Proteomic Analysis Of Differential Expression Proteins In Acute Spinal Cord Injury Treated With Tetramethylpyrazine In Rats

OBJECTIVE Compare the differential expression of proteins in spinal tissue between rats treated with tetramethylpyrazine(TMP) and rats treated with normal sodium(NS) after acute spinal cord injury(ASCI) by using proteomic technology and ASCI model.Identify the differential proteins related to the effect of TMP. Analyze the primary function of the differential proteins.Reveal protein molecular mechanism of the curative effect of TMP in rats after ASCI.METHODS A total of 12 healthy adult Sprague Dawley rats were used to establish the ASCI models according to Allen's weight drop methed. These models were divided randomly into two groups:(1) Normal sodium treatment group(group NS,n=6);(2)Tetramethylpyrazine treatment group (group TMP, n=6).Two groups animals were further divided into two sub-groups(n=3 per sub-group), each at 3 and 7 days of survival before euthanasia.200mg/kg of TMP was administered in the TMP group intraperitoneally and the NS group were treated with the same volume of normal solution 30 minutes after injury. Each rat was given the same dosage once a day until euthanasia or survived for 5 days following the spinal cord injury if the rats survived for more than 5 days. At the corresponding time points following the injury, the rats of two groups were killed and the spinal cord tissue samples were collected from the injured segment with the length of 0.6cm. The spinal cord protein's lysate was extracted and the concentrations of proteins were detected by Bradford method. Then the proteins were separated by two-dimensional gel electrophoresis(2-DE).The differential expression proteins were detected by PDQuest-8.0.1 software after the gels were stained with colloidal coomassie blue.These proteins were digested with trypsin into peptides.And the resulting tryptic peptides were analyzed by matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-Q-TOF).The mass spectrometric data were used to identify the proteins by searching SWISS-PROT and NCBInr protein sequence database through MASCOT software.The primary function of the differential proteins was analyzed by literature reviewing.RESULTS High resolution gel maps were produced by 2-DE.The difference of gel maps between two groups were significant. The differential expression proteins were successfully identified by MALDI-Q-TOF.3 days after ASCI, a total of 15 differential expression proteins were identified. Up-regulated proteins were serum albumin,neurofilament light polypeptide,vimentin,glial fibrillary acidic protein,tubulin alpha-1A chain,tubulin beta-2A chain,tubulin beta-2C chain,leucyl aminopeptidase,dihydropyrimidinase-related protein 2; down-regulated proteins were alpha-internexin,60 kDa heat shock protein,heat shock cognate 71 kDa protein,heat shock related 70 kDa protein 2,heat shock protein beta-1,phosphatidylethanolamine-binding protein 1.7 days after ASCI, a total of 19 differential expression proteins were identified. Up-regulated proteins were serum albumin,tubulin alpha-1A chain,tubulin beta-2A chain,neurofilament light polypeptide,neurofilament medium polypeptide,aldehyde dehydrogenase,guanine nucleotide binding protein subunit beta-1,beta-actin,vimentin,glial fibrillary acidic protein;down-regulated proteins were heterogeneous nuclear ribonucleoprotein H2,parkinson disease protein 7 homolog,vesicle-associated membrane protein-associated protein B,ubiquitin-conjugating enzyme E2N,pyruvate dehydrogenase E1 component subunit beta,isocitrate dehydrogen ase [NAD] subunit alpha,creatine kinase B-type,alpha-internexin,60 kDa heat shock protein. By literature reviewing, the majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction.CONCULUSION There are proteomic difference between the expression proteins of TMP group and NS group.The majority of differential expression proteins have been reported to participate in neuron growth, apoptosis and stress-reaction. The data will be helpful to illuminate the mechanism involved in the recovery of ASCI and the curative effect of TMP in rats after ASCI.