Comparative Proteomics Study Of Dl-3-n-butylphthalide Protect PC12Cells From Hypoxia-induced Damage

Objective: Ischemic stroke is one of the most commoncerebrovascular disease, its mortality and morbidity are higher, andserious harm human health and the quality of life. The basic mechanismis that an occlusion of arteries supplying intracranial or extracranialarteries leads to a complex cascade of pathophysiological events thatdamage the brain tissue due to the mismatching between local brain tissuemetabolism and blood supply,including: the cellular energy metabolismdisorders, acidosis, calcium loss steady, radical toxicity, inflammatoryreaction. DL-3-n-butylphthalide(NBP)can increase blood flow of thebrain ischemia area, improve the energy metabolism of whole brain afterischemia and the microcirculation of brain ischemia area, protect themitochondrial function, restrain inflammatory reaction and internal flowof calcium, protect the mitochondrial function, alleviate the damageextent of nerve function, and reduce focal cerebral ischemia area afterinfarction, which has preferable treatment protection to ischemic stroke.Many studies have been elaborated the mechanism of NBP from differentaspects, but we have yet to see any research about the mechanism of NBPin proteomics level. Systematacially recognizing specific protein changesin response to drug administration in humans will have the potential forthe development of personalized medicine. Such changes can beidentified by proteomics approach based on2-DE and MALDI-TOF/TOF.Proteomics technologies has become extremely active in the field of lifesciences disciplines. Comparative proteomics focuses on the changes anddifferences of proteomics under different conditions aiming to identifythe variant components and provide a fully understanding aboutmolecular mechanism and cell metabolism on the proteome level. Drug discovery, new target identification and research on multi-target-directeddrugs will all be benefited from the further advances in proteomicsapproaches and methods, and the proteomics has the potential to greatlyenhance the efficiency of the drug discovery process. PC12cells arepheochromocytoma cells come from rat adrenal, in which themorphological and physiological, biochemical, etc close to neurons, andhave characteristics of neuroendocrine cells and neurons, are often usedin neuronal cell death way and the neurotoxicity injury research. In thisstudy, by applying comparative proteomics to PC12cells ofoxygen-glucose deprivation leading by sodium hydrosulfite, we haveidentified and isolated a panel of proteins that are differently representedin model control and NBP groups so as to deeply understand ischemiccerebrovascular disease pathogenesis and neuroprotective of NBP inischemic cerebrovascular disease.Mathod:1. Made cell model of hypoxia. PC12cells were impaired byserum-free culture, and media with sodium hydrosulfite (Na₂S₂O₄) wereused as the cell model of hypoxia.2. NBP treated on PC12cells inducedby hypoxia injury. MTT (methyl thiazolyl tetrazolium) was used to assaythe viability of the PC12cells. We observed the protective effects of NBPon damages of PC12cell induced by Na₂S₂O₄.3. Extracted proteins ofPC12cells, then run immobilized pH gradient (IPG) isoelectric focusingelectrophoresis as the first dimension, and vertical SDS-PAGE as thesecond dimension. The map was visualized by colloidal coomassive blueand analysised with ImageMaster2D Elite software. The proteins ofinterest were digested and identified using MALDI-TOF/TOF tandemmass spectrometry and database retrieval.Results：1. The influence of the survival rate of PC12cells by differentconcentration of Na₂S₂O₄.We joined different concentration of Na₂S₂O₄, the growing of PC12cells induced different degree suppression, cell boundaries defected, the body atrophied and falled off, and cells aggregated. As the increase ofNa₂S₂O₄concentration, the cells average absorbance value graduallyreduced. When Na₂S₂O₄concentration was equal or greater than5mmol/L, absorbency value have obvious difference compared with thecontrol group (P <0.01).2. Protective effects of NBP on damages of PC12cell induced byNa₂S₂O₄.PC12cells were given5mmol/LNa₂S₂O₄, at the same time givendifferent doses of NBP, as the increase of concentration NBP, cell averageabsorbance value gradually increased. When concentration of NBP isequal or greater than10μmol/L, absorbency value have obviousdifference compared with model group (P <0.01).3.2-DE gels protein profile of PC12cells with the model of hypoxiagroup and NBP group.By the comparison of2-DE gels protein profile of PC12cells with themodel of hypoxia group and NBP group, we identified21kinds ofdifferences protein, which raised15, down6.4. Mass spectrometry analysis and appraisal result.21protein spots were differentially expressed. Among them,15up-regulated proteins in NBP group were identified as d (1—3spots),actin, Heat shock cognate71kDa protein, Cyclin G-associated kinase,Annexin A6, Peripherin, Hypothetical protein, Elongation factor1-gamma, similar to serine/threonine-protein phosphatase2A regulatorysubunit B, Peroxiredoxin-2, Transitional endoplasmic reticulum ATPase,Ubiquitin carboxyl-terminal hydrolase isozyme L1,6down-regulatedproteins in NBP group were identified as V-type proton ATPase subunit B,Phosphoribosylformylglycinamidine Synthas, Hypoxia up-regulatedprotein1, Pirin, N(G),N(G)-dimethyl-arginine dimethyla-minohydrolase2, Heat shock protein beta-1.Conclusion：1. PC12cells impaired by serum-free culture media with sodium hydrosulfite (Na₂S₂O₄) are builded a successful cell model of hypoxia,through observe the MTT metabolism rate of oxygen injury PC12cellstreated by NBP, we assay the viability of the PC12cells, which showsthat NBP exerts a potent neuroprotective effect against ischemia-inducedbrain injury.2. In this study, we identify21differentially-expressed proteins thatinvolve in various types such as metabolic enzymes, molecular chaperoneprotein, signal transduction protein and cytoskeletal related protein using2D-PAGE. Further research of these proteins in depth, has importantsense for greater understanding of NBP neuroprotection mechanismagainst oxygen-glucose deprivation.