| Background and objective:Cerebrovascular disease (CVD), one of three most serious high-mortality diseases, is the main cause of disability for the middle-aged and the aged people. Among it, ischemic stroke accounts for 75%–80%. With the rapid development of aging process, it become more and more severe to threaten and harm people's health. At present, there is still no effective medcine for neuron protection. So more and more attention has been paid to the research on the pathophysiological mechanisms and the prevention of the ischemic CVD (ICVD).Nowadays many researches focus on the exploitation of cerebral protective methods, however, except for large doses of albumin and hypothermia cerebral protection, most methods only have effect on animal models. This phenomenon is due to unclear mechanisms and no suitable drug targets for cerebral protection. Recently, reversing the neurons in apoptotic status in ischemic penumbra became a good idea for cerebral protection. In recent years, it is regarded that the mitochondria plays a pivotal role in the activation of the apoptotic signals transduction and the determination of neurons fate during cerebral ischemia. So protecting mitochondria by various mechanisms might be a new approach to treat ICVD. The mitochondrial different proteins between normal and ischemic neurons are the material foundation for the definite effect of mitochondria on apoptosis, so these differentially expressed proteins are very important for the investigation of medicine targets for cerebral protection, and they are meaningful for the prevention and treatment for ischemic neuronal injury. 3-n-butylphthalide (NBP) is a mitochondrial protective agent which can prevent neuron from apoptosis by blocking pathological procedures during cerebral ischemia, while the mechanism of its protective effect still remains unclear. Differential proteomics provides a platform for systematic comparative research at protein level. The strategy combining two-dimensional electrophoresis(2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is most common in practice. It has been proven to be very valuable in the screening and identification of function-associated molecules, biomarkers and drug targets.To explore the pathological molecular differences of cerebral ischemia and identify the specific proteins related to neuronal injury, differential proteins of mitochondria were detected and identified in normal and MCAo rat cortex by differential proteomics technology. At the same time, the dynamic changes of neuron ultrastructure and mitochondrial respiratory function were observed. We also applied NBP to MCAo rats to explore the agent targets through proteomics technologies.Methods:Adult male SD rats were randomized into normal group, MCAo group, sham operation group and NBP treatment group. Nylon monofilament is used to estahlish middle cerebral artery occlusion (MCAo) models. Each group was further divided into 4 subgroups, that is, 6-hour subgroup, 24-hour subgroup, 48-hour subgroup and 72-hour subgroup. NBP was administered to MCAo rats 15 min after MCAo. For each subgroup, cerebral cortex were collected and mitochondria were extracted and purified at above mentioned time points. After two dimensional electrophoresis and PDQuest for gel image analysis, the MS/MS of the peptides from the differential proteins digested by tripsin were analyzed by HPLC-ESI-MS/MS. The raw data was pre-processed by Spectrum Mill, then searched in NCBInr. Transmission electron microscopy was used to assess the neuronal ultrastructure. Respiratory state III (ST3), Respiratory state IV (ST4), oxidative phosphorylation (OPR), and respiratory control rate (RCR) of mitochondrial respiratory chain were tested to estimate the mitochondrial respiratory function.Results:1. We have established high resolution electrophoregram using 2-DE technique, which provides a theoretical basis and a technical support for the research of pathological cerebral cortex mitochondria proteins.2. Specific protein spots in MCAo group were identified as TUC-4b, CKB, HS1 binding protein, mitochondrial ribosomal protein S27, dihydrolipoamide dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase(NAD+)alpha, dihydrolipoamide S-acetyl transferase, cytochrome c oxidase subunit Vla, succinate-coenzyme A ligase, ubiquinol- cytochrome c reductase core protein, ATP synthase beta subunit, heat shock 70KD protein, heat shock 60KD protein, and acyl-coenzyme A dehydrogenase family. The expression of HS1 binding protein was decreased in 6 h after occlusion, reached to the minimum in 24 h, then recovered close to the normal lever in 72 h. Other spots were all increased in 6 h MCAo, reached the lowest in 24 h, then came back to the normal lever in 72 h.3. The ultrastructure of neurons was damaged seriously in 6 h after cerebral ischemia, reached to the worst in 24 h and recovered close to the normal in 72 h. ST3, RCR and OPR in cerebral cortex mitochondria were decreased significantly at 6h, 24h, 48h and 72 h after cerebral ischemia compared to the normal control group, but that of ST4 was increased evidently. ST3 was decreased significantly in MCAo 6-hour group (P<0.01), reached to the bottom in MCAo 24-hour group (P<0.01) and recovered close to the normal level in MCAo 72-hour group(P<0.01). RCR and OPR had a similar tendency with that of ST3. ST4 was increased significantly, difference was observed in 6 h and 72 h subgroup(P<0.05).4. The protein expressions in NBP treatment group changed vigorously compared with those of the proteins in MCAo 6-hour group. The identified specific protein spots were as same as those in MCAo 6-hour group compared with normal group. HS1 binding protein which was decreased significantly in MCAo 6-hour group was increased in NBP treatment group and reached close to the normal level. Other proteins increased in MCAo 6-hour group were decreased close to the normal levels in NBP treatment group.Conclusions:1. Our results show that ischemia will affect the proteins related to cerebral cortex mitochondria, which suggests that these proteins may be correlated with the energy metabolism and neural apoptosis.2. In the acute stage of cerebral ischemia, mitochondria shows structural and functional damages. These damages will be recovered with the elapse of time, which is consistent with the changes of protein expressions. It may be of protective effect on cerebral ischemic injuries by improving mitochondrial respiratory chain function. 3. NBP can protect cortex mitochondria from cerebral ischemia by regulating the differentially expressed proteins. |
