Proteomics Study Of Hypothalamus Tissue In Model Of Intracerebral Hemorrhage In Rat

nervous system diseases,including hemorrhage in parenchyma, which is caused by parenchyma of arteries, veins and capillaries.It takes up 20 percent to 30 percent of the acute cerebral-vascular disorders, with high rates of mortality and disability,severely threatening the quality of life and human health. Recently, hypothalamus vegetative nerve dysfunction,complicated by cerebral hemorrhage,has drawn worldwide attention,since it has a serious effect on the prognosis of ICH patients. Generally,the vegetative nerve dysfunction includes stress ulcer,central hyperpyrexia,fluid electrolyte imbalance, and et al.The stress ulcer is one of the most serious complications of acute stroke.Thus,to a certain degree,effective prevention and treatment will improve the patients′prognosis.And the central hyperpyrexia caused by cerebral hemorrhage always indicates the aggravation of the illness and thus increasing dramatically the mortality rate.Hypothalamus,acting as an integration center, regulates the function of the autonomic nervous system by contacting nerve fibres in hypophysis and brainstem,involving nervous incretion,the regulation of temperature,eating and drinking,the sleep-wakefulness cycle,and reproduction. So the research done on hypothalamus will clarify the pathophysiological mechanisms of the ICH-complicated hypothalamus vegetative nerve dysfunction.As everyone knows,proteins are the final executor of gene expression,as well as the embodiment of vital complexity and diversity.The technology of proteomics is a sign that life has entered into post-genome era.It studies the feature of the proteins as a whole,including the expression of proteins,the modification of the post-translation of proteins,the interaction between proteins,and et al,in order to explore the rules between protein functions and life.Differential proteomics,a specialized subdivision of the technology,is used to analyze the differences of protein expression in different organisms at different moments and under different states,which can provide the possibility for the research on molecular mechanism in cell and for tracing the treatment drones.In this paper,in order to find the metabolic proteins and come up with an understanding of the mechanism for ICH-complicated vegetative nerve functional disturbance in hypothalamus,we focused the differential proteomics on normal hypothalamus and abnormal hypothalamus which had suffered from cerebral hemorrhage for 72 hours,by preparing rat models with ICH.In this way,we expected to explore the pathological and physiological mechanisms of ICH-complicated vegetative nerve functional disturbance,and to provide further evidence for screening the disease-specific markers and intervention targets.Methods:1.We made models of rat ICH.Kill the rats at 72 hour and got the tissues of the hypothalamus.Got the corresponding part in controls.2. We extracted the whole tissue proteins, then compared the differences in the protein expressions of normal hypothalamus and abnormal hypothalamus, using 2-dimension electrophoresis(2-DE):isoelectric focusing electrophoresis being the first dimension,and vertical SDS-PAGE electrophoresis being the second dimension.The gels were visualized by colloidal coomassive blue staining and analysed with ImageMaster 2D Elite software.The proteins of interest were in-gel digested and identified,using MALDI-TOF/TOF tandem mass spectrometry.3. We used Western blot technology to verify the alteration of protein VDAC1 and DPYL2.Results:We made models of ICH in rat successfully.Our data showed that the levels of 29 proteins in hypothalamus tissue were significantly altered in ICH group compared with controls.14 up-regulated protein spots were found which were identified as Succinate dehydrogenase [ubiquinone] flavoprotein subunit, Ubiquitin carboxyl-terminal hydrolase isozyme L1,Voltage-dependent anion-selective channel protein 1,14-3-3 protein epsilon,Pyruvate kinase isozymes M1/M2,Cytochrome b-c1 complex subunit 1, Spectrin alpha chain(brain), 4-aminobutyrate aminotransferase(mitochondrial),Dihydrolipoyl dehydrogenase(mitochondrial),Rab GDP dissociation inhibitor alpha, Alpha-internexin,Neurofilament light polypeptide,Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2,Glutamine synthetase. While 15 down-regulated proteins were identified as Creatine kinase B-type, Malate dehydrogenase(mitochondrial),Peroxiredoxin-2,Myelin basic protein S,Phosp- hoglycerate mutase 1,Fructose-bisphosphate aldolase C,Triosephosphate isomerase,Glyceraldehyde-3-phosphate dehydrogenase,N(G),N(G)-dimethyla- rginine dimethylaminohydrolase 1,Dihydropyrimidinase-related protein 2,Pyr- uvate dehydrogenase E1 component subunitbeta,(mitochondrial),Phosphatidy- lethanolamine-binding protein 1,ATP synthase subunit alpha,Glutamate dehydrogenase 1(mitochondrial), Alpha-enolase.Conclusion:1.We got 2-DE maps of the whole hypothalamus tissue proteins from both the ICH group and the control group.2.The identified proteins involved cytoskeleton proteins, energy metabolic enzymes, antioxidant proteins and neurotransmitter enzyme. We concluded that these proteins may be involved in the pathogenesis of ICH-complicated vegetative nerve functional disturbance, laying the foundation for further screening of the disease-specific markers and intervention targets.3.We used Western blot technology to verify the alteration of VDAC1 and DPYL2 , which makes it possible to study the participation of these two proteins in function network.