion time, at reperfusion 24h reachedthe lowest level of 49.09±21.63U/mgprot, the contrast with control groups wassignificant (P<0.01). In flow cytometry analysis on cell cycle phase at differentreperfusion time, in OGD 10h/R groups with prolonged reperfusion time, thepercentage of G0/G1 phase increased at first and decreased later with its peak of74.09±2.62% at R24h, which was significantly elevated compared with controlgroups at the same time except R0h (P < 0.05 or P < 0.01). The percentage ofG2/M phase reached its peak of 26.85±1.35% at R0h and declined gradually later, which significantly increased compared with control groups at the sametime before R24h (P < 0.01). The percentage of S phase declined gradually andreached the lowest level of 19.2±1.58% at R24h, which was lower significantlythan control groups at the same time (P < 0.05 or P < 0.01). The apoptotic ratedetectation through flow cytometry at different reperfusion time showed, inOGD/R groups, the apoptotic rate increased gradually with prolongedreperfusion time and reached its peak of 26.85±1.25% at R24h, then declined to20.16±2.81% and 19.11±1.58% at R48h and R72h, the contrast with controlgroups was significant (P < 0.001). Mean 1800±156 protein spots were displayedon 2D-DIGE image. In OGD10h/R24h group, 30 protein spots were detecteddifferentially expressed compared with control group (P < 0.05 or P < 0.01), ofwhich 22 spots were up-regulated and 8 spots were down-regulated. Seven ofthe 30 protein spots were identified as: Crystal structure of the XiapCaspase-7complex, Usurpin-gamma, Proto-oncogene serine/ threonine protein kinase Pim-1,Squalene synthase, transglutaminase-3, Zinc finger protein DPF3 and Sptin2,which in function are associated respectively with inhibitation of apoptosis, cellsmetabolism, insult defence, transcription regulating, cystoskeleton building.Conclusion: Oxygen and glucose deprivation 10h and reperfusion (OGD10h/R) to mimic cerebral ischemia and reperfusion in vitro is a reliable modelthat induces SH-SY5Y cells apoptotosis. By employing proteomics techniques,seven valuable differential proteins were found at the first time in this model,which provided new clues for studies of neuronal apoptotic mechanisms andnew candidates for potential treatment targets of neuronal apoptosis.