Proteomic Analysis Of The Cerebral Cortex During The Acute Stage Of Intracerebral Hemorrhage

Objective Intracerebral hemorrhage is a destructive way of stroke, the incidence of which was found to increase 2-3 times each year in Asian countries. Compared to the white race, the Chinese were reported to have a higher incidence of intracerebral hemorrhage. Intracerebral hemorrhage is usually divided into primary injury and secondary injury. During the acute stage of intracerebral hemorrhage, bleeding as a rapidly expanding intracranial mass directly damage the structure of neurons and compress the surrounding tissue to damage it. In this period it is very crucial to timely treat, which can prevent the tissue from further damage. It became critical point to find biomarkers which indicate the condition’s change during the acute phase. At present the research on the biomarker about intracerebral hemorrhage was a lot, but still none of biomarker is definite and recognized in clinical. Based on the above situation, we will carry out proteomic research on the samples in each time point during the acute stage of intracerebral hemorrhage and obtain different proteins. At last the marked protein will be verified by western-blot and to explore the possible mechanism during the acute stage of intracerebral hemorrhage.Methods We make intracerebral hemorrhage model by injecting autologous blood(caudal artery blood 50ul)into the brain under the stereotactic. The cortex tissue at the same side of the hematoma will be taken off on the 2hours, 4 hours, 6 hours, 8 hours(5 rats per group) after intracerebral hemorrhage, and the normal rat brain cortex tissue also will be collected. All the cortex tissue must be carried on proteolytic cleavage(450μl 8M Urea and 50μl phosphatase inhibitor)、 purity determination(Bradford method)、 reductive alkylation、 the liquid pre-separation(RIGOL L-3000 high-performance liquid chromatography)、mass spectrometry(Thermo Mass Spectrometer),then by the database retrieval system(Proteome Discoverer 1.4; search engine: MASCOT; databases: NCBI-rat ref-sequence protein database(60122 protein, updated on 07-2015)and statistical analysis to obtain different proteins. The up-regulated protein neurofibromin1 and down-regulated protein Ras-related protein Rab15 will be verified, which are selected from the differential proteins. The cortex tissue at the same side of the hematoma will be taken off on the 2hours, 4 hours, 6 hours, 8 hours(5 rats per group) after intracerebral hemorrhage, and the normal rat brain cortex tissue also will be collected. All the tissue will be carried on tissue cleavage with the lysates, then by western-blot to see whether its result is consistent with the expression of the selected proteins.Results According to mass spectrometry and database searching, the 807 differentially expressed proteins are obtained. By the statistical analysis and comparison with the normal group, the 167 up-regulated proteins and 173 down-regulated proteins are selected, by Ratio value and P value in which 3 up-regulated proteins: NF1, Vgf, Ncs1 and 8 down-regulated proteins: Rab15, Slcla4, Eps15, Arhgap21, Npdc1, Mycbp, Nrn1, Tp53 rk are focused on. The second result: The up-regulated protein neurofibromin1 and the down-regulated protein Ras-related protein Rab15 were chosen to carry out western-blot, the result shows the neurofibromin1 protein expression within 8 hours after intracerebral hemorrhage are gradually increased, however the Ras-related protein Rab15 protein expression is gradually low.Conclusion 1、during the acute phase of intracerebral hemorrhage the protein expression of brain cortical neurons is regularly, in which the most up-regulated proteins inhibit the regulation and the most down-regulated proteins undertake transport, construction and positioning. 2、the up-regulated protein neurofibromin1 and the down-regulated protein Ras-related protein Rab15 can be selected as biomarkers about the disease evolution or targets about drug therapy during the acute phase; 3、the NF1-RAS pathway may be one of the possible mechanism during the acute phase of intracerebral hemorrhage.