Proteomic Analysis Of Neuroendocrine-immune Network Of Liver-Yang Hyperactivity Syndrome And Liver Yang Forming Wind Syndrome In Rat

Objective:(1)To establish the model of hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome which have the characteristic of combined Disease and Syndrome.(2)To investigate the protein profile in the rat pituitary,adrenal gland, thyroid gland and splenic lymphocyte using proteomics techniques, and to elucidate the nature of Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome from the view of the neuroendocrine-immune system,which can be helpful to provide new field for approaching the nature of the two Syndromes.(3)To elucidate the essence of the "different syndrome complexes in the same disease" and "the same syndrome complex in different diseases" by analyzing the undifferential and differential expression proteins between Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome.Methods:(1)Randomly selected 15 rats from 70 male SD rats as normal control group,and the remnant 60 rats as hypertensive with Liver-Yang Hyperactivity Syndrome which was made through pouring "Aconite Decoction" and drinking 1.5%hypertonic saline for six weeks. Observing the degree of irritation and conjunctiva hyperemia and measuring the blood pressure and rotation time after six weeks.(2)The model of hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome was made through injecting TypeⅦcollagenase into the right globus pallidus(GP) of rats by stereotaxic method on the base of the model of hypertensive with Liver-Yang Hyperactivity Syndrome.Then,the brain tissues were made sections,stained by HE method,to evaluate the histological changes.Furthermore,neuropathological profile was observed at the same time.(3)The model of cerebral ischemia with Liver-Yang Forming Wind Syndrome was made through occluding the middle cerebral artery on the base of the model of hypertensive with Liver-Yang Hyperactivity Syndrome.Then,the brain tissues were made sections,stained by TTC method,to evaluate the histological changes.Furthermore, neuropathological profile was observed at the same time.(4) The total proteins of pituitary,adrenal gland,thyroid gland and splenic lymphocyte in the rats were separated by two-dimensional gel electrophoresis(2-DE) respectively.The gels of 4 groups were stained by Coomassie brilliant blue,scanned by ImageScanner and analyzed in PDQuest software.The undifferential and differential expression protein spots were identified by peptide mass fingerprint(PMF) based on matrix -assisted laser desorption/ionization time of flight mass spectrometry(MALDI- TOF-MS) and database searching,then some of them were validated by RT-PCR and western blotting.Results:(1)Copying rats model:Forty-eight rats were reproduced into a successful model of hypertensive with Liver-Yang hyperactivity Syndrome among 60 rats,with the incidence of 80.0%approximately. The 48 rats were randomly divided into three groups:hypertensive with Liver-Yang Hyperactivity Syndrome group including 14 rats, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome group including 17 rats and cerebral ischemia with Liver-Yang Forming Wind Syndrome group including 17 rats.(2)Evaluation of the models:①hypertensive with Liver-Yang hyperactivity Syndrome model group with high aggression and conjunctiva hyperemia and high blood pressure and shortened rotation time.Compared with the normal control group the difference was significant(P＜0.05).②Affer operation,the majority of the hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome rats showed up conjunctival congestion and the aggression degree further deepened.Compared with the normal control group and hypertensive with Liver-Yang hyperactivity Syndrome,the difference were significant(P＜0.05).The rotation time of hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group shortened and blood pressure heightened.Compared with the normal control group and hypertensive with Liver-Yang hyperactivity Syndrome,the difference were significant(P＜0.05).After injecting TypeⅦcollagenase,the neurological deficits score of 13 rats are more than 2 scores.After occluding the middle cerebral artery,the neurological deficits score of 12 rats are more than 2 score.Lymphocytes,other kinds of leukocytes, glial cells and lots of red blood cells were observed in the tissues of hematoma area after stained by HE in the hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome group. Under staining with 0.1%TTC,the infarcted tissues caused by middle cerebral artery occlusion(MCAO) were white in color on the injured side,while the normal brain tissues on the non-injured side and the sham-operated controls were pink in color in cerebral ischemia with Liver-Yang Forming Wind Syndrome groups.(3) Establishment of 2-DE maps in the rat pituitary,adrenal gland,thyroid gland and splenic lymphocyte and image analysis:Well-resolved and reproducible 2-DE maps of rat pituitary,adrenal gland,thyroid gland and splenic lymphocyte from normal,hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group were acquired.As compared with the control 2-DE maps,the gels of the pituitary displayed approximately 1100±32 protein spots,the 2-DE maps of the adrenal gland displayed 800±56 spots,the 2-DE maps of the thyroid gland displayed 500±17 spots,and the 2-DE gels of the spleen lympholeukocytes displayed 1200±24 spots.The location and abundance of the majority of protein spots are consistent,mainly distributed in the pH4-8 and Mr20-60kD range.(4) Select the undifferential and differential expression proteins for further analysis by MALDI-TOF-MS.46 spots more than 2 folds differential expression protein spots were identified.The proteins were divided into seven groups based on their functions using information obtained from the Swiss-Prot and NCBInr websites:antioxidant,metabolic enzymes,protein biosynthesis and degradation,structural proteins, chaperone,thromboxane enzyme,and so on.Steroidogenic acute regulatory protein,ubiquitin carboxyl-terminal hydrolase isozyme L1, dihydropyrimidinase-related protein 2,peroxiredoxin may concerned with the neuroendocrine-immune network in Liver-Yang hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome.(5)Viladation of differentially expressed protein:①The results of RT-PCR:STAR mRNA,DRP-2 mRNA and PRDX2 mRNA is stronger in hypertensive with Liver-Yang Hyperactivity Syndrome, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.01) and it is also stronger in hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).UCH-L1 mRNA is weaker in hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.05) and it is also weaker in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).The expression level of STAR mRNA,DRP-2 mRNA,UCH-L1 mRNA and PRDX2 mRNA in pituitary and adrenal gland are identical.②The results of Western blotting:STAR is stronger in hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal contral group(P＜0.01) and it is stronger in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05).UCH-L1 mRNA is weaker in hypertensive with Liver-Yang Hyperactivity Syndrome, hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than the normal control group(P＜0.05) and it is weaker in hypertensive intracerebral hemorrhage(HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group than hypertensive with Liver-Yang Hyperactivity Syndrom group(P＜0.05).It showed no significant difference between hypertensive intracerebral hemorrhage (HICH) with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome group(P＞0.05). The results are consistent with the results of proteomics and RT-PCR. Conclusion:(1)Founded successfully animal models of hypertensive with Liver-Yang Hyperactivity Syndrome,hypertensive intracerebral hemorrhage with Liver-Yang Forming Wind Syndrome and cerebral ischemia with Liver-Yang Forming Wind Syndrome.The models have the characteristic of combined Disease and Syndrome.(2) The forming process of Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome is a complex pathological process which many proteins involved in.The neuroendocrine-immune system was obviously changed,which suggest that the neuroendocrine - immune network of the two syndromes are obvious disturbed.(3) There are undifferential and differential proteins between Liver-Yang Hyperactivity Syndrome and Liver-Yang Forming Wind Syndrome which suggest that two syndrome have the same basic material and different nature connotations.