| The bovine Pancreatic Typsin inhibitor (BPTI) is a representative inhibitor of Kunitz family.BPTI is a serine protease inhibitor.BPTI is a kind of nonspecific and broad-spectrum serine protease inhibitor which affects known serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein. It can protect blood platelet against bleeding, treat the patient with cruor impediment, in which situation the hemorrhage may occur. BPTI can be used in cardiac surgery as being extracorporal circulated to decrease the incidence of bleeding and the inflammatory reaction caused by the surgery. But infusion of high doses of these nonmammalian inhibitor could result in immunologic reactions in patients undergoing repeat use. So we need find a substitute which is preferable ,small ,potent and of human sequence origin.The amyloidβ-protein precursor contains a domain homologous to Kunitz-type serine protease inhibitors.The sequence of KPI/AβPP which is composed of 57 amino has 43% sequence identity with that of BPTI. The tertiary fold, reactive center and mechanism of function of KPI/AβPP is very similar to that of the Kunitz inhibitor BPTI.The KPI containing isoforms (APPIs) show ability to inhibit a variety of serine proteases such as factor XIa, epidermal growth factor (EGF)-binding protein, theγ-subunit of nerve growth factor, especially the inhibition constant Ki of KPI/AβPP to factor XIa is higher than BPTI. The APPI is an effective cell adhesion molecule, which has growth activity. They probably play a role in the happening of series of events that lead to tissuerepair at vascular wound sites and protect neurocytes. The pathological and physiological role of this protease inhibitor domains which concerns with abnormal metabolism of rhKPI/AβPP are not well understood. So we need to produce large quantity of rhKPI/AβPP to discover and study the possible mechanism on pharmacodynamics.Increasing evidence has shown that brain injury can develop as a result of cerebral ischemia-reperfusion due to stroke and other cardiovascular diseases. However, the mechanism on cerebral injury is not understood. The aim of this study is to investigate the protective effect and mechanism of on rhKPI/AβPP cerebral injury induced by brain ischemia/reperfusion of rats.It can provide a theory basis for its clinical application on cerebral visculardiseases.1. The construction and identification of rat cerebralischemia-reperfusion model According to the method, middle cerebral artery occulusion (MCAO), formed by Zealonga, to construct the rat cerebral ischemia-reperfusion model. Afer the operation,observed if the rat appeared the following symptom to judge if the model constructed successfully.①Symptom of Hornor.②The left limbs of the rats are nerveless, the symptom is the left forelimb adduction crooked while lift the rat's tail, and the rats tilted to the left or turn around counter-clockwise while crawling. Their tails turned coiled while they were still.2.The study of the neural protective effect of rhKPI/AβPP on ischemia reperfusion injury in rat brain The purpose is to investigate the protective effects of rhKPI/AβPP on cerebral ischemia-reperfusion injury in rats. Rats were pretreated with rhKPI/AβPP for 1h and then subjected to cerebral ischemia/reperfusion injury induced by a middle cerebral artery occlusion (MCAO). After 3h ischemia and 24h reperfusion, the neurological outcome was evaluated by the Longa'smethod; the infarct volume was assessed by TTC staining and the cerebral water content was measured by dry-weight method. rhKPI/AβPP (8,16 mg·kg-1) significantly reduced the cerebral infarct volume and water content, and ameliorated the neurological deficit (P<0.05 or P<0.01). These results showed that pretreatment with rhKPI/AβPP could decrease brain injury after cerebral ischemia/reperfusion.1)The assay of neurological functionThe neurological function of model group impaired seriously, the death rate is 27.1% and the neurological function of the Nimodipine group is better than that of the model group(P<0.05),but the death rate is still higher than that of the rhKPI/AβPP group ( P<0.05 ) .The neurological function of the rhKPI/AβPP groups are better than that of the model group(P<0.01),and on the test extent the more dose of rhKPI/AβPP, the higher score of the neurological function. The death rate of rhKPI/AβPP groups are lower than that the model group(P<0.05).2) The assay of pathologyThe results of pathology stained by HE showed that the neurons of the fake operation group and non-ischemia area of the operation groups are normal.The ischemia area of the operation groups are light stained and the brain tissues were liquefied and necrotized. The room between the cells turned larger and the edema was obvious. There were many PMN cells. Compared to medel groups,there were less necrotized neurons in the Nimodiphine group and the rhKPI/AβPP groups, and the edema around the necrotized tissues lessened.3)The assay of the area of cerebral infarct and edemaAfter 3h ischemia and 24h reperfusion, the neurological outcome was evaluated by the Longa's method; the infarct volume was assessed by TTCstaining and the cerebral water content was measured by dry-weight method. rhKPI/AβPP (8,16 mg·kg-1) significantly reduced the cerebral infarct volume and water content, and ameliorated the neurological deficit (P<0.05 or P<0.01). These results showed that pretreatment with rhKD/APP could decrease brain injury after cerebral ischemia/reperfusion.4)The assay of the expression of ICAM-1 and E-selectinTo investigate the effects of rhKPI/AβPP on neutrophil migration and adhesion molecules after middle cerebral artery occlusion,rats were pretreated with rhKPI/AβPP for 1h and then subjected to cerebral ischemia/reperfusion (I/R) injury induced by a middle cerebral artery occlusion. The expressions of adhesion molecules (ICAM-1 and E-selectin) were determined by immunohistochemistry. The results showed that in rhKPI/AβPP group (8,16 mg·kg-1), the cells expressed ICAM-1and E-selectinwere were obviously less(P<0.05 or P<0.01, vs model group).To sum up,we have constructed rat cerebral ischemia-reperfusion model successfully,and treated rat cerebral ischemia-reperfusion model with rhKPI/AβPP to observe the protective effection of rhKPI/AβPP on the rat brain ischemia-reperfusion injury. The results showed that rhKPI/AβPP could improve the neural fuction,reduce the infarct volume,relieve the cerebral edma,enhance the endurance of the rat's brain to the ischemia and reduce the death rate. It is relative to rhKPI/AβPP can obviously inhibit the expressions of the adhesion molecules (ICAM-1, E-selectin) after cerebral ischemia-reperfusion.
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